Structure and conformations of the bovine ATP synthase bysingle-particle electron cryomicroscopy
by
Anna Zhou
A thesis submitted in conformity with the requirementsfor the degree of Master of Science
Graduate Department of Medical BiophysicsUniversity of Toronto
© Copyright 2016 by Anna Zhou
Abstract
Structure and conformations of the bovine ATP synthase by single-particle electron
cryomicroscopy
Anna Zhou
Master of Science
Graduate Department of Medical Biophysics
University of Toronto
2016
Adenosine triphosphate (ATP), the energy currency of biology, is synthesized primarily
by the mitochondrial ATP synthase in most eukaryotes. Proton translocation in the
membrane-bound FO region leads to ATP synthesis in the catalytic F1 region through
rotation of a central rotor. The generation of rotation is proposed to occur through a
Brownian ratchet mechanism requiring two offset half-channels in the FO region. Here,
using single-particle electron cryomicroscopy (cryo-EM), seven distinct conformations
within three rotational states of the ATP synthase are presented at sub-nanometre
resolution. An average of the FO region of all seven maps provided a detailed view of the
proton-translocating a subunit, allowing an atomic model to be built using evolutionary
co-variance. The arrangement of subunits in the membrane region suggests two half-
channels for proton translocation. These channels, along with an observed oscillation
of the c8-ring in the FO region between substates in each rotational state, supports the
Brownian ratchet mechanism for the generation of rotor rotation and ATP synthesis.
ii
Acknowledgements
I would like to thank:
My supervisor, John Rubinstein, for his constant support and encouragement, and for
providing me with opportunities to learn and grow in my graduate studies and beyond.
My supervisory committee members, Mitsu Ikura and John Brumell, for their helpful
ideas and feedback.
My labmates, the Molecular Structure & Function community at the SickKids Research
Institute, and the students and faculty of the Department of Medical Biophysics for all of
the laughs, good food, and great conversations.
My family and friends, for always being there for me during my graduate school journey.
iii
Contents
List of Figures viii
List of Movies viii
List of Abbreviations viii
1 Introduction 1
1.1 The ATP synthase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.1 Biological context . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1.2 Structure and composition . . . . . . . . . . . . . . . . . . . . . . 3
1.1.3 Rotary catalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.1.4 The IF1 inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.1.5 Brownian ratchet mechanism for proton translocation . . . . . . . 11
1.1.6 Anchoring of the peripheral stalk . . . . . . . . . . . . . . . . . . 14
1.1.7 Oligomerization and membrane curvature . . . . . . . . . . . . . . 15
1.2 Single particle electron cryomicroscopy (cryo-EM) . . . . . . . . . . . . . 16
1.2.1 Introduction to biological cryo-EM . . . . . . . . . . . . . . . . . 16
1.2.2 Microscopy theory . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.2.3 Protein structure determination with single particle cryo-EM . . . 18
1.2.4 Specimen preparation . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.2.5 Image processing . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
1.2.6 Near-atomic resolution with single particle cryo-EM . . . . . . . . 24
iv
1.2.7 Study of the structure of the ATP synthase using single particle
cryo-EM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
1.3 Thesis objectives and overview . . . . . . . . . . . . . . . . . . . . . . . . 27
1.4 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2 Methods 29
2.1 Protein purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2 Specimen preparation and microscopy . . . . . . . . . . . . . . . . . . . . 29
2.3 Image processing and 3D map refinement . . . . . . . . . . . . . . . . . . 30
2.4 Map analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.5 Building of atomic models . . . . . . . . . . . . . . . . . . . . . . . . . . 35
3 Results and Discussion 37
3.1 Three rotational states of the ATP synthase . . . . . . . . . . . . . . . . 37
3.2 Membrane curvature and a novel feature in the FO region . . . . . . . . . 44
3.3 The arrangement of subunits in the FO region . . . . . . . . . . . . . . . 47
3.4 An atomic model of the a subunit . . . . . . . . . . . . . . . . . . . . . . 51
3.5 A model for proton translocation . . . . . . . . . . . . . . . . . . . . . . 55
3.6 Conformational changes between substates . . . . . . . . . . . . . . . . . 60
3.7 Implications of flexibility in the ATP synthase . . . . . . . . . . . . . . . 63
4 Conclusions and future directions 66
4.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.2 Strategies for higher resolution of the intact ATP synthase . . . . . . . . 67
4.3 Unanswered questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.3.1 The catalytic cycle of the ATP synthase . . . . . . . . . . . . . . 69
4.3.2 Dynamics of the intact ATP synthase . . . . . . . . . . . . . . . . 70
4.3.3 Structure of FO subunits . . . . . . . . . . . . . . . . . . . . . . . 71
v
List of Figures
1.1 Structure of the mitochondrion . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 The mitochondrial electron transport chain . . . . . . . . . . . . . . . . . 4
1.3 Structure of the mitochondrial ATP synthase . . . . . . . . . . . . . . . . 5
1.4 Binding change mechanism and rotary catalysis. . . . . . . . . . . . . . . 9
1.5 IF1 inhibitor protein bound to F1. . . . . . . . . . . . . . . . . . . . . . . 12
1.6 Brownian ratchet mechanism . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.7 Single particle cryo-EM sample preparation . . . . . . . . . . . . . . . . . 20
2.1 Program for masking contamination in micrographs. . . . . . . . . . . . . 32
2.2 Particle image collection and processing. . . . . . . . . . . . . . . . . . . 33
3.1 Three main rotational states of the ATP synthase. . . . . . . . . . . . . . 38
3.2 Seven conformations of the ATP synthase. . . . . . . . . . . . . . . . . . 39
3.3 Fourier shell correlation (FSC) curves. . . . . . . . . . . . . . . . . . . . 40
3.4 Local resolution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.5 Atomic models fit into maps. . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.6 Features in the FO region. . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.7 Segmentation of the FO region. . . . . . . . . . . . . . . . . . . . . . . . 48
3.8 Evolutionary co-variance constraints. . . . . . . . . . . . . . . . . . . . . 53
3.9 Model of the a subunit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3.10 Proposed proton translocation channels. . . . . . . . . . . . . . . . . . . 57
3.11 Disease-causing mutations in the a subunit . . . . . . . . . . . . . . . . . 59
3.12 Differences between substates. . . . . . . . . . . . . . . . . . . . . . . . . 62
vii
List of Movies
Movie 1. Conformational changes during the rotary cycle
Movie 2. Conformational changes between substates (Side view)
Movie 3. Conformational changes between substates (Top view)
Movie 4. Brownian ratchet mechanism
List of Symbols and Abbreviations
2D two–dimensional3D three–dimensionalA angstromADP adenosine diphosphateATP adenosine triphosphateCMC critical micelle-forming concentrationcryo-EM electron cryo-microscopyCTF contrast transfer functionDAPIT Diabetes-Associated Protein in Insulin-sensitive TissuesDDM dodecylmaltoside∆x translation along x axis∆y translation along y axisEMDB Electron Microscopy Data BankETC electron transport chainFSC Fourier shell correlationFT Fourier transformIF1 inhibitory factor 1IF11-60 ATP synthase inhibitor with residues 61 onwards deletedIMS intermembrane spacekDa kilodaltonLHON Leber hereditary optic neuropathyLS Leigh syndromeMC5DM1 Mitochondrial complex V deficiency, mitochondrial 1MIBSN Mitochondrial infantile bilateral striatal necrosisMLASA3 Myopathy, lactic acidosis, and sideroblastic anemia 3MPTP mitochondrial permeability transition pore
viii
MSA multiple sequence alignmentNADH nicotinamide adenine dinucleotideNARP Neuropathy, ataxia, and retinitis pigmentosaNMR nuclear magnetic resonanceOSCP oligomycin-sensitivity conferring proteinOTR orthogonal tilt reconstructionPDB Protein Data Bankϕ Euler angle describing rotation about the z” axisPi inorganic phosphatePSF point spread functionψ Euler angle describing rotation about the z axisRCT random conical tiltROTAN rotational analysisSNR signal-to-noise ratioθ Euler angle describing rotation about the -y’ axis
ix
Chapter 1
Introduction
1.1 The ATP synthase
1.1.1 Biological context
The mitochondrion is the organelle found in most eukaryotic cells where most adenosine
triphosphate (ATP), the energy currency of biology, is generated from the oxidation of
nutrients. Due to the relative cellular concentrations of ATP, adenosine diphosphate
(ADP) and inorganic phosphate (Pi), free energy is released when ATP is hydrolyzed
by the cell to ADP and Pi. ATP hydrolysis is therefore used to drive many cellular
processes. However, the production of ATP is energetically costly. Cells have evolved
proteins that work together to store the energy from metabolism in the chemical form of
ATP. In eukaryotes, these proteins make up the electron transport chain (ETC), a series of
metalloprotein complexes located in the inner membrane of mitochondria. In addition to
this inner membrane, which has highly folded cristae structures (Fig. 1.1), mitochondria
also have an outer membrane. The double membrane encloses the intermembrane space
(IMS) between its layers and surrounds the matrix.
During oxidative phosphorylation, substrates that were reduced during the breakdown
of nutrients (eg. carbohydrates and fatty acids), including nicotinamide adenine dinu-
1
Chapter 1. Introduction 2
Intermembrane space
Cristae
Inner membraneOuter membrane
Matrix
Figure 1.1: Structure of the mitochondrion. The mitochondrion is the organelle where ATP isproduced in most eukaryotes. It has a double membrane, which surrounds the matrix. Betweenthe membranes is the intermembrane space (IMS). The inner membrane forms highly-foldedstructures called cristae.
Chapter 1. Introduction 3
cleotide (NADH) and succinate, pass their electrons to the mitochondrial ETC protein
complexes (Fig. 1.2). Complex I (NADH dehydrogenase) receives electrons directly from
NADH. Complex II (succinate dehydrogenase) receives electrons directly from succinate.
Electrons are then transferred from both complex I and complex II to complex III (cy-
tochrome bc1 complex), then to cytochrome c, and finally complex IV (cytochrome c
oxidase). During these electron transfers, energy from the oxidation-reduction reactions
are used by complexes I, III and IV to pump protons across the inner mitochondrial
membrane from the matrix to the IMS. An electrochemical gradient or the proton motive
force (PMF)is established, which is required for ATP synthesis. The ETC culminates with
the reduction of molecular oxygen to water by complex IV. In the final step of oxidative
phosphorylation, the ATP synthase uses the PMF, or more specifically the energy from
the flow of protons down the proton gradient from the IMS to the matrix, to synthesize
ATP from ADP and Pi.
1.1.2 Structure and composition
The ATP synthase (Fig. 1.3) is a ∼550 kDa reversible molecular motor. It can be
divided into four functional parts: the soluble F1 (Fraction 1) region that catalyzes ATP
synthesis; the membrane-bound FO (Fraction binding Oligomycin) region through which
proton translocation occurs; the central rotor that couples proton translocation with ATP
synthesis; and the peripheral stalk that holds the F1 region stationary relative to FO. The
ATP synthase is oriented in the inner mitochondrial membrane with the F1 region in the
matrix.
The F1 region is made of subunits α3β3γδε [46, 1]. These subunits are conserved across
eukaryotes. The α and β subunits are nearly identical in their structures despite only
sharing ∼20% sequence identity, differing mostly in their C-terminal 40 residues. There
are six nucleotide binding sites in the α3β3 hexamer, three of which are catalytic sites and
three of which are non-catalytic. The nucleotide-binding site at the catalytic interface
Chapter 1. Introduction 4
¹⁄2 O2H2O
ADP + Pi ATPNADH NAD+ + H+
succinate fumarate
Complex I
Complex II
QComplex III Complex IV
cyt c
ATP synthase
Intermembrane space
MatrixH+H+
H+
H+
H+H+
H+ H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
Figure 1.2: The mitochondrial electron transport chain. The electron transport chain (ETC) islocated in the inner mitochondrial membrane. Electron transfer from substrates reduced duringmetabolism to the metalloprotein complexes in the ETC is used to form an electrochemicalgradient across the inner membrane. The proton motive force (PMF) that is established isharnessed by the ATP synthase (blue) to generate ATP.
Chapter 1. Introduction 5
F1
FOafA6L e, g
b
OSCP
α β
γ
δε
d
F6
c8
Figure 1.3: Structure of the mitochondrial ATP synthase. The mitochondrial ATP synthaseis found in the inner membrane of mitochondria. It has a catalytic F1 region and a proton-translocating FO region, connected by a central rotor and peripheral stalk. Crystal structures ofthe bovine ATP synthase are shown (PDB IDs: 2WSS, 2XND, 2CLY [129, 166, 41]). There areno crystal structures for the membrane subunits except the c8-ring. Scale bars 25 A.
Chapter 1. Introduction 6
is formed predominantly by the β subunit, with the adjacent α subunit providing an
essential “arginine finger” (Arg373 in Escherichia coli and Saccharomyces cerevisiae).
The binding site at the non-catalytic interface is formed mainly by the α subunit. The
specific residues involved in catalysis and the binding of substrates were first determined
mostly from mutational studies of the E. coli enzyme [109] and later confirmed by crystal
structures of the F1 region from various organisms [105, 82, 80]. Crystal structures of the
F1 region also revealed that the non-catalytic sites are different from the catalytic sites in
that they lack an equivalent residue in an appropriate position to act as a catalytic base
(Glu188 in the β subunit is replaced by Gln208 in the α subunit). The non-catalytic sites
are unable to cycle between different conformations during the catalytic cycle (described
in section 1.1.3).
