Regulation of the Mammalian ElongationCycle by Subunit Rolling: A Eukaryotic-Specific Ribosome RearrangementTatyana V. Budkevich,1,2,3 Jan Giesebrecht,1 Elmar Behrmann,1 Justus Loerke,1 David J.F. Ramrath,1 Thorsten Mielke,1,4

Jochen Ismer,1 Peter W. Hildebrand,1 Chang-Shung Tung,5 Knud H. Nierhaus,1,2 Karissa Y. Sanbonmatsu,5,6

and Christian M.T. Spahn1,*1Institut fur Medizinische Physik und Biophysik, Charite–Universitatsmedizin Berlin, Chariteplatz 1, 10117 Berlin, Germany2Max-Planck Institut fur Molekulare Genetik, Abteilung Vingron, AG Ribosomen, 14195 Berlin, Ihnestraße 73, Germany3Institute of Molecular Biology and Genetics, Group of Protein Biosynthesis, 03143 Kiev, Ukraine4Max-Planck Institut fur Molekulare Genetik, UltraStrukturNetzwerk, 14195 Berlin, Ihnestraße 73, Germany5Theoretical Biology and Biophysics Group, Theoretical Division, Los Alamos National Laboratory, MK710, Los Alamos, NM 87545, USA6New Mexico Consortium, 4200 West Jemez Road, Suite 301, Los Alamos, New Mexico 87544, USA

*Correspondence: christian.spahn@charite.de

http://dx.doi.org/10.1016/j.cell.2014.04.044

SUMMARY

The extent to which bacterial ribosomes and thesignificantly larger eukaryotic ribosomes sharethe same mechanisms of ribosomal elongation isunknown. Here, we present subnanometer resolut-ion cryoelectron microscopy maps of the mam-malian 80S ribosome in the posttranslocational stateand in complex with the eukaryotic eEF1A,Val-tRNA,GMPPNP ternary complex, revealing sig-nificant differences in the elongation mechanismbetween bacteria and mammals. Surprisingly, andin contrast to bacterial ribosomes, a rotation of thesmall subunit around its long axis and orthogonalto the well-known intersubunit rotation distinguishesthe posttranslocational state from the classical pre-translocational state ribosome. We term this motion‘‘subunit rolling.’’ Correspondingly, a mammaliandecoding complex visualized in substates beforeand after codon recognition reveals structural dis-tinctions from the bacterial system. These findingssuggest how codon recognition leads to GTPaseactivation in the mammalian system and demon-strate that in mammalia subunit rolling occurs duringtRNA selection.

INTRODUCTION

Genetic informationwithinmRNA is translated into protein during

the elongation phase of translation (Voorhees and Ramak-

rishnan, 2013). Ribosomes decode one codon of the mRNA

sequence per elongation cycle by using tRNA substrates and

append the encoded amino acid to the nascent peptide. An elon-

gation cycle can be subdivided into three steps: (1) delivery of

aminoacyl-tRNA (aa-tRNA), which involves decoding and ac-

commodation, (2) peptide-bond formation, and (3) tRNA translo-

cation. Peptide-bond formation is catalyzed by the ribosome’s

peptidyl transferase center and is a fast and spontaneous step.

However, during decoding and translocation the ribosome has

to overcome large activation energy barriers. Accordingly, the

pretranslocational (PRE) and the posttranslocational (POST)

states of the ribosome are relatively stable (Schilling-Bartetzko

et al., 1992). These activation energy barriers are reduced by

translational GTPase elongation factors, which are responsible

for speed and accuracy of protein synthesis.

The bacterial elongation cycle and its substeps have been

extensively studied over the past decades using a battery of

functional, genetic, and structural methods (Frank and Spahn,

2006; Voorhees and Ramakrishnan, 2013). An elongation cycle

starts when an aa-tRNA is delivered to the POST state ribosome

carrying a peptidyl-tRNA in the P-site and a deacylated tRNA

in the E-site. A-site occupation is a complex, multistep proc-

ess (Schmeing et al., 2009; Schuette et al., 2009; Voorhees

and Ramakrishnan, 2013). In the initial decoding step the aa-

tRNA,EF-Tu,GTP ternary complex binds to the ribosome.