The membrane-bound FO region is made of subunits a, c8, e, g, f, A6L, Diabetes-
Associated Protein in Insulin-sensitive Tissues (DAPIT), 6.8 kDa proteolipid, and two
transmembrane α-helices from the b subunit [33]. The core membrane-bound subunits a,
b and c are found in all ATP synthases and are the only subunits necessary for proton
translocation in the minimal versions of the ATP synthase in bacteria [30, 59, 67]. The
additional mitochondrial ATP synthase subunits, A6L, f, e, g, DAPIT and 6.8 kDa
proteolipid, are each predicted to have one transmembrane α-helix [18]. The functions
of the essential A6L and f subunits are unknown [43, 96]. The non-essential e and g
subunits are predicted to be involved in oligomerization of the mitochondrial ATP synthase
(discussed in section 1.1.7) [7]. DAPIT and 6.8 kDa proteolipid are also non-essential
and are proposed to be involved in maintaining the populations of ATP synthase in
mitochondria [114, 51].
The F1 and FO regions contain parts of the central rotor and peripheral stalk, which
connect the two regions. The central rotor is made of subunits γδε in the F1 region [1] and
the c8-ring in the FO region. The γ subunit is bound to the c8-ring [166]. The peripheral
stalk that holds the catalytic F1 subunits stationary relative to the FO region is made of
Chapter 1. Introduction 7
subunits d, F6, oligomycin-sensitivity conferral protein (OSCP) and the soluble portion
of the b subunit [35, 34]. Despite its name, OSCP does not bind oligomycin, an ATP
synthase inhibitor. Oligomycin binds the FO region to inhibit proton translocation [155]
and requires OSCP. While the sequence identity of similar peripheral stalk proteins in
the bovine and yeast enzymes is low (∼14.5% between the bovine F6 subunit and the
similar h subunit in yeast), their functions appear to be conserved, as deletion of the h
subunit in yeast can be complemented with cDNA encoding the bovine F6 subunit [164].
The structure of the peripheral stalk of the eukaryotic ATP synthase is quite different
from the structure of the E. coli ATP synthase peripheral stalk, which is composed of
subunit δ (the bacterial equivalent to the mammalian OSCP subunit) and two copies of
the membrane-bound bacterial b subunit [45].
Crystal structures of the F1 region and peripheral stalk of the bovine ATP synthase
have been determined, providing important insight into the architecture of these protein
complexes [41, 129] (Fig. 1.3). There are structures of F1 with and without the c8-ring
[166, 129] and a structure of a portion of the peripheral stalk with segments of subunits b,
d and F6 [41]. The only high-resolution structures available for subunits in the FO region
are of the c-ring. c-rings with eight subunits are predicted to be conserved in all animals
[166]. Although the numbers of c subunits in the crystallized c-rings of different organisms
differ from 8 to 15 [166, 152, 124], all c subunits were found to have an outward-facing
acidic residue on helix 2 that is located near the middle of the membrane bilayer. This
residue is conserved and proposed to be involved in proton binding (section 1.1.5).
The difference in the number of c subunits means that different organisms may have
different bioenergetic costs of producing ATP. This cost is associated with the ratio of the
number of ATP molecules produced to the number of oxygen atoms reduced by the ETC,
termed the P/O ratio [166]. The P/O ratio depends, in part, on the number of c subunits
that translocate protons for each full turn of the rotor that results in the synthesis of three
ATP molecules. For the c-ring of the mammalian mitochondrial ATP synthase with eight
Chapter 1. Introduction 8
c subunits, theoretically, the maximum P/O ratio is 2.7 for electrons from NADH (that
enter the ETC at NADH dehydrogenase) and 1.6 for electrons from succinate (that enter
the ETC at succinate dehydrogenase) [166]. P/O ratios of 2.5 and 1.5, respectively, have
been observed [89, 64]. For yeast, which have a different ETC protein complex I that does
not translocate protons, and which have ten c subunits in the c-ring of its ATP synthase,
the P/O ratio is around 1.3 for both NADH and succinate [81]. The bioenergetic cost of
ATP production also depends on the [ATP]/[ADP] ratio in the cell.
1.1.3 Rotary catalysis
ATP is synthesized through a rotary catalytic mechanism. Proton translocation across
the FO region is driven by the transmembrane PMF, and occurs from the IMS to the
matrix at the interface between the c8-ring and the a subunit. The movement of protons
is coupled to rotation of the central rotor. Since the central rotor is asymmetric and
extends into the α3β3 hexamer [152], a sequence of concerted conformational changes is
induced in the three catalytic αβ sites as the rotor turns. These changes are described by
the binding change mechanism first hypothesized by Paul Boyer [22], and are illustrated
in Figure 1.4. Depending on the position of the rotor, each αβ catalytic site assumes one
of three conformations: loose, tight or open. The loose conformation binds ADP and
Pi, the tight conformation contains ATP, and the open conformation does not contain
nucleotide. During ATP synthesis, each site cycles through the three states, binding ADP
and Pi and releasing ATP, resulting in the production of three ATP molecules per 360◦
rotation of the rotor. The position of the peripheral stalk with respect to the catalytic
subunits is fixed, resulting in at least three distinct conformations of the ATP synthase.
The structure of the soluble F1 region is well-characterized by crystal structures of the
region with and without substrate analogs and inhibitors [80, 21, 52, 56]. Crystallization
has been facilitated by the ease of dissociation of the F1 region and central rotor from the
FO region and peripheral stalk in vitro. A combination of this high-resolution structural
Chapter 1. Introduction 9
T
LO
O
TL
L
O
T
ATP hydrolysis
ATP synthesis
ADP
ADP
ADP
ATP ATP
ATP
Figure 1.4: Binding change mechanism and rotary catalysis. Cross-sections through the α3β3hexamer are illustrated with α subunits in red, β subunits in yellow, the central rotor in blue,and the peripheral stalk in green. Each catalytic site in the F1 region assumes either an open(O), tight (T) or loose (L) conformation, depending on the position of the asymmetric rotor.The sequence of conformational changes in the catalytic sites is reversed between ATP synthesisand ATP hydrolysis. Due to the fixed position of the peripheral stalk, the enzyme has at leastthree distinct conformations.
Chapter 1. Introduction 10
information [21, 105], computational studies [63, 116] and single molecule experiments
[112, 2, 6, 101, 161] has provided a detailed understanding of catalysis in the F1 region.
These experiments were done by observing ATP hydrolysis by the isolated F1 region,
which does not require the presence of a PMF across membranes to occur. During ATP
hydrolysis in the intact enzyme, the binding of ATP and release of ADP and Pi in the F1
region result in conformational changes in the catalytic sites that drive rotor rotation.
Although not confirmed experimentally, the order of the structural changes observed
during the hydrolytic cycle are assumed to be the opposite during ATP synthesis.
1.1.4 The IF1 inhibitor
Energy released through ATP hydrolysis by the ATP synthase can be coupled to the
maintenance of a proton gradient across the inner mitochondrial membrane. However,
wasteful ATP hydrolysis, as would occur due to an uncoupling agent that dissipates
the proton gradient, dissociation of F1 from FO, or production of free F1 during the
assembly of the ATP synthase [128] is detrimental to the cell because it compromises
oxidative phosphorylation. ATP hydrolysis during ischemia, or lack of oxygen in tissues,
during which the PMF collapses, leads to cell death even after reperfusion (termed
ischemia/reperfusion injury). In mitochondria, futile ATP hydrolysis is inhibited by
inhibitory factor 1 (IF1) [126]. The bovine IF1 is a naturally-occurring 84-residue protein
that binds the ATP synthase in conditions that promote ATP hydrolysis [138]. Its
C-terminal 49-81 residues are involved in dimerization, and its N terminus binds and
inhibits the ATP synthase. At pH >6.5, IF1 exists as an α-helical, autoinhibited tetramer
[28]. At pH <6.5, protonation of His49 leads to the dissociation of the tetramer into two
active dimers held together by a coiled coil [29]. The IF1 homodimer is able to inhibit
pairs of ATP synthases.
An IF1 mutant containing only residues 1-60 and lacking the dimerization region
(IF11-60) is a potent, monomeric inhibitor with a Ki of 30 nm−1 [17]. A crystal structure
Chapter 1. Introduction 11
of IF11-60 bound to the bovine F1 region in a ratio of 1:1 showed that IF1 binds at the
loose (ADP-containing) catalytic site [56]. Another crystal structure where the IF11-60
mutant is bound at all three catalytic sites provided insight into the mechanism through
which IF1 binds the ATP synthase to inhibit ATP hydrolysis [15] (Fig. 1.5). The N
terminus of IF1 is disordered in solution before it interacts with a catalytic αβ site in
the open conformation. The interaction is mediated through polar/charged interactions
that induce folding. ATP hydrolysis leads to the formation of additional IF1:F1 contacts.
Hydrolysis of a second ATP molecule results in the final inhibited form of IF1:F1, in which
IF1 is stabilized in an α-helix via numerous electrostatic and hydrophobic interactions in
the active site of the αβ heterodimer in the loose conformation.
1.1.5 Brownian ratchet mechanism for proton translocation
While the cooperative conformational changes in the F1 region that couple rotor rotation
to ATP synthesis are well understood, the lack of atomic models for almost all of the
subunits in the FO region means that the mechanism through which proton translocation
is coupled to rotor rotation is still unclear. Movement of protons in FO is believed to
be coupled to rotation of the central rotor by the Brownian ratchet model (Fig. 1.6)
[77, 78]. In this model, there are two offset aqueous half-channels near the a subunit/c-ring
interface, each allowing access to different sides of the membrane. A proton will enter one
half-channel in the direction of the proton gradient to protonate the negatively charged
Glu58 residue on one c subunit (bovine ATP synthase numbering). Once the Glu58
residue is protonated and neutralized, it can no longer interact favourably with the highly
conserved, positively charged Arg159 on the a subunit. Instead, the protonated Glu58
residue will partition into the hydrophobic lipid bilayer and the c-ring will rotate. While
one Glu58 residue becomes protonated, a previously protonated Glu58 residue on an
adjacent c subunit becomes aligned with the second half-channel. The Glu58 residue
becomes hydrated, and the proton is released. Since the deprotonated Glu58 residue is
Chapter 1. Introduction 12
O
T
L
O
T
L
Figure 1.5: IF1 inhibitor protein bound to F1. Structure of the F1 region of the ATP synthasewith IF11-60 bound at all three catalytic sites (PDB ID:4Z1M [16]), looking in the directionfrom the FO region towards the F1 region. IF11-60 is in purple, α subunits in red, β subunits inyellow and the central rotor in blue. The conformations of the catalytic pairs are indicated aseither open (O), loose (L) or tight (T). IF1 is in its final inhibitory conformation in the loosecatalytic site. This conformation is the most α-helical and has the most interactions with F1.Scale bar 25 A.
Chapter 1. Introduction 13
Arg
a subunit c-ring
-COO- -COOHproton
Figure 1.6: Brownian ratchet mechanism. The Brownian ratchet mechanism for coupling ofproton translocation in the FO region to rotor rotation requires two offset half-channels atthe interface of the a subunit (green) and c-ring (yellow). Each half-channel is only accessiblefrom one side of the membrane. Rotation is generated by the protonation and deprotonationof conserved acidic residues on the c-ring, as described in section 1.1.5. The path of protonsis indicated by black arrows. An acidic residue on a c subunit becomes protonated at onehalf-channel, and moves through the membrane bilayer before becoming deprotonated at theother half-channel. The essential and conserved Arg residue on the a subunit that interacts withdeprotonated acidic residues on the c-ring is indicated in blue.
Chapter 1. Introduction 14
now negatively charged, it cannot partition back into the lipid bilayer, and will interact
with the positively charged Arg159 residue. Glu58 residues protonated at one half-channel
travel through the lipid bilayer before reaching the second half-channel and releasing the
proton.
The random movement of particles due to collisions with the surrounding liquid or
gas molecules is called Brownian motion. Electrostatic and hydrophobic forces prevent an
unprotonated, charged acidic residue on a c subunit from entering the lipid bilayer due
to the large energetic penalty. Thus, Brownian oscillations of the central rotor relative
to the other FO subunits, induced by the small random motions of the water and lipid
molecules surrounding it, will only result in rotation by one c subunit upon the binding of
protons to the c-ring at an unprotonated conserved acidic residue. The direction of rotor
rotation when external forces such as ATP hydrolysis are not present is determined by
the electric field, and therefore the side of the membrane more likely to deliver a proton
to a half-channel to bind the c-ring. Thus, in the presence of the PMF established by
the ETC, net rotation is in the direction of ATP synthesis (clockwise when viewed from
the IMS). Structural information for the FO region is required to determine the exact
locations of the half-channels and to trace the path of proton translocation.
1.1.6 Anchoring of the peripheral stalk
While rotating within the catalytic α and β subunits, the central rotor applies a torque
to drive ATP-generating conformational changes. To counteract this force and keep
the catalytic subunits stationary relative to the FO region, which is necessary for the
efficient coupling of proton translocation to ATP synthesis, the peripheral stalk anchors
the α3β3 hexamer to the FO region. How and where the peripheral stalk may change
in conformation to accommodate the torque from rotor rotation has not been shown.
Identifying regions of flexibility in the peripheral stalk can elucidate how it contributes to
the efficient coupling of the F1 and FO motors during ATP synthesis.
Chapter 1. Introduction 15
Cross-linking has shown that the b subunit is in direct contact with both the a subunit
and c-ring in the E. coli ATP synthase [40, 76]. These three subunits (a, b and c)
are the only FO subunits necessary for proton translocation in bacterial ATP synthases
[30, 59, 67], suggesting that the transmembrane α-helices of the b subunit are involved in
the transport of protons. A previous 18 A map of the bovine ATP synthase [14] showed
the approximate location of where the b subunit enters the membrane region near the a
subunit. However, where the two transmembrane α-helices of the b subunit are located in
relation to other FO subunits and their interactions with other subunits remain unclear.
Determining the structure of the membrane-bound segment of the b subunit in the intact
ATP synthase will provide insight into how it anchors the peripheral stalk and contributes
to proton translocation.
1.1.7 Oligomerization and membrane curvature
F1 regions form characteristic rows of “lollipop” shapes on cristae that can be used to
identify ATP synthase molecules in electron micrographs of mitochondrial membranes
[119]. Rows of ATP synthase dimers were first observed with electron microscopy on
freeze-fractured and deep-etched Paramecium mitochondria [5]. Dimers were subsequently
observed to be formed by yeast, bovine, chloroplast and fungal ATP synthases [7, 39, 144].
Electron tomography of mitochondria and mitochondrial membranes showed that these
“dimer ribbons” are located at regions of high membrane curvature, suggesting that ATP
synthase dimers are responsible for creating and maintaining cristae [120, 153, 39]. More
recently, single particle cryo-EM [14] and electron tomography of two-dimensional (2D)
crystals [71] of the bovine ATP synthase showed that ATP synthase monomers are
sufficient for inducing high membrane curvature. From this observation, dimerization
was proposed to be driven by the resulting reduction in the membrane strain caused by
the presence of ATP synthase monomers [14]. Subunits e and g in the FO region are
necessary for dimer formation. Yeast strains that lacked these subunits had abnormal
Chapter 1. Introduction 16
cristae structure and did not have detectable ATP synthase dimers [7, 9]. However,
specific protein-protein interactions that mediate dimer and oligomer formation have not
been determined and the mechanism of dimerization is still unknown.
1.2 Single particle electron cryomicroscopy (cryo-EM)
1.2.1 Introduction to biological cryo-EM
Transmission electron microscopy (EM) is used to study biological specimens at cellular to
near-atomic scales. When applied to studying individual protein molecules, the technique
is referred to as single particle EM. One method of specimen preparation in single particle
EM involves fixing and staining the sample, which increases the resistance of the specimen
to radiation damage when exposed to an electron beam in the microscope, and also
increases electron scattering and image contrast. However, this method dehydrates and
flattens proteins, distorting their structure [62]. A method that preserves proteins in
native-like states has also been developed, called single particle cryo-EM. Using this
technique, frozen, hydrated specimens are prepared for electron microscopy. Samples
are applied to a layer of holey amorphous carbon (and more recently, gold [139], see
section 4.2), and flash frozen in a thin (<100 nm) layer of vitreous ice. Vitreous ice is a
non-crystalline type of ice that preserves protein structure for imaging. This technique
allows for the use buffers with diverse chemical components that provide native-like
conditions for the proteins of interest, including detergents for solubilizing membrane
proteins such as the ATP synthase.
1.2.2 Microscopy theory
Visualization of protein specimens in cryo-EM is possible due to the scattering of high-
energy electrons when they interact with biological matter. Contrast in images is generated
by two phenomena: amplitude contrast and phase contrast. Amplitude contrast derives
Chapter 1. Introduction 17
mainly from the scattering of electrons at high enough angles that they become excluded
from the objective aperture. The number of these lost electrons depends on a sample’s
molecular weight and specimen thickness [147]. Amplitude contrast is produced by
the difference in the number of scattering events that occur at each position in the
frozen specimen. However, few electrons are scattered by biological material because
it is composed primarily of light elements (hydrogen, carbon, oxygen and nitrogen).
Additionally, the density of protein is not much higher than that of the ice in which it is
embedded for cryo-EM [135, 44], resulting in only a small difference in scattering. Thus,
amplitude contrast contributes little to the contrast seen in cryo-EM images. Increase
in amplitude contrast can be achieved by using heavy metal stains, but as mentioned in
section 1.2.1, this has the disadvantage of damaging protein structure.
For cryo-EM images, the ability to see protein particles relies primarily on phase
contrast. Phase contrast in images arises from the change in phase of the scattered
electron beam before re-combining with the unscattered electron beam. Specifically, when
electrons interact with the specimen during cryo-EM, they undergo a phase shift of 90◦
[130] compared to electrons that travel through vacuum. When the microscope is in
focus, there is very little phase contrast because the difference in amplitude between the
unscattered beam and the unscattered beam combined with the scattered beam with
a 90◦ phase shift is small. To increase the contrast in cryo-EM images, the microscope
is over- or under-focused in practice. This “defocus” gives additional phase shifts to
scattered waves according to their scattering angle, and thereby increases the changes in
amplitude and image contrast upon combination of scattered and unscattered beams.
Microscope images are approximate 2D projections of macromolecules [55]. Image
formation involves the contrast transfer function (CTF), which describes the signal
contrast as a function of frequency. The specimen projection image is convoluted by
the microscope’s point spread function (PSF), and the CTF is the Fourier transform
of the PSF [55]. The effects of the CTF oscillations, which decrease in amplitude and
Chapter 1. Introduction 18
increase in frequency with increasing resolution, must be corrected computationally in
order to recover high resolution information. The CTF depends on the defocus used when
acquiring images. Because there are many points in a CTF where it is equal to zero and
there is no information for the image, a range of defocus values must be used in order
to collect information at all resolutions. It is simplest to correct for the CTF in Fourier
space, such as by multiplication of the Fourier transform of the image with a Weiner filter
[50].
1.2.3 Protein structure determination with single particle cryo-
EM
Single particle cryo-EM involves acquiring many images of individual proteins that have
been frozen in random orientations in vitreous ice. These images are processed com-
putationally to produce a three-dimensional (3D) density map. An advantage of using
cryo-EM for structure determination is that the treatment of images of individual proteins
means that different conformations that are present simultaneously in a sample can po-
tentially be separated into homogeneous datasets through image analysis. This separation
allows for more accurate structure determination for each conformation identified. Also,
single particle cryo-EM does not require protein crystals, which are difficult to obtain for
large macromolecular complexes that are dynamic, flexible and have unstable subunit
interactions. Crystallization is especially difficult for membrane proteins due to the added
complication of hydrophobic domains that need to be kept soluble in aqueous buffers.
Cryo-EM is a more straightforward technique for membrane proteins, because once they
have been purified, solubilised in detergent buffer, and flash-frozen in vitreous ice, they
can be imaged directly. Different detergents will exert different effects on proteins, and
the detergent that results in the highest quality images for each sample is determined
empirically. Another advantage of single particle cryo-EM in comparison to other structure
determination methods like X-ray crystallography is the requirement of relatively low
Chapter 1. Introduction 19
concentrations of protein (∼0.02 to 2 µM).
The signal in single particle cryo-EM improves with molecular weight, making the
technique well-suited for studying the structures of large macromolecular complexes.
Atomic structures of subcomplexes and subunits from other structural biology methods,
such as nuclear magnetic resonance (NMR) and crystallography, can be docked into
cryo-EM maps of large complexes to learn about their overall structure. The structures
of large molecules with high internal symmetry have been studied most effectively with
single particle cryo-EM, with icosahedral viruses first reaching near-atomic resolution
[70, 93, 170, 171]. It has been more difficult to use single particle cryo-EM to study
proteins less than ∼300 kDa in size. More recently, with the development of direct electron
detector technology, as well as improvements in image processing algorithms, structures
of smaller specimens lacking internal symmetry have also been determined to near-atomic
resolution (discussed in section 1.2.6).
1.2.4 Specimen preparation
Specimen preparation for single particle cryo-EM starts with the application of several
microlitres of the sample to a ∼10-50 nm thick layer of amorphous carbon supported
by a 3 mm copper/rhodium mesh grid (Fig. 1.7). The grids are glow-discharged to
increase their hydrophilicity. There are small holes of up to a few µm in diameter in the
carbon layer. After the sample has been applied and allowed to adsorb, the grid is blotted
to remove excess material, leaving a thin layer of sample across the holes. The grid is
then plunge-frozen in an ethane/propane mixture kept near liquid nitrogen temperature
(∼-196◦C). The specimen is frozen in microseconds, preventing water molecules from
crystallizing and damaging protein structure. Liquid nitrogen itself is not used to flash
freeze grids because it is close to its boiling point at ambient pressure, and heat transferred
from a grid would cause a layer of nitrogen gas to form around the specimen. The slowing
of heat transfer due to this protective gas layer would give enough time for water molecules
Chapter 1. Introduction 20
3 mm 60 μm 1 μm 1 μm
90°
protein particlesice
EM Grid Grid square Hole in carbon film Cross-section of hole in carbon film
Figure 1.7: Specimen preparation for single particle cryo-EM. A metal mesh grid supports athin layer of amorphous carbon with small holes in it. The sample is adsorbed to the grid, andproteins are flash frozen in a thin layer of ice in the holes.
Chapter 1. Introduction 21
in the sample to crystallize before freezing. This effect does not occur with short-chain
hydrocarbons at liquid nitrogen temperature because they are far from their boiling point.
A continuous, rather than holey, layer of carbon can also be used as a specimen support
in cryo-EM. The advantages of using a continuous carbon layer include the requirement
of lower protein concentrations and the ability to exchange buffers after protein has been
adsorbed to a grid. Disadvantages include the induction of preferred orientations of the
protein particles on the grid, as well as an increase in background noise in images from the
carbon itself. Having preferred orientations of particles on grids results in the collection
of more information for some views of the protein particle than others, and prevents
3D maps from being built with isotropic resolution. Extra background in the images
lowers the signal-to-noise ratio (SNR) and decreases the ability of programs to detect
and align particle images accurately. These effects can hinder the ability to determine
protein structure to high resolution (see section 1.2.5 for a detailed description of image
processing). Like the use of different detergents, the decision to use holey carbon or
continuous carbon support is dependent on the behaviour of the sample and is determined
empirically.
1.2.5 Image processing
Since cryo-EM images must be acquired with a low exposure of electrons to limit radiation
damage to specimens, they have low SNRs. Thus, thousands of particle images must
be averaged together to improve SNRs enough to access high resolution information.
Assuming Gaussian noise, the SNR of averages of particle images improves proportionally
with the square root of the number of images averaged. The SNR of averages is also
affected by the quality of images, including any background density caused by buffer
components or contamination, and by the ability to accurately separate the images into
homogeneous datasets before averaging.
To produce 2D averages, particle images are aligned by in-plane rotations and transla-
Chapter 1. Introduction 22
tions, and then classified into different subsets using multivariate statistical analysis. 2D
classification can be done with or without a reference [50]. All images in a class are then
averaged. 2D class averages that do not represent 2D projections of the specimen can be
discarded at this stage of image processing as they likely include other image features
and/or particle images that cannot be aligned well due to poor quality.
Following 2D classification, selected images are used to produce 3D maps. Since
the images acquired with an electron microscope are approximate 2D projections of
the protein [55], the Fourier projection theorem is used. This theorem states that the
Fourier transform of a 2D projection of an object is a central slice through the 3D
Fourier transform of the object [55]. The orientation of this central slice is related to the
orientation of the protein recorded in the image. By capturing 2D projections of a protein
in many random orientations, a 3D map of the protein can be computed. The resolution
of the 3D map depends, in part, on how well Fourier space is sampled, or, equivalently in
real space, how thoroughly the different views of the protein have been covered by the
acquired particle images.
In order to construct a 3D map using the Fourier projection theorem, the projection
direction must be determined for each image so that transformations can be applied
to align the coordinate system of each particle image with an arbitrary, fixed reference
coordinate system. Transformations are described by three Euler angles (ϕ, θ, ψ) and two
in-plane translations along the x- and y-axes (∆x and ∆y). Since these transformations are
not known a priori, they must be first determined roughly, often using 2D class averages
with higher SNRs than the noisy individual particle images, before further refinement.
There are several methods for the determination of initial transformations:
� Rotational analysis (ROTAN) [12, 137] was designed for analysis of proteins that
often assume a particular type of orientation on cryo-EM grids. This can be due to
the existence of a long axis in the protein’s overall shape, thin ice, interactions with
the air/water interface, and/or preferential adsorption of one side of the protein to
Chapter 1. Introduction 23
carbon. For example, the long axis of the ATP synthase leads to mainly side views
in cryo-EM particle images. The change in position of asymmetric features for class
averages sharing a single rotation axis is used to determine the relative orientations
of the class averages.
� The common lines method, employing the Fourier projection theorem, requires
three images with different projection angles. Their Euler angles are determined by
the three common lines shared by the images. High SNRs are required in the images
because lines of single pixels are used in the analysis. The real space method using
the same approach is known as angular reconstitution [162]. This method does not
work for images of the ATP synthase because particles are primarily rotated around
one axis (the long axis of the enzyme), and the images lack the third orthogonal
line needed for unambiguous assignment of Euler angles [12].
� Tilt methods for initial 3D map building require the same specimen to be imaged
at different angles relative to the electron beam. Depending on the specific method
chosen, orthogonal tilt reconstruction (OTR) [92] or random conical tilt (RCT)
[127], pairs of images are either collected at ± 45◦, or 90◦ and ∼30◦, respectively.
Untilted images are first aligned and classified to determine the in-plane rotation
angle, ϕ. The orientations of tilted images corresponding to the identical projections
can then be determined based on the microscope tilt and in-plane rotations of their
corresponding untilted image.