Recognition of the cognate codon leads to a complex rearrange-

ment of the tRNA. In the resulting A/T-state the tRNA can bind to

the A-site of the ribosomal 30S subunit and remains simulta-

neously bound to EF-Tu, which in turn interacts with the ribo-

some’s factor-binding site. The conformational rearrangements

of the ternary complex, combined with changes in the ribosome,

transmit decoding signals from cognate codon-anticodon inter-

actions to EF-Tu-stimulating GTP hydrolysis and subsequent

dissociation of EF-Tu,GDP. Decoding is completed when the

body of the tRNA swings into the ribosome through the accom-

modation corridor and establishes interactions with the A-site of

the 50S subunit during accommodation (Sanbonmatsu et al.,

2005). After peptide-bond formation, the resulting PRE complex

contains a peptidyl-tRNA in the A-site and a deacylated tRNA in

the P-site. In order to reset the ribosome for the next round of

elongation, the EF-G-dependent translocation reaction moves

Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc. 121

the tRNA2,mRNA complex by one codon through the ribosome,

establishing the POST state (Voorhees and Ramakrishnan,

2013).

While the structure and function of the ribosome is sub-

stantially conserved across the domains of life, comparatively

little is known about the detailed mechanism of eukaryotic

translation. The eukaryotic (80S) ribosome is significantly lar-

ger and more complex than its bacterial counterpart (Anger

et al., 2013; Ben-Shem et al., 2011; Klinge et al., 2011; Spahn

et al., 2004a) and mechanistic investigations of the transla-

tion mechanisms in higher organisms are lacking. Substantial

disparities in the translation mechanism between eukaryotic

and prokaryotic systems are implied by the elaborated initia-

tion, termination, and recycling mechanisms, requiring num-

erous additional factors in eukaryotes (Melnikov et al., 2012).

In contrast, the elongation phase is thought to be highly

conserved across domains of life because core elements of

the ribosome, including the substrate-binding sites and the

general elongation factors, are largely conserved. Conversely,

the existence of ribosome-targeting antibiotics that display

domain specificity (Wilson, 2009) and evidence of essential,

domain-specific translation factors (Andersen et al., 2006), indi-

cates that aspects of the translation mechanism must differ. A

first analysis of the mammalian PRE complex revealed distinc-

tions of the mammalian 80S ribosome with respect to the dy-

namic behavior of the complex and the exact nature of the

tRNA-binding sites (Budkevich et al., 2011). Here, we explore

the mammalian elongation cycle through structural investiga-

tions of the mammalian 80S ribosome decoding complexes

and the POST state from rabbit liver using cryoelectron micro-

scopy (cryo-EM). Strikingly, substantial differences to analo-

gous bacterial complexes can be observed, revealing that the

bacterial and the mammalian elongation cycle diverge more

than previously thought.

RESULTS

Cryo-EM Maps for Mammalian POST, PRE,and Decoding ComplexesIn order to provide the structural foundation for the A-site

occupation/decoding step in the mammalian system, we

used cryo-EM to analyze ribosomal decoding and POST com-

plexes. Both specimens were prepared in vitro from mamma-

lian components (Budkevich et al., 2011; Budkevich et al.,

2008). To yield the decoding complex the ternary complex

Val-tRNA,eEF1A,GMPPNP was stalled by the nonhydrolyzable

GTP analog. For the POST complex, PRE ribosomes were

translocated by eEF2 to move N-acylated Lys-tRNALys3 and

deacylated tRNAPhe from A- and P-sites to P- and E-sites,

respectively (Experimental Procedures).

The resulting complexes were analyzed by multiparticle cryo-

EM (Loerke et al., 2010). The compositional heterogeneity of the

POST specimen was larger than expected from the biochemical

data and the subpopulation corresponding to the desired 80S

POST complex consisted of only about 35% of ribosomal com-

plexes (Figure S1A available online and Extended Experimental

Procedures). This major subpopulation (236,113 particle images)

representing a POST complex with two tRNAs in classical P- and

122 Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc.

E-sites (Figure 1A) was further refined to a resolution of 6.9 A

(Figure S1B).

The classical PRE state is the final product of the ribosomal

A-site occupation/decoding step. We made use of the presence

of significantly populated classical PRE states within the POST

data set (Figure S1A) to furthermore obtain cryo-EM maps of

the 80S ribosome in classical-1 (66,618 particle images) and

classical-2 (73,951 particle images) PRE states (Figures 1B and

1C) at 7.6 A and 7.5 A resolution, respectively (Figure S1B).