� The random initial starts method makes use of 3D shapes such as spheres or
ellipsoids as starting references for projection matching, in which each individual
particle image is compared to a set of projections calculated from the reference.
Particle images are then assigned transformation parameters according to the
projection of the starting model that it most represents. In a similar (but not
random) method, a low-pass filtered map of a similar protein or the protein of
Chapter 1. Introduction 24
interest, when available, can be used as a reference.
Once rough estimates for the projection directions of images or class averages are
obtained, they are refined by several rounds of alignment and map building. In an iterative
process, maps built using refined alignments become the reference for the next round of
alignment. Alignment algorithms make use of projection matching, as described above.
This process is most easily and effectively implemented in Fourier space. Alignments can
be ranked using different algorithms, including amplitude-weighted phase residual [60]
and SNR-weighted correlation coefficient [151].
When alignments and the 3D map no longer change with additional rounds of refine-
ment, the resolution to which the map can be interpreted is determined. Most commonly,
the Fourier shell correlation (FSC) [50] is used to determine the resolution of maps in
cryo-EM. The FSC method for resolution determination involves calculating the normal-
ized cross-correlation coefficient at different spatial frequencies between two maps that
were processed using independent halves of the dataset. The highest resolution shell
that is statistically significant and above the noise level is the overall resolution of the
3D map. An FSC threshold of 0.143 is most frequently used to determine resolution,
and is equivalent to the figure-of-merit used in X-ray crystallography to indicate the
interpretability of crystal structures [133].
1.2.6 Near-atomic resolution with single particle cryo-EM
As previously mentioned, in order to limit radiation damage that causes structural
changes in proteins, low electron exposures must be used during imaging in cryo-EM.
The resulting low SNR of images means that it is often difficult to determine accurate
orientation parameters for particle images. In addition, CTF parameters such as defocus
and astigmatism may be computed incorrectly, resulting in blurring of particle images
when CTF correction is applied. Such errors during map building limit the resolutions
of the constructed maps. The nature of different samples leads to varying degrees of
Chapter 1. Introduction 25
difficulty in particle image alignment. It is often easier to reach high resolution with
complexes that are relatively larger, more rigid, and more homogeneous, and that have
symmetry and/or distinctive and easily aligned features. Sub-nanometre resolutions are
difficult to reach with complexes that are flexible, small, and heterogeneous.
Despite these challenges, near-atomic resolution structures from cryo-EM have become
increasingly feasible [84, 37, 10]. This is due in large part to the development of direct
electron detectors that are capable of capturing images with higher SNRs than was
possible previously, especially for high resolution information [104]. These detectors allow
image processing programs to more accurately align images for map building. They have
also contributed to the ability of programs to classify different protein conformations in
a single sample into homogeneous datasets for refinement [141, 95], which increases the
resolution of maps because protein features are not blurred by the averaging of many
different conformations in a single 3D class.
As the fast read rates of direct electron detectors allow for movies instead of single
images to be collected, the frames of these movies can be aligned before averaging [23] to
remove the blurring that would otherwise occur in a single image due to any specimen
movement during imaging (eg. due to stage drift or beam-induced motion). Image
processing programs have also been developed that are able to estimate and remove
local beam-induced motion of individual particle images [142, 136], which varies across
the imaged area and cannot be corrected by the whole-frame drift correction of movies.
Localized blurring in images is believed to occur due to radiation damage to the specimen
that causes it to distort during imaging [54], and electric charging of the sample that
causes deflection of electrons. Correction of these effects allows for recovery of high
resolution information from individual particle images.
Chapter 1. Introduction 26
1.2.7 Study of the structure of the ATP synthase using single
particle cryo-EM
The structure of the intact ATP synthase has been studied previously with single particle
cryo-EM. A 32 A map of the bovine ATP synthase showed the overall shape of the complex
[137], but did not provide information on the arrangement of subunits in the membrane
region. A structure of the S. cerevisiae ATP synthase, which loses subunits e and g when
extracted from mitochondria with detergent buffer, was then determined at 24 A [86].
Comparison of the two maps allowed localization of the e and g subunits in the FO region.
A 18 A map of the bovine ATP synthase [14] revealed the arrangement of subcomplexes
in the FO region, as well as where the peripheral stalk enters the FO region. However,
the arrangement, interactions, and secondary structure of individual membrane-bound
subunits remained unclear. Since different conformations of the enzyme could not be
separated when the 18 A map was built, unique rotational positions of the central rotor
and different conformational states of the αβ catalytic sites were not resolved.
More recently, the technological advances described in the previous section have
allowed for maps of rotary ATPases to be resolved to subnanometre resolutions using
single-particle cryo-EM. Three rotational states of the S. cerevisiae V-type ATPase were
resolved to 6.9-8.3 A [172], allowing for the analysis of flexibility in the enzyme. In these
maps, two highly tilted α-helices in the a subunit at its interface with the c-ring were also
revealed. A 6.2 A map of a dimer of the colourless green alga Polytomella sp. F-type ATP
synthase also showed two highly tilted α-helices in the proton-translocating a subunit
that are in contact with the c-ring [4]. For the mammalian mitochondrial ATP synthase,
different conformational states of the intact enzyme, as well as secondary structure in
the FO region, have not been shown to date. Thus, a clear understanding of both the
mechanism that couples proton translocation to rotation of the central rotor in the FO
region and the dynamic changes in conformation in the ATP synthase during its catalytic
cycle are lacking.
Chapter 1. Introduction 27
1.3 Thesis objectives and overview
Although the catalytic F1 region of the ATP synthase has been extensively studied and
the mechanism of ATP synthesis in the α3β3 hexamer is well understood, there is a lack
of high resolution structural information for the FO region in the context of the intact
ATP synthase. This means that how proton translocation in FO generates rotor rotation
remains unknown. In addition, regions of flexibility in the enzyme that are required for
efficient coupling of the mismatched three-step F1 and eight-step FO motors have not
been analyzed. Thus, the objectives for this thesis were:
� to better understand the coupling of proton translocation with rotation of the
central rotor by resolving the secondary structure and arrangement of subunits in
the FO region, and
� to investigate how the symmetry-mismatched F1 and FO regions are coupled effi-
ciently by identifying and analyzing different conformations of the ATP synthase
and determining regions of flexibility.
In this thesis, using single-particle cryo-EM, we present maps of the bovine mitochondrial
ATP synthase in seven distinct conformations. These maps were refined to 6.4-7.4 A
resolution, and show three rotational states with two to three substates within each
rotational state. The conformational changes that occur in the transitions between
substates reveal flexibility throughout the enzyme, particularly in the peripheral stalk,
that is important for efficient coupling of the symmetry-mismatched F1 and FO motors
during catalysis. By averaging the FO regions of all seven maps, α-helices of membrane-
bound subunits were resolved. We show that the mammalian F-type ATP synthase a
subunit, like the S. cerevisiae V-type ATPase and algal F-type ATP synthase a subunits
[172, 4], has two highly tilted α-helices that are in contact with the c8-ring. We built an
atomic model of the a subunit based on map density using constraints from evolutionary
co-variance analysis. This model has since been corroborated by a crystal structure of
Chapter 1. Introduction 28
the α-proteobacterium Paracoccus denitrificans ATP synthase [108]. The structure of the
a subunit in complex with two transmembrane α-helices of the b subunit suggests the
location of two half channels for proton translocation, supporting the Brownian ratchet
mechanism for the generation of rotation required for ATP synthesis.
This work has been published in eLife [173]. The EM maps of the seven conformations
are available in the Electron Microscopy Data Bank (EMDB) under accession codes
EMD-3164, EMD-3165, EMD-3166, EMD-3167, EMD-3168, EMD-3169 and EMD-3170.
The EM map of the average of the seven maps aligned at their FO regions is deposited
under the accession code EMDB-3181. The fitted atomic structures are available on the
Protein Data Bank (PDB) under PDB IDs 5ARA, 5ARE, 5ARH, 5ARI, 5FIJ, 5FIK and
5FIL.
1.4 Acknowledgements
Bovine ATP synthase was purified by John V. Bason and Martin G. Montgomery in the
laboratory of Prof. Sir John Walker (Medical Research Council’s Mitochondrial Biology
Unit, Cambridge, United Kingdom). Microscopy and map building was done by Alexis
Rohou in the laboratory of Prof. Nikolaus Grigorieff (Janelia Research Campus, Virginia,
United States). Analysis of evolutionary co-variance and generation of the atomic model
of the a subunit was done by Daniel Schep in the Rubinstein laboratory.
Chapter 2
Methods
2.1 Protein purification
Bovine ATP synthase was purified by John V. Bason and Martin G. Montgomery as
described previously [138]. Briefly, metal (Ni-NTA) affinity chromatography was used
to isolate ATP synthase in complex with residues 1-60 of heterologously expressed IF1
with a C-terminal His6-tag. After elution from the column, the complex was PEG-
precipitated and resolubilised in buffer containing 20 mM Tris-HCl (pH 7.2), 100 mM
NaCl, 10% (vol/vol) glycerol, 0.05% (wt/vol) dodecylmaltoside (DDM), 2 mM ATP and
0.02% (wt/vol) NaN3. Glycerol was removed from purified ATP synthase using 7000
MWCO Zeba Spin Desalting Columns (Thermo Scientific) according to the manufacturer’s
instructions before sample preparation for cryo-EM.
2.2 Specimen preparation and microscopy
Preparation of grids was done by Anna Zhou. Nanofabricated grids [99] were glow-
discharged for 2 minutes in air and loaded into a Vitrobot plunge freezing device (FEI
Company, Eindhoven, Netherlands) at 100% humidity and 4◦C. 2 µl of purified ATP
synthase at ∼8 mg/mL were applied onto the grids, followed by equilibration for 5 s,
29
Chapter 2. Methods 30
blotting for 27 s and freezing by plunging into a liquid ethane/propane (30/70% vol/vol)
mixture.
Microscopy was done by Alexis Rohou. Movies were recorded from three grids using
a Titan Krios (FEI) operating at 300 kV. An area of 2.5 µm diameter was illuminated
with a parallel beam at 3 e−/A2/s and using a 70 µm objective aperture. Micrographs
were recorded at 18000× nominal magnification on a K2 Summit direct detector device
(Gatan Inc.) used in super-resolution counting movie mode, with a calibrated physical
pixel size of 1.64 A and super-resolution pixel size of 0.82 A. An exposure of 8 e−/pixel/s
without a specimen was used for 20.1 s and 67 frames, resulting in a total exposure of the
specimen area of 60.3 e−/A2. Data was automatically collected using SerialEM [102].
2.3 Image processing and 3D map refinement
Movie processing, alignment and averaging were done by Alexis Rohou. Distortion in
each frame due to magnification anisotropy [172] measured previously using the program
mag distortion estimate [58] was corrected with the program mag distortion correct [58].
Fourier-space cropping was used to downsample frames to the physical pixel size of 1.64
A. To correct for whole-frame movement, frames were aligned using Unblur [58]. The
aligned sums were used to estimate CTF parameters with CTFFIND4 [132].
Automatic particle image picking was done by Anna Zhou. Imaging of the edges of
carbon holes occurred often in movies that were collected automatically using SerialEM.
Regions of carbon contamination were masked in the aligned averages of automatically
recorded movies with a novel program written by Anna Zhou, maskmicrograph.f90. This
program prevented the automatic particle-picking function in Relion [143] from picking
carbon and ice contamination features as particle images. The program first applies a
high-pass filter to the Fourier transform (FT) of an aligned average micrograph to remove
low frequency information (eg. ice gradients). It then uses a translating box to identify
Chapter 2. Methods 31
where areas in the image have a greater variance and/or lower mean than user-set intensity
threshold values, and finally masks these areas by setting them to the mean value of the
micrograph (Fig. 2.1).
Anna Zhou used Relion [141, 143] to determine coordinates for 408,934 candidate
proteins in masked micrographs automatically (Fig. 2.2A). Local beam-induced motion in
particle images was corrected by Alexis Rohou using alignparts lmbfgs [136] (Fig. 2.2B).
195,233 candidate particle images were selected by Anna Zhou for further analysis from
5,825 micrographs after 2D classification with Relion [141] (Fig. 2.2C).
Map building was done by Alexis Rohou. 256 × 256 pixel filtered and aligned [58, 136,
13] particle images were down-sampled by a factor of 2 for determination of orientation
parameters. Using an 18 A map of the bovine ATP synthase filtered to 20 A resolution
as a reference [14], initial particle orientation parameters were determined with five
rounds of mode 3 in FREALIGN (systematic parameter search) [61]. Particles were
then classified into different 3D maps with a likelihood-based algorithm [95], alternating
between orientation parameter refinement and class occupancy refinement every three or
four rounds.
2.4 Map analysis
Map analysis was done by Anna Zhou. Segmentation of 3D maps into subunits and
subcomplexes of the ATP synthase was performed manually with UCSF Chimera [57, 121].
The Segger plugin [121] in UCSF Chimera was first used to segment the maps [121].
Segments generated by Segger were ungrouped to the regions initially produced by
the watershed method employed by Segger. Ungrouped segments were then combined
manually to generate the segments for individual FO subunits and subcomplexes according
to known and predicted structures [172, 4, 90, 113].