Both maps exhibited density for tRNAs in classical A-, P-, and

E-sites. They agree well with our previously reported structures

(Budkevich et al., 2011) but are significantly improved in terms

of map quality and resolution. Moreover, the PRE and the

POST maps were obtained from the same sample, thus facili-

tating a direct comparison of both states. For the ribosomal de-

coding complex, our multiparticle approach resulted in two

maps (Figures 1D and 1E) in subtly different conformations

(see below) with resolutions of 8.7 A and 8.9 A, respectively

(Figure S1B).

In total, we obtained five cryo-EM maps delineating the de-

coding process from the POST state via two decoding states

to the classical-1 and -2 PRE states (Figure 1). All maps show

typical features expected at subnanometer resolution such as

major and minor grooves of RNA double helices, a helices, and

extended protein tails. To interpret our maps in molecular terms,

we created a cryo-EM-based homology model of the mamma-

lian 80S ribosome. As secondary structure maps for rRNAs

from rabbit are not available, and rabbit and human ribosomes

are very similar (Budkevich et al., 2011; Spahn et al., 2004b),

we chose the human ribosome as target for our modeling. The

high-sequence conservation (see Extended Experimental Pro-

cedure) also suggests that a human-based model is a valid

approximation for the presented cryo-EM maps. Importantly,

our model will aid subsequent studies in the human system.

Recent X-ray structures of the ribosomal 40S and 60S subunits

from Tetrahymena thermophila (Klinge et al., 2011; Rabl et al.,

2011) and the yeast 80S ribosome (Ben-Shem et al., 2011) allow

homologymodeling not only for the evolutionary conserved inner

core of the ribosome but also for many of the eukaryotic-specific

components (Figure 1A, Table S1). Recently, a model for the

human 80S ribosome in the rotated state became available

based on a cryo-EM map at 5.4 A resolution (Anger et al.,

2013). The model presented here is in good overall agreement

with this model regardless of our use of an alternative sequence

for the ribosomal RNAs.

The Path of the mRNA in Eukaryotic Ribosomes andCodon-Anticodon Interactions with P- and E-Site tRNAsThe presented cryo-EM map of the mammalian POST complex

allows a direct visualization of an mRNA fragment with a length

of approximately 34 nucleotides (Figures 1A, 2A, and 2B). The

modeled mRNA ranges from position -15 on the 50 side to posi-

tion +19 on the 30 side. This agrees well with the part of the

mRNA that is shielded by the ribosome according to mRNA pro-

tection studies (Steitz, 1969) and recent ribosome profiling data

(Ingolia et al., 2011). As in bacterial ribosomes (Jenner et al.,

2010), the mRNA enters the mRNA groove of the 40S subunit

from the solvent side through the mRNA entry tunnel, wraps

Figure 1. Reconstruction of Eukaryotic

80S POST, Classical-1 PRE, Classical-2

PRE, Codon Sampling and Codon Recogni-

tion/GTPase Activation Complexes from

Rabbit Liver

(A–E) Reconstruction of eukaryotic 80S POST (A),

PRE (classical-1) (B), PRE (classical-2) (C), codon

sampling (D), and codon recognition/GTPase

activation (E) complexes from rabbit liver. Left:

Overall view of the cryo-EM reconstructions, dis-

playing the 60S subunit (blue), 40S subunit (yel-

low), A-tRNA (pink), P-tRNA (green), E-site tRNA

(orange), A/T-tRNA (dark violet), eEF1A (red), and

mRNA (blue). Middle and right: Individual mesh

representation of the subunit maps with docked

models, showing the ribosomal ligands relative to

the 40S subunit (ribosomal RNA yellow and S

proteins gray) and the 60S subunit (rRNA blue and

L proteins orange), respectively. Landmarks are

denoted for 40S: beak (bk), left foot (lf), right foot

(rf), head (h), shoulder (sh), and 60S: central pro-

tuberance (CP), L1 stalk (L1), stalk base (SB) and

stalk (St). See also Figure S1 and Table S1.

around the neck of the 40S subunit, where it interacts with the

tRNAs, and leaves the 40S through the mRNA exit tunnel. On

the 50 side the overall path of the mRNA through the mRNA

exit tunnel deviates significantly from the bacterial system (Jen-

ner et al., 2010), whereas on the 30 side, the path of the mRNA

through the mRNA entry tunnel is remarkably similar (Figure 2B).

However, there is a kink in the mRNA at the solvent side of the

entry tunnel and about four nucleotides of mRNA (+16 to +19),

which are not visible in the bacterial system, lead upward toward

the 40S head and 18S rRNA helix 16 (h16).