Atomic structures were flexibly fit into 3D maps using UCSF Chimera and NAMD as
Chapter 2. Methods 32
A B C D
E
Figure 2.1: Program for masking contamination in micrographs. The effects of each step of themaskmicrograph.f90 program on a micrograph with carbon and ice contamination are shownhere. A) Original micrograph with carbon and ice contamination. B) The micrograph afterhigh-pass FT filtering. C) Identification of areas of carbon and ice contamination based onuser-set variance and mean intensity thresholds. D) Micrograph with areas identified to containcontamination masked with the mean intensity value of the micrograph. E) Comparison of theresults from Relion automatic particle picking [143] for the unmasked and masked micrographs,showing a decrease in the number of features picked from areas of carbon and ice contaminationfor the masked micrograph. Particle-like features identified by the Relion autopicking programare circled in blue. Scale bar 200 A.
Chapter 2. Methods 33
A B
C
0
500
1000
1500
2000
2500
3000
3500
0 500 1000 1500 2000 2500 3000 3500
pixe
l pos
ition
(y-d
irect
ion)
pixel position (x-direction)
0
10
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30
40
50
60
70
mov
ie fr
ames
Figure 2.2: Particle image collection and processing. After particle images were picked usingRelion [141], local beam-induced movement was corrected, and particle images were 2D classified.A) A representative micrograph with examples of bovine ATP synthase particle images circled inblue. Scale bar 200 A. B) Trajectories of particle images and other image features during movieacquisition determined by the alignparts lmbfgs algorithm [136]. Trajectories are exaggerated5-fold. C) Averages of 2D classes selected for 3D classification and map building. Scale bar 100A.
Chapter 2. Methods 34
described in the Molecular Dynamics Flexible Fitting (MDFF ) tutorial [159, 160]. For
crystal structures of various subcomplexes of the ATP synthase [56, 129, 41, 166], different
regions of the cryo-EM maps corresponding to the subcomplexes were first manually
segmented in UCSF Chimera. The crystal structures were then rigidly docked into the
map segments using the Fit in Map function in UCSF Chimera. For flexible fitting, a map
segment was converted to an MDFF potential using the command mdff griddx in VMD
[69]. Using the VMD plugin AutoPSF, the crystal structure to be fit into the density
was prepared for NAMD by generating a PSF file with information about structure
connectivity and partial charges. A PDB file with scaling factors for each atom was then
prepared with mdff gridpdb. Secondary structure restraints, including conservation of
peptide bonds in their cis/trans configuration in the given structure and conservation of
the hand of chiral centres, were applied to preserve the secondary structure of the crystal
structure during flexible fitting. Two consecutive NAMD configuration files were then
prepared for MDFF simulations. The first used a small scaling factor (-gscale option)
of 0.3 for 50 ps, and the second used a higher scaling factor of 10 for 2 ps for energy
minimization. Higher scaling factor values meant stronger forces were used to fit the
crystal structure into the map density. After the MDFF simulation was run using NAMD,
the trajectory was viewed in VMD. The final structure of the trajectory, representing the
fitted atomic structure, was saved as a PDB file to be used in analysis of conformational
changes between the different states.
Conformational changes between substates were analyzed by linear interpolation in
UCSF Chimera (Moviemaker). To approximate and quantify the change in conformation
observed between substates, the rotation of the α3β3 hexamer in the F1 region was
measured. This was done by matching the α3β3 hexamer of the fitted structure of the
first substate of a transition to the α3β3 hexamer of the fitted structure of the second
substate of the transition using the Fit in Map function in UCSF Chimera. Using the
measure rotation command, the magnitude of rotation, as well as the axis of rotation
Chapter 2. Methods 35
between the first and second substates were determined.
To increase the SNR in the FO region, the FO regions of the seven maps were aligned
in UCSF Chimera with Fit in Map and averaged in real space using volaver in the Situs
package [167]. Local resolution of maps was assessed using ResMap with maps that were
downsized by a factor of 2 [83].
2.5 Building of atomic models
An atomic model of the a subunit was built by Daniel Schep using the a subunit map
density and analysis of evolutionary co-variance constraints. Co-varying residues in the a
subunit were determined using EVcouplings [65] without the assumption of transmembrane
α-helices and using all other default settings. The top 90 co-varying residue pairs were
used to build a atomic model of the a subunit. For analysis of co-varying residues between
the a subunit and c subunit, GREMLIN [118] was used with Jackhmmr to generate
multiple sequence alignments using an E-value threshold of 1 × 10−10.
Straight, ideal α-helices built in UCSF Chimera according to MEMSAT-SVM [113]
were manually arranged in the average a subunit density to satisfy co-variance constraints.
Loops to connect the α-helices were built using Modeller [47] in UCSF Chimera, and fit
into the a subunit map segment with MDFF. Rosetta [131] was used to model the loop
between residues 115 and 148 (loopmodel command [42]) with the quick ccd method, and
to idealize the bond lengths and angles in the output structure with the lowest energy out
of 100 output structures (idealize.jd2 command). The idealized model was then energy
minimized using UCSF Chimera.
The N terminus of the b subunit crystal structure [41] was extended by Anna Zhou
into the FO region by first building the two transmembrane α-helices in UCSF Chimera
based on transmembrane α-helix prediction from MEMSAT-SVM [113]. The α-helices
were docked rigidly into the b subunit map segment from the FO region, then connected
Chapter 2. Methods 36
using Modeller. The extended b subunit structures were then fit into the maps using
MDFF as described in the previous section.
Calculations with Relion and MDFF were performed using the Scinet cluster [94] and
SickKids High Performance Computing Facility.
Chapter 3
Results and Discussion
3.1 Three rotational states of the ATP synthase
The resolution of the previous 18 A cryo-EM map of the bovine ATP synthase was limited
by the inability to separate different rotational positions of the central rotor within the
α3β3 catalytic hexamer, and did not allow for the unambiguous assignment of the three
αβ catalytic sites to different conformational states [14]. Here, three rotational states of
the ATP synthase in which the central rotor is rotated ∼120◦ relative to each other were
initially identified by 3D classification of 195,233 particle images (Fig. 3.1). The three
αβ catalytic pairs assume different conformations in accordance with rotor position in
these three states.
Further 3D classification identified two to three substates in each rotational state.
State 1 (43,039 particles) had two substates (States 1a and 1b), State 2 (48,053 particles)
had three substates (States 2a, 2b and 2c) and State 3 (46,257 particles) had two substates
(States 3a and 3b) (Fig. 3.2). The resolutions of the seven maps of the ATP synthase
were between 6.4 and 7.4 A (Fig. 3.3), with higher resolution in the F1 region compared
to the FO region (Fig. 3.4). The difference in resolution between the two regions was
expected due to the decrease in SNR in the FO region and blurring of the periphery of
37
Chapter 3. Results and Discussion 38
TightLoose Open
A
BState 1 State 2 State 3
Figure 3.1: Three main rotational states of the ATP synthase. A) Maps of states 1, 2 and 3. B)Map sections through the F1 region as indicated by the blue and purple arrows in (A). ∼120◦
turns of the two α-helices of the γ subunit in the central rotor (indicated by orange arrows) isseen in map sections boxed in blue, while the changes between loose, tight, and open statesof the three αβ catalytic sites is seen in map sections boxed in purple. The state of the αβcatalytic pair boxed in red is indicated. Scale bars 25 A.
Chapter 3. Results and Discussion 39
State 1a6.7 Å20,104 particles (10.3%)
State 1b7.4 Å22,935 particles (11.7%)
State 2a7.2 Å19,250 particles (9.9%)
State 2b7.4 Å17,610 particles (9.0%)
State 2c7.4 Å18,899 particles (9.7%)
State 3a6.4 Å24,140 particles (12.4%)
State 3b7.1 Å22,117 particles (11.3%)
F1
FO
Figure 3.2: Seven maps of the ATP synthase. Each map shows the ATP synthase in a distinctconformation. All maps have the structural features discussed below, including a novel featureextending from the bent FO region. The bend is indicated by the dashed line. Percentagesare relative to the dataset of 195,233 particle images selected from 2D classification for 3Dclassification. Scale bar 25 A.
Chapter 3. Results and Discussion 40
A
B
C
0.143
0
0.25
0.5
0.75
1
30 15 10 8 7 6 5 4
FSC
Resolution (Å)
state 1astate 1b
0.143
0
0.25
0.5
0.75
1
30 15 10 8 7 6 5 4
FSC
Resolution (Å)
state 2astate 2bstate 2c
0.143
0
0.25
0.5
0.75
1
30 15 10 8 7 6 5 4
FSC
Resolution (Å)
state 3astate 3b
Figure 3.3: Fourier shell correlation (FSC) curves for maps of each state. FSC curves for mapsof states 1a and 1b (A), states 2a, 2b and 2c (B), and states 3a and 3b (C).
Chapter 3. Results and Discussion 41
4.0 4.8 5.6 6.4 7.2 8.0 8.8 9.6
detergent micellec8-ring
a subunit
Figure 3.4: Local resolution. Local resolution of State 1a as determined by Resmap [83] iscoloured according to the legend, showing higher resolution in the F1 region than in the FO
region. Scale bar 25 A.
Chapter 3. Results and Discussion 42
membrane-bound proteins by the detergent micelle [87]. In addition, the extraction of the
ATP synthase from its native membrane environment may have decreased the stability of
the FO region, producing heterogeneity in this region that lowers resolution. The uneven
number of substates in each rotational state suggests that the conformations we were able
to refine to sub-nanometre resolution are a sampling of many conformations that may
exist in solution. Other conformations may not have been present in sufficiently large
populations in our dataset to be identified.
The ability to separate different rotational states and substates was possible in this
work due to the advances in direct electron detector technology and image processing
described in section 1.2.6. Because particle images were separated into near-homogeneous
datasets during image analysis, their orientations were refined more accurately, and the 3D
maps reached higher resolution than when image analysis incorrectly combines different
conformations into one class. The ability to separate distinct conformations present in
the sample also allowed for the analysis of dynamics of the ATP synthase, which will be
discussed further in section 3.5.
The particle images were distributed almost equally between the three rotational states,
unlike what was observed for the rotational states of the S. cerevisiae V-type ATPase
[172]. The near equal distribution of states may have been due to the IF11-60 inhibitor,
which would be expected to primarily stabilize the ATP synthase in three different states
depending on which of the αβ catalytic sites it binds to. Additionally, the presence of the
IF11-60 inhibitor may have limited the substates that were identified in the sample to
those that were most stable when it is bound. The addition of the IF11-60 inhibitor also
meant that the ATP synthase was not visualized during its catalytic cycle. Consequently,
the substates that we identified are not representative of catalytic intermediates of the
rotary cycle. They appear to be the result of Brownian motion based on the difference in
orientation of the c8-ring relative to the a subunit between substates (discussed further in
section 3.6), which suggests that they are conformations that are energetically similar or
Chapter 3. Results and Discussion 43
State 1a State 1b
OSCP
β
αγεδ
c8-ring
F6
b
d
a
Figure 3.5: Atomic models of F1 and the peripheral stalk (PDB IDs 2WSS, 2CLY, 2XND[129, 41, 166]) and models of the a (green) and b (red-orange) subunits created in this thesiswere fit into maps of States 1a and 1b, showing the arrangement of all subunits except A6L, f, eand g in the intact ATP synthase. A region of unassigned density near the peripheral stalk isindicated by the black arrow. Scale bar 25 A.
Chapter 3. Results and Discussion 44
equivalent.
At the resolutions of the maps, it was possible to fit crystal structures accurately
into map density for analysis of conformational changes (Fig. 3.5). Linear interpolation
between a combination of crystal structures fit into the maps of the three rotational states
(including the F1:peripheral stalk complex [129], peripheral stalk alone [41], F1:c-ring
complex [166], and atomic models of the a and b subunits built in this work and described
in sections 3.3 and 3.4) shows the large conformational changes in the central rotor and
catalytic α3β3 hexamer that occur during the full rotary cycle (Movie 1, see caption in
Appendix A). As the central rotor turns, concerted conformational changes are observed
in each of the three αβ catalytic sites. Each of the sites changes from what appear to
be the open (empty), to loose (ADP-binding), to tight (ATP-bound), then back to the
open conformation in the catalytic cycle. These changes are described by the binding
change mechanism as discussed in section 1.1.3. Large conformational changes in the
peripheral stalk can also be observed in this movie. Implications of this flexibility will
be discussed further in section 3.7. It is important to note that movies made by flexible
fitting of atomic structures into cryo-EM maps at sub-nanometre resolution followed by
linear interpolation may include small changes in conformation that may be artifacts of
the fitting. These changes can arise due to the low resolution in some areas of the map,
especially in the FO region, that allow for erroneous changes in crystal structures during
flexible fitting.
3.2 Membrane curvature and a novel feature in the
FO region
High membrane curvature of the inner mitochondrial membrane increases the surface area
that is available for oxidative phosphorylation and ATP synthesis. Electron tomography
of mitochondrial membranes showed that ATP synthases form rows of dimers that
Chapter 3. Results and Discussion 45
induce high membrane curvature, producing characteristic cristae structures [153, 39,
38]. The dimerization interface is located in the FO region, between the peripheral
stalks of ATP synthase monomers. As was previously observed in the 18 A map and
membrane-reconstituted 2D crystals of the bovine ATP synthase [14, 71], as well as
electron tomograms of mitochondrial membranes [153, 39], the FO regions in our maps
have a bend in the portion farthest from the c8-ring where the dimerization interface
is expected to be located (Fig. 3.6A). This portion is thought to contain the e and g
subunits, since a corresponding density is not present in a map of the S. cerevisiae ATP
synthase lacking these subunits [14, 86]. Subunit f is expected to be located near subunits
e and g [18], and may also be located in this bent density.