In the P-site the three-nucleotide helix formed by codon-anti-

codon interaction can be directly observed (Figure 2A). Interest-

ingly, we also observe a shorter interaction interface between the

mRNA codon and the tRNA anticodon in the E-site, indicating

Cell 158, 121

one or at most two base pairing inter-

actions (Figure 2A). This is consistent

with bacterial X-ray structures where

Watson-Crick base pairing of the E-site

tRNA anticodon with the first nucleotide

of the E-site codon was described (Jen-

ner et al., 2010). Thus, codon-anticodon

interaction at the E-site may be a feature

of the mammalian POST complex, but

weakened with respect to the P-site.

Interactions of tRNA and mRNAwith Ribosomal Proteins Specificto EukaryotesThe domain-specific differences in the

path through the mRNA exit tunnel on

the mRNA’s 50 side can be explained by

the presence of the eukaryotic-specific

ribosomal proteins eS26 and eS28 (we

use the new system for naming ribosomal

proteins according to Ban et al., 2014).

The N- and C-terminal parts of eS26 block the bacterial path of

the mRNA and at the same time shield the 30-end of 18S rRNA

preventing formation of Shine-Dalgarno-like interactions. More-

over, eS26, eS28, and also uS11 (rpS14) appear to interact with

the eukaryotic mRNA and thus line out an alternative path of the

mRNA exit (Figure 2B).

At the solvent side of themRNA entry tunnel, a eukaryotic-spe-

cific contact with the 30 side of the mRNA may also take place,

facilitated by the C-terminal part of protein eS30. Interestingly,

eS30 wraps around the shoulder, and reaches into the decoding

center (Rabl et al., 2011), where its N terminus has been pro-

posed to interact with the A-site tRNA in the mammalian PRE

complex (Budkevich et al., 2011). Thus, eS30 could provide a

structural link between the outer and inner ends of the mRNA

–131, July 3, 2014 ª2014 Elsevier Inc. 123

Figure 3. 40S Subunit Rolling

(A and B) Comparison of the 40S subunit positions in classical-1 state (orange)

to the POST state (yellow) represented by (A) cryo-EM maps and (B) ribbons.

Comparisons are based on a common 60S alignment. Arrows indicate the

direction of movement during transition between the two different states. The

distance changes in the 40S subunit positions resulting from the rigid body

transformation are color-coded in A units. Landmarks for 40S are denoted:

head (h), body (b) and beak (bk). See also Figure S3 and Movie S1.

Figure 2. Eukaryotic-Specific Features of the mRNA Path Visible

from Intersubunit Space and Solvent Side of 40S Subunit

(A) Interaction of the P- and E-site tRNAs of the 80S POST complex (trans-

parent gray) with mRNA.

(B) Comparison of eukaryotic (transparent gray) and prokaryotic (red ribbon,

Jenner et al., 2010, PDB ID 3I8G) mRNA paths. The model for the additional

four nucleotides on the 30-end of the eukaryotic mRNA is shown in cyan.

Densities for the ligands were segmented from the cryo-EM map presented in

Figure 1A. The models for tRNAs, mRNA and ribosomal proteins from the

homology model of the human 80S ribosome are presented in this paper.

Ribosome orientations are indicated by orientation aids.

See also Figure S2.

entry tunnel. Hence, our structure suggests a functional role for

the eukaryotic ribosomal proteins uS11, eS26, eS28, and eS30

in escorting the mRNA through the 40S subunit. Furthermore,

eukaryotic-specific contacts also contribute to the tRNA-binding

sites (Figure S2).

40S Subunit Rolling: A Novel Mode of IntersubunitRearrangementThe presented POST complex contains classically configured

tRNAs in the P/P- and E/E-sites (Figure 1A). It is therefore ex-

pected to correspond, in terms of overall ribosome conforma-

tion, to the classical-1 PRE state that also carries classically

configured tRNAs (Figure 1B). Unexpectedly, the subunit

arrangement of the POST state differs markedly from that of

the classical-1 PRE state (Figure 3A, Movie S1). The underlying

conformational change, which we term ‘‘subunit rolling,’’ can

be described as a �6� rotation of the 40S subunit toward the

L1 stalk around the long axis of the small subunit. The axis co-

localizes approximately with the upper part of h44 of 18S rRNA

and is roughly orthogonal to the well-known intersubunit rotation

(Figure S3A).

Interestingly, subunit rolling shapes the openings to the inter-

subunit space and causes reciprocal opening and closing of

the A- and E-site regions (Movie S1). The distance between

the 40S and 60S subunits on the A-site side of the 80S rib-

osome decreases by about 13–15 A during subunit rolling

from the POST state to the classical-1 PRE state. As a conse-

quence, the A-site region is more widely open in the POST

state. The opposite is observed for the E-site, which is narro-

wer in the POST state than in the classical-1 PRE state. How-

ever, due to the smaller distance from the rotation axis, the

underlying movements are only in the range of 6–7 A at the

E-site. Subunit rolling also affects the interactions between

124 Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc.

the 40S and the 60S subunit, i.e., the intersubunit bridges

(Figure S3D). Most of the conserved bridges are present in

the current POST 80S state with the exception of B6 and B7

(Figure S3D, in green), which are, however, found in the

mammalian classical-1 PRE state. Subunit rolling provides an

explanation for this observation as it results in movement on

the order of �5–7 A in the lower part of 40S (Figure 3B). More-

over, subunit rolling is expected to affect tRNA positions.

Indeed, the elbow region of the P/P-tRNA in the PRE state is

shifted by �6 A toward the E-site in comparison with the

mammalian POST state (and also the bacterial PRE state; Fig-

ures S3E and S3F). Thus, some differences in tRNA positioning

exist for the mammalian 80S ribosome between the classical

PRE and POST states, with either a deacylated or peptidyl-

tRNA being present in the P-site, respectively.

Cryo-EM Analysis of the Mammalian Decoding ComplexThe observed difference in the subunit arrangement of the 80S

ribosome in POST and classical PRE states has consequences

for themechanismof tRNAselection. It implies that subunit rolling

has to occur when the POST complex is converted into the PRE

complex. To exploremammalian A-site occupation, we analyzed

a mammalian decoding complex. Unexpectedly, we were able

to observe two subpopulations of the 80S,Val-tRNA,eEF1A,GMPPNP complex (Figures 1D and 1E). Both cryo-EM maps

display clear density for the ternary complex (Figures 4A and

4B) but are distinguished by more subtle changes in position,

conformation and interaction patterns between the ternary com-

plex and the 80S ribosome (Figures 4, 5, 6, and S4 and S5).

In terms of subunit configuration, both substates of the

80S,Val-tRNA,eEF1A,GMPPNP complex are similar to the

POST complex. When the larger substate of the decoding com-

plex (79,705 particle images) and the smaller one (52,686 particle

images) are compared to the POST complex only small rotations

of �0.5� and �1�, respectively, between the 40S subunits were

Figure 4. Conformation of the Mammalian

Ternary Complex and Differences from the

Bacterial Counterpart

(A and B) Overall fitting of crystallographic mo-

dels of Aeropyrum pernix aEF1A (Kobayashi

et al., 2012) (red ribbon, PDB ID 3VMF)

and T. thermophilus A/T-tRNA (pink ribbon,

modified from PDB IDs 2XQD and 1TTT for ASL

and tRNA body, respectively) to the ternary com-

plex cryo-EMmaps (transparent gray) of (A) codon

sampling state and (B) codon recognition/GTPase

activation state. Densities for the ternary complex

are extracted from the cryo-EMmaps presented in

Figure 1D and E.

(C–E) Superposition of the codon sampling state

(transparent gray) and codon recognition /

GTPase activation state ternary complex molec-

ular models on the 40S subunit surface (C). The

alignment was based on the 40S subunit densities.

(D and E) Comparisons of the mammalian

(D) codon sampling state and (E) codon recogni-

tion/GTPase recognition state (E) ternary com-

plexes with the bacterial ternary complex stalled

by GTP analog (Voorhees et al., 2010) (PDB ID

2XQD; transparent gray). The alignment was

based on conserved parts of the 18S/16S rRNA.

Ribosome orientations are indicated by orientation

aids. (C–E) The distances between positions of

the ternary complexes are color coded (capped at

6 A). We note that these distances essentially

reflect rigid body transformations of eEF1A/EF-Tu

and ASL or body of tRNA.

See also Figure S4 and Movie S2.

found. In contrast, comparing the two subpopulations of the

decoding complex with the classical-1 PRE complex reveals

respective rotations of �5.6� and �5.0�. Thus, subunit rollingoccurs after the decoding step during accommodation of the

tRNA from the A/T- to the A/A-state. By contrast, structural in-

vestigations in bacteria did not show evidence for such a major

subunit rearrangement (Schmeing et al., 2009; Schuette et al.,

2009; Voorhees et al., 2010).