Subunits e and g are associated with dimer formation and normal mitochondrial
morphology because their deletion was shown to result in the formation of balloon-like
cristae in yeast mitochondria [38]. The bent region of FO was measured in the 18 A map
to be ∼40◦ in relation to the long axis of the enzyme [14]. Since the e and g subunits
of dimerized ATP synthases have been biochemically shown to be in close contact [24]
and likely lying in the same plane, this measurement was in good agreement with the
80◦ angle measured between the long axes of dimerized ATP synthases in sub-tomogram
averages of intact bovine mitochondria [39] and further supported the role of subunits e
and g in dimerization.
Deletion of the e subunit [7], mutation of the GxxxG motif in the transmembrane
α-helix of either the e or g subunit [8, 27], or deletion of the first transmembrane α-helix
of the b subunit [146] results in the loss of the g subunit and abnormal mitochondrial
morphology. In contrast, deleting the g subunit or the first transmembrane α-helix of the b
subunit does not result in loss of the e subunit, but does result in abnormal morphological
phenotypes [7, 146]. These experiments suggested that the e and b subunits are involved
in anchoring the g subunit, and the g subunit is involved in maintaining normal cristae
structure. Subunit g is predicted to have three soluble α-helices in addition to its single
Chapter 3. Results and Discussion 46
State 1a Average of FO region from all seven maps
A B C
Figure 3.6: Features in the FO region. A) The bent portion of FO region furthest from thec-ring is boxed in red. The novel feature extending from this portion and thought to be fromthe e subunit is indicated by the red arrows on the map and corresponding slices in (B). Slicesthrough the FO region of state 1a (B) and of the average FO region (C) show the increased SNRin the average, with better resolution of α-helices in the a subunit indicated by the blue arrows.A low density feature predicted to be from lipids or disordered protein is indicated by the whitearrows. Scale bars 25 A.
Chapter 3. Results and Discussion 47
transmembrane α-helix [25, 75]. These amphipathic α-helices may embed themselves in
one membrane layer in a mechanism known to be used to induce membrane curvature by
proteins involved in vesicle trafficking [103], cell division [140, 145], and viral infection
[97, 134]. It was proposed in previous work [11] that the bending of the membrane by the
g subunit of monomeric ATP synthases would lead to strain in the membrane bilayer, and
the dimerization of ATP synthase would be favoured because it would reduce this strain.
In this model, dimerization would occur without the need for protein-protein interactions.
However, it remains unknown if protein-protein interactions mediate the formation of
ATP synthase dimers or higher order oligomers.
In this work, an extramembranous feature that has not been described previously for
maps of the ATP synthase [14, 4, 86, 137] is observed extending towards the c8-ring from
the bent FO density predicted to contain the e and g subunits (Fig. 3.6A). Previously,
a bridge-like feature was observed in bovine ATP synthase dimers in the same location
with negative-stain EM, and was proposed to be formed by the interaction between the
soluble, conserved coiled coil regions of the e subunits of two ATP synthase monomers
[48, 107]. While the novel feature in our maps extends from the density corresponding
to the expected location of the e subunit, its position in these maps does not suggest a
clear mechanism for dimerization. Thus, it can only be speculated that this feature is
involved in protein-protein interactions that form dimers, and perhaps also higher order
oligomers. The mechanism of dimerization is unclear and remains to be determined by
higher resolution crystal structures or EM maps of the dimerized complex.
3.3 The arrangement of subunits in the FO region
By aligning and averaging the FO region of the seven maps to increase the SNR of similar
features in the region (Fig. 3.6C), we were able to resolve secondary structure and
assign density to various membrane-bound subunits (Fig. 3.7). Assignment of density to
Chapter 3. Results and Discussion 48
a subunitb subunit
A6L
e and g subunits
90˚ 90˚
Figure 3.7: Segmentation of the FO region. Segments for the a subunit (green), two transmem-brane α-helices from the b subunit (red), a transmembrane α-helix from the A6L subunit (blue),and the region predicted to contain the e and g dimerization subunits (orange) are shown. Thec8-ring is omitted for clarity. Scale bar 25 A.
Chapter 3. Results and Discussion 49
individual subunits was done manually by first segmenting and removing the detergent
micelle in the maps, then identifying α-helices for each subunit based on biochemical
and structural data [4, 172, 90, 14, 88, 113]. This process was inexact because the DDM
micelle surrounding the FO region obscures the ends of transmembrane α-helices within
the detergent micelle. Also, α-helices in the dimerization region remained unresolved,
which meant that the secondary structure and locations of the e, g and f subunits could
not be determined. Despite these limitations, we were able to learn new information from
the segments presented in Figure 3.7.
The c8-ring was the easiest to segment because its density was mostly isolated from
that of the other FO subunits, except for a small contact point with the a subunit. This
limited contact between the c-ring and a subunit was also observed in the membrane-
bound regions of maps of other rotary ATPases [87, 14, 4]. Its importance is discussed
more in section 3.4. In the segment for the proton-translocating a subunit, we observed
two highly tilted α-helices that are in contact with the c8-ring (Fig. 3.7, green density),
as was also seen in the a subunits of the Polytomella sp. F-type ATP synthase, the S.
cerevisiae V-ATPase [4, 172], and most recently, the P. denitrificans ATP synthase [108].
This segment allowed us to predict and model the fold of the a subunit, which will be
described in the next section.
The average map of the FO region shows where the N terminus of the b subunit
enters the membrane region and forms two transmembrane α-helices in contact with the
a subunit, revealing exactly where the peripheral stalk is anchored in the FO region to
allow it to resist the torque from the rotor during catalysis (Fig. 3.7, red density). The
location of these b subunit α-helices shows that, in agreement with previous cross-linking
studies [40] and evolutionary co-variance analysis [66] (described in depth in the following
section) with the E. coli ATP synthase, it is in close proximity with the a and c subunits.
Density for two α-helices in the same position was also observed in the P. denitrificans
ATP synthase crystal structure [108] and was also attributed to the N termini of the b
Chapter 3. Results and Discussion 50
and b’ subunits of its peripheral stalk.
The proximity of the b subunit to the a subunit/c-ring interface, along with studies
that have shown it is essential for proton translocation in bacterial ATP synthases, support
its involvement in the proton-translocating mechanism that generates rotor rotation in
FO. Comparison of the map density containing the a and b subunits with the maps of the
membrane-bound VO regions from yeast and T. thermophilus V-ATPases [86, 87] shows
that the arrangement of α-helices is conserved across rotary ATPases. As will be discussed
further in our model for proton translocation channels in section 3.5, this conservation
of structure also supports the role of the b subunit in forming half-channels for proton
translocation. Based on the segmented density, the two predicted transmembrane α-helices
from the b subunit were extended from the available crystal structure [41] in accordance
with transmembrane α-helix predictions [113] (Fig. 3.5A, red-orange subunit). The b
subunit is expected to have a sharp kink in its structure where it enters the FO region,
and where there are two of each of the helix-breaking residues, glycine and proline, in
its sequence. Indeed, a sharp bend in the peripheral stalk is observed in the 18 A map
[14] and in our maps. The predicted break in the α-helix was not modelled here since its
exact location is not known. High resolution structures of the intact enzyme will show
where it occurs.
An additional transmembrane α-helix that is in contact with the a subunit at a separate
interface from the b subunit is assigned to the A6L subunit due to its predicted proximity
to the peripheral stalk from cross-linking [90] (Fig. 3.7, blue density). In the bent FO
region, density for the e and g (and possibly f) subunits and the novel extramembranous
feature was segmented (Fig. 3.7, orange density). However, the resolution in this area did
not allow for a clear separation of protein from the detergent micelle due to their similar
densities (1.36 g/ml for protein [135] and 1.19 g/ml for DDM [158]), and the segment
likely contains some detergent density. Near the density from which the novel feature
extends, an additional low density feature is observed (Fig. 3.6B,C, white arrow). It is
Chapter 3. Results and Discussion 51
unclear what this feature may be. It could arise from endogenous lipids remaining in
the complex after purification, or a disordered or flexible portion of a protein subunit.
Since the purification did not include the necessary phospholipids for the association of
DAPIT and 6.8 kDa proteolipid, these subunits are not expected to be present in the maps
described here [32, 31]. Structural studies with ATP synthase complexes that include
these subunits will be required to determine their locations relative to, and interactions
with, other FO subunits.
Higher resolution will be required to determine the structures of each of the subunits
in the FO region. Knowledge of the structures of individual subunits will show how
membrane curvature is generated by the FO region. It will also clarify the roles of the f
and A6L subunits, which have been suggested to be involved in organization and structural
support of the proton translocating subunits [175]. Notably, there was an additional
unassigned density in the seven maps that appeared to wrap around the peripheral stalk
just above the FO region and near where the b subunit enters the membrane (Fig. 3.5,
black arrow). This density may be from the d subunit, since the crystal structure of the
peripheral stalk [41] that was modelled here does not include the full subunit. It could
also be from either the f or A6L subunits that are predicted to be near the peripheral
stalk [90], as the apparent winding of the density around the base of the peripheral stalk
suggests that it could be involved in structural support and reinforcement. The origin of
this feature would be determined with improved resolution of the FO region.
3.4 An atomic model of the a subunit
An atomic model of the a subunit was built to fit into the segmented map density using
analysis of evolutionary co-variance. Evolutionary co-variance predicts that residues
that are in close proximity in a structure will evolve together in a way that maintains
energetically favourable interactions [65, 118]. Thus, sequences related by evolution can
Chapter 3. Results and Discussion 52
be used to determine residue pairs that have evolved in concert, and that likely interact
in a protein structure. Co-variance constraints from this analysis can be used to orient
α-helices in a structural model by indicating which sides of the α-helices may have contacts
with each other. The use of this technique for the mitochondrial ATP synthase a subunit
was robust because thousands of related sequences (20,637) were used in the analysis. The
pattern of evolutionary couplings and the predicted fold of the bovine a subunit looked
similar to what was predicted for the E. coli a subunit [66] (not shown), indicating the
relevance of cross-linking and aqueous accessibility experiments done in the E. coli enzyme
[49] to the bovine enzyme. This analysis was informative for the proton translocation
model that is presented in section 3.5.
Using the top 90 co-variance constraints shown in Figure 3.8A, we could trace the
α-helical sequences unambiguously in the a subunit within the segmented density from
the average FO region map (Fig. 3.9A). Our model was further supported by evolutionary
co-variance analysis that predicted interactions between residues on the two highly tilted
α-helices of the a subunit expected to be in contact with the c8-ring [172, 4] and residues of
c subunit helix 2 that face outwards on the c8-ring (Fig. 3.8B). This a/c subunit covariance
analysis also supported our placement of the two α-helices in relation to the different
sides of the membrane, as described below. Our model violates 6% of the constraints
(defined here as having Cα-Cα distances greater than 15 A), which is within the expected
false discovery rate of the method [98]. Since the loops connecting the α-helices of the
model were built only to fit into the density as described in Materials and Methods, and
were not determined experimentally, they were not interpreted.
The model of the a subunit consists of six α-helices, five of which are transmembrane
α-helices (Fig. 3.9B). Because detergent molecules in the buffer adhere to hydrophobic
regions of membrane-embedded portions of protein complexes, the detergent micelle
present in our maps around the FO region indicates the relative location of the membrane
bilayer. The N terminus of the a subunit is located in the IMS. α-helix #1 spans the
Chapter 3. Results and Discussion 53
A
B
a subunit
a subunit
c subunit helix 2
MNENLFTSFITPVILGLPLVTLIVLFPSLLFPTSNRLVSNRFVTLQ
QW
MLQ
LVSKQM
MSIH
NSKG
QTWTLM
LMSLILFIGSTNLLGLLPHSFTPTTQLSMNLGMAIPLWAGAVITGFRNKTKASLAHFLPQGTPTPLIPMLV
IIETISL
FIQPM
ALAV
RLTA
NIT
AGH
LLIH
LIG
GAT
LALM
SIST
TTAL
ITFT
ILIL
LTILE
FAVA
MIQ
AYVFTLLVSLYLHDNT1
50
100
15020
0
226
MNENLFTSFITPVILGLPLVTLIVLFPSLLFPTSNRLVSNRFVTLQ
QW
MLQ
LVSKQM
MSIH
NSKGQTW
TLMLM
SLILFIGSTNLLGLLPHSFTPTTQLSMNLGMAIPLWAGAVITGFRNKTKASLAHFLPQGTPTPLIPMLVIIE
TISLFIQ
PMAL
AVRL
TAN
ITAG
HLL
IHLI
GG
ATLA
LMSI
STTT
ALIT
FTIL
ILLT
ILEFA
VAMIQ
AYVFTLLVSLYLHDNT
1
50
100
15020
0
226
Transmembrane prediction(MEMSAT-SVM)
LFSYAILG
FALSEA
MG
LFCL MV A
FLI LF
46
72
Highly conserved residues
Figure 3.8: Evolutionary co-variance constraints. A) The top 90 evolutionary co-varianceconstraints between residues in the a subunit (shown in a numbered circle) are indicated by blacklines. Transmembrane α-helices predicted in the a subunit by the MEMSAT-SVM algorithm[113] are indicated in green in the sequence, and by the dark blue rectangles in the circular bardiagram surrounding the a subunit sequence. A soluble α-helix is indicated by the light bluerectangle. B) The top six evolutionary co-variance constraints between residues in the a subunitand the outward-facing α-helix of the c subunit are indicated by black lines. Highly conservedresidues in the a subunit are in red. This figure was created by Daniel Schep and is published ineLife [173].
Chapter 3. Results and Discussion 54
A B
C terminus
N terminus
Matrix
IMS
1
2 3
4
6
5
Figure 3.9: Model of the a subunit. A) The top 90 evolutionary co-variance constraints betweenresidues in the a subunit (green) are indicated by red lines. B) The model of the a subunit iscoloured from blue (N terminus) to red (C terminus). The α-helices in the model are numberedfrom 1 to 6. Arg159 on α-helix #5 is coloured blue. The approximate boundaries of the lipidbilayer are indicated by black lines. IMS, intermembrane space. Scale bar 25 A.
Chapter 3. Results and Discussion 55
lipid bilayer perpendicular to the membrane plane. It is followed by α-helix #2, which
travels along the micelle surface. Moving in the direction towards the c8-ring, α-helix #2
is followed by a tilted hairpin consisting of α-helices #3 and #4. This hairpin does not
completely cross the lipid bilayer, as was observed for the Polytomella sp. F-type ATP
synthase [4]. The structure ends with a longer hairpin that is in contact with the c8-ring
consisting of α-helices #5 and #6, which is analogous to the highly tilted hairpin observed
in the S. cerevisiae V-type ATPase [172] and Polytomella sp. F-type ATP synthase [4] a
subunits. The C terminus is located in the matrix. The bundle of four α-helices created
by the two hairpins was also recently observed in a 4.0 A crystal structure of the P.
denitrificans ATP synthase [108], and the same sequence of α-helices was assigned. The
full a subunit was not resolved in this crystal structure and, as in our maps, did not show
side-chain density.
Our model places the conserved Arg159 that is essential for proton translocation in
the middle of α-helix #5 near the IMS side of the membrane, in contrast to the proposed
model of the Polytomella sp. ATP synthase a subunit [4]. It is near the predicted
location of the Arg159 residue that the a subunit has a small point of contact with the
c8-ring. This, as discussed further in section 3.7, is important to the function of the ATP
synthase. Firstly, the minimal contact between the a subunit and the c8-ring allows for
more water and lipid molecules to be inserted that can induce Brownian oscillations in the
c8-ring. Fewer protein-protein interactions can also mean there is less friction to hinder
the movement of the c8-ring, contributing to more smooth and rapid rotor rotation.
3.5 A model for proton translocation
The a subunit, b subunit and c-ring are the only subunits essential for proton translocation
in the minimal ATP synthase structures found in bacteria [30, 59, 67], suggesting that
these subunits may also be sufficient for forming the channels for proton translocation in
Chapter 3. Results and Discussion 56
eukaryotic ATP synthases. However, the path of proton movement remains unclear. As
described in section 1.1.5, the Brownian ratchet mechanism for generation of rotation by
proton translocation is contingent on two offset half-channels in the FO region [77, 78].
Brownian fluctuations from water and lipid molecules in contact with the c-ring, and
possibly also from protein-protein interactions, are proposed to cause small, random
oscillations of the c-ring. Rotation of the c-ring by a full c subunit occurs upon binding
of a proton to a conserved acidic residue located near the centre of the membrane bilayer
when it is aligned with one aqueous half-channel. Protons are released to the other side of
the lipid bilayer through the other half-channel. From the Polytomella sp. ATP synthase
structure, it was proposed that the aqueous half-channel on the lumenal side (equivalent
to the IMS side in mitochondria) is formed in part by α-helices of the a subunit, while the
half-channel on the matrix side involves an interface between the a subunit and c-ring [4].
This is consistent with a previous model for the location of the half-channels proposed
based on cysteine mutation and ion accessibility studies for the E. coli ATP synthase a
and c subunits [49]. Here, we propose a model in which the transmembrane α-helices of
the b subunit also participate in proton translocation, in addition to the a subunit and
c-ring. This model is compelling because of the striking conservation of the architecture
of the bovine ATP synthase FO region with that of the S. cerevisiae V-ATPase VO region,
despite the lack of sequence similarity of the bovine ATP synthase a subunit to the
V-ATPase a subunit [172].
In this model (Fig. 3.10), the opening of the matrix half-channel is formed by the
matrix ends of a subunit α-helices #5 and #6 at the interface with the c8-ring, as was
proposed for the Polytomella sp. and E. coli ATP synthases [4, 49]. The opening of the
IMS half-channel is formed at the interface of the IMS ends of the b subunit transmembrane
α-helices and a subunit α-helices #5 and #6. These openings would channel protons
towards and away from where the acidic Glu58 residues on the c8-ring interact with the
conserved Arg159 residue on the a subunit, near the middle of the membrane bilayer.
Chapter 3. Results and Discussion 57
H+ H+
Matrix
IMS
A B
half-channel
1half-channel
2
Figure 3.10: Proposed proton translocation channels. Arrows indicate a potential path of protontranslocation during ATP synthesis. The intermembrane space half-channel, consisting of thetwo transmembrane α-helices from the b subunit and the intermembrane space ends of a subunitα-helices #5 and 6, is in red. The matrix half-channel, consisting of the matrix ends of a subunitα-helices #5 and 6 at their interface with the c8-ring, is in purple. The conserved a subunitArg159 is indicated in blue, and the c8-ring is shown in a pink surface representation. Theapproximate boundaries of the lipid bilayer are indicated by black lines. IMS, intermembranespace. Scale bar 25 A.
Chapter 3. Results and Discussion 58
Notably, human disease-causing mutations identified in the a subunit to date are all
located on α-helices #5 and #6 in our model, near the proposed half-channel openings
(Fig. 3.11). Conserved residues in the hairpin composed of α-helices #3 and #4, and
not just in α-helices #5 and #6, are also likely to participate in proton translocation
since the bundle of four α-helices is highly conserved in rotary ATPases. High-resolution
structures by cryo-EM or X-ray crystallography are ultimately required to determine the
specific residues that are involved in proton translocation.
Chapter 3. Results and Discussion 59
Leu220Leu222Leu217
Leu156
Ser148
Thr192Arg159
Matrix
IMS
Figure 3.11: Disease-causing mutations in the a subunit. Mutations in the a subunit that havebeen identified in Neuropathy, ataxia, and retinitis pigmentosa (NARP), Leber hereditary opticneuropathy (LHON), Leigh syndrome (LS), Mitochondrial infantile bilateral striatal necrosis(MIBSN), Mitochondrial complex V deficiency, mitochondrial 1 (MC5DM1) and Myopathy,lactic acidosis, and sideroblastic anemia 3 (MLASA3) are coloured red on our atomic model ofthe a subunit [74, 26, 36, 85, 157]. The sequences of the human and bovine a subunits are highlyconserved at the C-terminal α-helices. The strictly conserved a subunit Arg159 is indicatedin blue. The approximate boundaries of the lipid bilayer are indicated by black lines. IMS,intermembrane space. Scale bar 25 A.
Chapter 3. Results and Discussion 60
3.6 Conformational changes between substates
Using linear interpolation of the flexibly fitted crystal structures described in section 3.1,
the changes in conformation during transitions between substates of each rotational state
were analyzed to determine regions of flexibility in the ATP synthase. Conformational
changes were observed in nearly all of the subunits during the transitions between substates,
as was observed in transitions between the three rotational states of the S. cerevisiae
V-ATPase [172]. In the peripheral stalk specifically, there were two main regions of
flexibility: one close to the OSCP subunit near the C terminus of the b subunit, and one
close to the FO region. During the transition between states 1a and 1b (Movie 2, panel
A), and between states 2a and 2b (Movie 2, panel B), a bending of the peripheral stalk
is observed near the OSCP subunit, while between states 2b and 2c (Movie 2, panel C),
and 3a and 3b (Movie 2, panel D), a bending of the peripheral stalk is observed near the
FO region. Movie 3 shows the same transitions between the substates viewed from the F1
region towards the FO region. Here, again, it is seen that the transitions between states
1a and 1b (Movie 3, panel A) and states 2a and 2b (Movie 3, panel B) are similar, and
the transitions between states 2b and 2c (Movie 3, panel C) and states 3a and 3b (Movie
3, panel D) are similar.
From the previous 18 A map of the bovine ATP synthase [14], two contact points
between the peripheral stalk and the F1 region, other than the interaction of the OSCP
subunit with the N terminus of the α subunit farthest from the peripheral stalk, were
observed. One of these contact points was near the N terminus of the closest α subunit,
and the second was halfway along the interface of a non-catalytic αβ pair. It was found
that docking of a crystal structure with a truncated peripheral stalk [129] into the 18
A map required flexibility in the peripheral stalk somewhere between these two contact
points. This finding is consistent with the flexible region near the OSCP subunit observed
in our analysis of conformational changes between substates. In addition, the two regions
of flexibility that we identified correspond to the two poorly resolved regions in the
Chapter 3. Results and Discussion 61
peripheral stalk of the P. denitrificans ATP synthase crystal structure [108], which further
confirmed that the regions are mobile.
The rotation of the α3β3 hexamer in the F1 region in relation to the rest of the
enzyme could be used to approximate and quantify the conformational changes observed
between substates (Fig. 3.12). The magnitude of the rotations ranged between 10◦ and
16◦. Although the axes of rotation for the α3β3 hexamer in the four transitions were
all different, the axes for the movements from state 1a to 1b and from state 2a to 2b
are similar, and the axes for the movements from state 2b to 2c and from state 3a to
3b are similar. The first two movements (Fig. 3.12A and B) have tilted axes that pass
through the central rotor and peripheral stalk, and the second two movements have axes
that are almost parallel to the long axis of the enzyme and near the central rotor (Fig.
3.12C and D). Differences in the axes of rotation of the α3β3 hexamer for what appear
to be overall similar movements in Movies 2 and 3 are expected because of the different
positions of the central rotor. In addition, the conformations captured in our maps are
likely a sampling of a continuum of conformations that the ATP synthase can assume in
solution, and they may not capture the full range of the observed motions, or all possible
kinds and combinations of motions.
Although only two points of flexibility in the peripheral stalk were obvious from the
analysis of our maps, this does not exclude the possibility of other regions of flexibility
that also contribute to smoothing catalysis. The points that we observed may have simply
been the most pronounced for the states that we were able to identify. The ability to
resolve more states may reveal other points of flexibility, and can also reveal a larger range
of motion than was seen in our analysis. Higher resolution for the current states and
other states will also mean that more subtle motions can be detected and flexibility can
be described more accurately. The use of linear interpolation between atomic structures
fit into cryo-EM maps will likely become more prevalent as a means to explore protein
dynamics as it becomes easier to separate different conformations of a protein within a
Chapter 3. Results and Discussion 62
A B D
C
States 1a and 1b States 2a and 2b
States 2b and 2c
States 3a and 3b
Figure 3.12: Differences between substates. The differences between substates are shown byoverlaying their 3D maps, as well as their corresponding fitted atomic structures. The axis ofthe movement of the α3β3 hexamer used to approximate the overall movement is indicated as ablack rod. There is a rotation of 10◦ between states 1a (red) and 1b (green) (A), 11◦ betweenstates 2a (red) and 2b (green) (B), 12◦ between states 2b (red) and 2c (green) (C), and 16◦
between states 3a (red) and 3b (green) (D). Scale bar 25 A.
Chapter 3. Results and Discussion 63
single sample by cryo-EM.
3.7 Implications of flexibility in the ATP synthase
Flexibility in the peripheral stalk may be involved in accommodating conformational
changes in the α and β subunits during the catalytic cycle to improve enzymatic efficiency.
In addition, it may be a site of transient energy storage while resisting torque generated
by the rotation of the rotor within the stationary F1 region. The observed flexibility in the
peripheral stalk of the mitochondrial ATP synthase is different from what was observed
for the E. coli ATP synthase, in which the peripheral stalk is composed of a homodimer
of b subunits [45], and also from what was observed for the T. thermophilus V/A-type
ATP synthase, in which the two peripheral stalks are each composed of two different
subunits that form a right-handed coiled coil around each other [91]. For the E. coli ATP
synthase, single molecule magnetic bead experiments showed that the peripheral stalk
was rigid in comparison to the central rotor [165], and for T. thermophilus, the agreement
of the crystal structure of the peripheral stalk [91] with the cryo-EM map of the intact
enzyme [87, 88] also suggested a rigid structure. Because the composition of the bovine
ATP synthase is very different from these organisms, with four distinct subunits that
interact with each other to form a complex structure, it is possible that flexibility in the
peripheral stalk is a feature that evolved in obligate aerobes for more efficient catalysis
compared to archael and bacterial enzymes. The analysis of flexible regions through linear
interpolation between fitted atomic models of substates was also extended to the rotor,
which appeared to be much more rigid than the peripheral stalk. Higher resolution and
biophysical experiments will be required to confirm this result.
In addition to the conformational changes observed in the peripheral stalk, the c8-
ring rotates slightly against the a subunit during transitions between substates of each
rotational state. Since this motion is smaller than the conformational changes observed
Chapter 3. Results and Discussion 64
elsewhere in the enzyme during the transitions, it is unlikely that it indicates a disruption
of the interface of the c8-ring and the a subunit in the sample. Rather, it further illustrates
the inherent flexibility of the enzyme. In addition, the rotation of the c8-ring against
the a subunit between substates, even in sample conditions that limit rotary catalysis,
indicates a lack of rigid interaction between the c8-ring and a subunit that is consistent
with the Brownian ratchet model. In this model, as described in section 1.1.5, due to
Brownian motion, the c8-ring oscillates as a proton competes with the conserved a subunit
Arg159 residue to interact with a c subunit Glu58 residue. The c8-ring only rotates when
a proton binds a Glu58 residue. Movie 4 shows this rotational oscillation of the c8-ring
between states 2a and 2c, with the Glu58 residues on the c8-ring changing between the
proton-locked conformation [123] in the lipid bilayer to the open conformation [122, 156]
near the proposed locations of the two half-channels and the conserved Arg159 on the a
subunit.
While the c8-ring oscillates between substates, the α-helices of the γ subunit in the
central rotor do not change their positions within the α3β3 complex. This suggests the
presence of flexibility in the rotor that can be involved in smoothing its rotation against
the stationary catalytic region during ATP synthesis, which has also been proposed
previously by molecular simulations and mechanical modeling of the γ subunit [115].
From this compliance in the central rotor, and our observation of two points of flexibility
in the peripheral stalk during the transitions between substates (Movies 2 and 3), it
appears that both the central rotor and peripheral stalk may contribute to the elastic
coupling of the F1 and FO domains for smooth power transition. Due to the symmetry
mismatch between the three-step motor of the α3β3 complex in the F1 region and the
eight-step motor of the c8-ring in the FO region, such compliance and elasticity in the
structure of the ATP synthase may enhance enzymatic efficiency [172, 174, 115, 165].
In addition to dimerization, the subunits in the FO region have been shown to
facilitate the opening of the mitochondrial permeability transition pore (MPTP), an
Chapter 3. Results and Discussion 65
inducible, non-specific pore located in the mitochondrial inner membrane [3, 53]. MPTP
opening leads to loss of membrane potential and cell death; it is thus implicated in
conditions with altered cell death, including muscular dystrophy, ischemic heart disease,
and neurodegenerative diseases [19]. The flexibility we observed in the peripheral stalk,
and throughout the structure of the ATP synthase, supports recently proposed models in
which the mitochondrial ATP synthase forms the MPTP. In one model, the dissociation of
the peripheral stalk from the FO region occurs upon binding of various MPTP modulators
to the ATP synthase. This dissociation results in uncoupling of the F1 region from the
c-ring, and allows the c-ring to expand and form the pore [73]. A second model proposes
that the MPTP is formed by ATP synthase dimers at the interface of the monomers.
The pore opens when Ca2+ binds to the catalytic sites in the α3β3 complex in place of
Mg2+ and causes conformational changes [19]. While the details of these specific models
remain to be tested, it has been shown that ATP synthase activity can be regulated
by the binding of allosteric modulators, as well as by post-translational modifications
[72, 150, 163, 19]. Thus, the flexibility we observed in the structure of the ATP synthase
may contribute to conformational changes that are involved in both the opening of the
MPTP and regulation of catalysis, in addition to smoothing the transition of power
between the mismatched F1 and FO motors.
Chapter 4
Conclusions and future directions
4.1 Conclusions
In this thesis, we presented cryo-EM maps of the intact bovine ATP synthase in seven
distinct conformations. A novel feature was shown to extend from the portion of the
FO region predicted to be involved in dimerization. Analysis of conformational changes
between the substates within each of the three rotational states indicated two main
regions of flexibility in the peripheral stalk that may contribute to the elastic coupling
of the F1 and FO motors during enzymatic activity. Additionally, an oscillation of the
c8-ring relative to the a subunit within each rotational state supports the Brownian
ratchet mechanism for generation of rotation in the FO region. Averaging the FO regions
from the seven maps revealed the locations of several membrane-bound subunits, as
well as the anchor point for the peripheral stalk in the FO region. Segmentation of the
proton-translocating a subunit from the average FO map allowed for the determination of
its fold when combined with analysis of evolutionary co-variance constraints. An atomic
model of the a subunit, along with the location of two transmembrane α-helices from the
b subunit, suggests two potential half-channels for proton translocation, also supporting
the Brownian ratchet mechanism of ATP synthesis.
66
Chapter 4. Conclusions and future directions 67
Several novel components were presented. A novel extramembranous feature in the
FO region was identified that is postulated to be involved in dimerization of the ATP
synthase. How the b subunit anchors the peripheral stalk in the membrane region and
how it may contribute to proton translocation was revealed. The first model for the fold
of the proton-translocating a subunit of a rotary ATPase was produced by combining
cryo-EM with analysis of evolutionary co-variance. Dynamics of the intact mitochondrial
ATP synthase were explored using cryo-EM through the separation of several distinct
conformations during image processing. This new information contributes to the overall
understanding of the function of the ATP synthase, from ATP synthesis to maintenance
of mitochondrial morphology.
4.2 Strategies for higher resolution of the intact ATP
synthase
An atomic or near-atomic model for the intact mitochondrial ATP synthase by cryo-EM
remains elusive. Several avenues through which this may be achieved in the near future
will now be discussed.
Map resolution depends on image quality, which determines the ability to recover
high resolution information and the accuracy of alignment of particle images during map
refinement. When imaging membrane proteins in detergent buffer, there is often low SNR
in particle images because protein-detergent complexes are most stable, and aggregation
is avoided at detergent concentrations close to the critical micelle-forming concentration
(CMC). At this concentration, detergent molecules produce high background noise in
microscope images. The use of detergent also decreases the ability to control ice thickness
due to the reduction in surface tension of the buffer, further lowering the contrast of
images with thick ice. There is often a thin layer of buffer in the middle of carbon
Chapter 4. Conclusions and future directions 68
holes that pushes protein into areas of thicker buffer at the edges of the holes. Because
the reduction in surface tension results in such uneven particle distribution, it can also
be difficult to image uniformly distributed and isolated protein particles in detergent
buffer. The quality of images of membrane proteins can be improved by using detergent
alternatives such as amphipols, which are amphipathic polymers that are able to stabilize
membrane proteins in solution but do not have the same detrimental effects to image
quality as detergent [125]. Detergents with lower CMCs can also be used that allow for
the removal of almost all free detergent molecules from the sample, thus mitigating the
effects of background noise and lowered surface tension. Other strategies to improve
particle image quality include the use of gold grids that reduce the crinkling of the carbon
support layer and the beam-induced motion of particles in ice [139], and per-particle CTF
estimation and correction, a method that is currently being explored in our laboratory.
While the IF1 inhibitor in our sample appeared to stabilize the three rotational states
of the ATP synthase, many conformational changes could still occur. The flexibility of
the ATP synthase structure that was observed in this work likely limited the resolution
of our maps. In order to improve the resolution achievable by single particle cryo-EM,
the sample can be altered in ways that fix the ATP synthase in one or a few distinct
states, rather than allowing for a continuum of conformations. Reduction in the number
of conformations present simultaneously in the sample can be achieved with cross-linking,
mutagenesis, or the addition of various substrate analogues or inhibitors that lock the
enzyme in a small number of states.
In an opposite approach, to determine the structure of many different conformations
in a heterogeneous sample at high resolution, the collection of large datasets will be
required. This will become more feasible and efficient with the ongoing development of
microscopes that streamline the cryo-EM pipeline with automated specimen-loading and
data collection capabilities. The maskmicrograph program that was written in this thesis
for masking carbon and ice contamination prior to automatic particle image selection
Chapter 4. Conclusions and future directions 69
may no longer be required when there are improved automated data collection schemes
that are able to avoid imaging carbon.
4.3 Unanswered questions
Several unanswered questions about the ATP synthase will now be discussed, which may
be addressed by using the strategies for reaching near-atomic resolution discussed above.
4.3.1 The catalytic cycle of the ATP synthase
Various models for the catalytic cycle in the F1 region currently exist based on studies
of the hydrolytic pathway (with assumption of its reversibility during ATP synthesis).
While three 120◦ steps in the full 360◦ cycle have been resolved in both the bacterial
and human enzymes [169, 154], different sizes and numbers of substeps between the
catalytic dwells that correspond to substrate release and product binding have been
proposed to make up each of the 120◦ incremental steps. These differences exist because
of the difficulty of capturing intermediate catalytic states of the enzyme by crystallization,
and of correlating available crystal structures to the states observed in single molecule
experiments [112, 2, 6, 101, 161]. The substeps during ATP hydrolysis for the human ATP
synthase (and likely also the bovine ATP synthase based on its high sequence identity and
crystal structure [1]) have been observed to be different from the eubacterial and bacterial
enzymes. In the human enzyme, three dwell positions were identified: 0◦ for ATP binding,
65◦ for Pi release and 90◦ for ATP hydrolysis [154]. In contrast, for the E. coli, Bacillus
PS3 and yeast enzymes, there were two dwells identified: 0◦ for ATP binding, and 80-90◦
for ATP hydrolysis [112, 169, 168, 111, 2, 149, 110, 117, 20, 148, 100]. The order of
phosphate/ADP release during ATP hydrolysis also appears to differ between human
and bacterial enzymes [16, 154, 79]. Furthermore, different types of rotary ATPases
have different catalytic cycles. For example, substeps were not observed within the 120◦
Chapter 4. Conclusions and future directions 70
incremental steps of the Na+-pumping V-ATPase of Enterococcus hirae [106].
With the ability to automatically acquire large cryo-EM datasets and to accurately
align and classify particle images with high SNR from direct electron detectors, it will be
possible to resolve all catalytic intermediate states of the ATP synthase at high resolution
and reveal the occupancy of the αβ catalytic sites at each catalytic dwell. Unlike the
sample used in this thesis, which included an inhibitor that limited the conformations
that were present and also meant that our maps did not represent catalytically relevant
states, experiments to visualize catalytic intermediates will need to be done with active
enzymes. The application of this approach to the study of ATP synthases from different
organisms and to various types of rotary ATPases will allow for a thorough comparison of
their catalytic cycles.
4.3.2 Dynamics of the intact ATP synthase
Another area that remains to be explored in depth is the dynamics of the ATP synthase
during its catalytic cycle. Although our work suggested that both the rotor and peripheral
stalk are flexible and contribute to the smooth transition of power between the F1 and
FO regions, biophysical experiments and molecular simulations with the bovine enzyme
can be used to confirm this. Dynamics can also be probed in more detail if many
conformations of the uninhibited ATP synthase are resolved at high resolution, and
linear interpolation between fitted atomic structures is used to reveal precisely where the
regions of flexibility in the active ATP synthase are located. For this type of analysis,
large cryo-EM datasets will be required to resolve the different conformations in the
sample at sub-nanometre resolution. A large number of particle images means that
classification and separation of the dataset into homogeneous populations for refinement
will not result in a resolution-limiting reduction in the number of particle images for each
conformation. Dynamics can then be explored with confidence that the resulting maps
represent meaningful conformations.
Chapter 4. Conclusions and future directions 71
4.3.3 Structure of FO subunits
At the resolution of our maps, it is still unknown which residues in the a, b and c subunits
are involved in proton translocation. Elucidating this would require structures of the FO
region at high enough resolution to observe bound water molecules that may indicate the
path of proton translocation [68]. In addition, although we were able to assign density
in the average FO region to different subunits (a, b and A6L), side chains and specific
interactions between subunits were not resolved. Improved algorithms and/or detectors in
cryo-EM may make it possible to determine the structures of individual membrane-bound
subunits or subcomplexes of subunits to near-atomic or atomic resolution in the near
future. Alternatively, X-ray crystallography or NMR can be employed. The resolution
of the maps from this thesis would allow atomic structures to be fit unambiguously
into a complete mosaic structure of the FO region. In addition to revealing the proton
translocation channels, high resolution information for the FO region will help elucidate
the yet unknown functions of several membrane-bound subunits, and provide insight
into how each of these proteins may support both the structure and function of the
proton-translocating subunits. Structures of the e and g subunits will also reveal how the
FO region induces membrane curvature and the mechanism of dimerization. These will
be important advancements in completing the structural picture of the ATP synthase.
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Appendix A
Movie captions
The movies in this thesis have been published in eLife [173].
Movie 1. Conformational changes during the rotary cycle. The transition by linear
interpolation between states 1a, 2a and 3a is shown, with pauses at each state. Large
conformational changes can be seen, including the turning of the c-ring (pink) against
the a subunit (green), the rotation of the rotor (blue, cyan and purple) within the α3β3
complex (red and yellow), and the conformational changes of each catalytic αβ pair. View
in loop.
Movie 2. Conformational changes between substates (Side view). A) The transition
by linear interpolation between states 1a and 1b, showing a bending of the peripheral
stalk near the OSCP (blue) and F6 subunit (magenta), around the C terminus of the b
subunit (red). B) The transition between states 2a and 2b, also showing a bending of the
peripheral stalk near the OSCP subunit. C) The transition between states 2b and 2c,
showing a bending of the peripheral stalk near the FO region. D) The transition between
states 3a and 3b, also showing a bending of the peripheral stalk near the FO region. Scale
bar 25 A. View in loop.
96
Appendix A. Movie captions 97
Movie 3. Conformational changes between substates (Top view). The same transitions as
in Movie 2, viewed from the F1 region towards the FO region. Scale bar 25 A. View in loop.
Movie 4. Brownian ratchet mechanism. The a subunit (green), b subunit (red) and
c8-ring (pink) are viewed in the direction of the matrix towards the intermembrane space.
Arg159 is shown as a blue sphere on the a subunit. The movement of the c8-ring between
substates (states 2a and 2c) is shown here, with the side chains of the Glu58 residues on
the c subunits represented as spheres. Glu58 on a c subunit changes from a proton-locked
conformation while in the lipid bilayer to an open conformation near Arg159, where it is
deprotonated. Upon re-protonation, it assumes the proton-locked position and can enter
the lipid bilayer again. The c8-ring oscillation is consistent with the Brownian ratchet
mechanism for coupling of proton translocation to rotor rotation. Scale bar 25 A. View
in loop.
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