Conformational Heterogeneity of the MammalianDecoding ComplexFor a molecular interpretation of the mammalian decoding com-

plexes we used the X-ray structure of aEF1A from the archaeal

EF1A/RF1 complex (Kobayashi et al., 2012), which we could fit

well as a rigid body into the cryo-EM maps of both subpopula-

tions (Figures 4A and 4B). Archaeal aEF1A and human eEF1A

Cell 158, 121

are closely related by sequence (Fig-

ure S4A). Remarkably, density for the

only major insertion helix 5 (aa 214–225)

(human numbering), which is present in

eEF1A but not in aEF1A, is observed in

our cryo-EM map and creates a signifi-

cant mismatch between model and map

at the C-terminal part of the G domain

of eEF1A before the junction to domain II

(Figure S4B). To account for the bend

between the anticodon-stem loop (ASL) and the D-stem, the

structure of the tRNA was flexibly docked (Figures 4A and 4B).

A comparison between both subpopulations of the mamma-

lian 80S,Val-tRNA,eEF1A,GMPPNP complex reveals a rota-

tional movement of eEF1A (and tRNA) around a hinge at the

interaction between the two evolutionary conserved 258GIGTV

and 289VKS/312VKN loops (human numbering) of eEF1A domain

II with the shoulder region of the 40S subunit (Figure 5A and

Movie S2). It closes an apparent gap between the G domain

(domain I) of eEF1A and the sarcin-ricin loop (SRL, H95 of 28S

rRNA) so that interactions between both elements can be

observed only in the smaller subpopulation (Figure 5B, right).

With 12 nucleotides, the SRL is the longest universally conserved

sequence of rRNAs and an essential part of the factor-binding

site. Because interactions between the SRL and the switch re-

gions of EF-Tu have been assigned a prominent role in the

–131, July 3, 2014 ª2014 Elsevier Inc. 125

Figure 5. Interactions of the Ternary Com-

plexes with the 80S Ribosome

(A) Contacts of eEF1A with the shoulder of the 40S

ribosomal subunit.

(B and C) Contacts of (B) eEF1A and (C) A/T-tRNA

inside of the ternary complexes with SRL of 28S

rRNA.

(D) Contact of the eEF1A domain III with the C

terminus of uS12. Left column represents the

codon sampling state, right column represents

the codon recognition / GTPase activation state.

Ribosome orientations are indicated by orienta-

tion aids. The UniProt numbering which includes

the leading methionine, was used, resulting in

a +1sequence shift, such that e.g., eukaryotic

His94 is His95 according to our numbering.

See also Figure S5.

molecular mechanism of GTPase activation in the bacterial sys-

tem (Voorhees and Ramakrishnan, 2013), we propose that the

smaller subpopulation of the 80S,Val-tRNA,eEF1A,GMPPNP

complex is at least close to the GTPase activation state.

This interpretation is corroborated by the appearance of the

ribosomal decoding center in our cryo-EM maps (Figure 6). For

the smaller subpopulation of the decoding complex (Figure 6D)

as well as for the classical-2 and -1 PRE complexes (Figures 6E

and 6F, respectively) the respective cryo-EM densities are

compatible with a fully flipped-out conformation of the bases

A1824 and A1825 of 18S rRNA at the top of h44 (A1492/A1493

in E. coli) allowing minor groove interactions with the codon-anti-

codon helix of the A/T- or A-tRNA. These minor-groove interac-

tions are at the heart of the ribosomal decoding mechanism and

126 Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc.

ensurefidelity of tRNAselection (Voorhees

and Ramakrishnan, 2013) (Figure 6A).

Because codon-recognition is a prerequi-

site for GTPase activation, the SRL inter-

actions and the state of the decoding

center both suggest that the smaller sub-

population of our mammalian 80S,Val-tRNA,eEF1A,GMPPNP complex repre-

sents the codon-recognition/GTPase

activation state of the mammalian sys-

tem, and is functionally equivalent to

the bacterial structure of the GDPCP

stalled 70S,tRNA,EF-Tu complex (Voo-

rhees et al., 2010).

In contrast, the POST complex (Fig-

ure 6B) and the larger substate of the

decoding complex (Figure 6C) do not

display density for stably flipped-out

18S rRNA bases A1824/A1825 indicating

a more dynamic state of the top of h44.

Therefore, and in line with the observa-

tion that eEF1A does not yet interact

with the SRL (Figure 5B, left), we suggest

that the larger substate of themammalian

decoding complex represents an initial

codon sampling state. Here, codon-anti-

codon interaction occurs but is not yet stabilized. So far such a

state of the ribosomal decoding complex has not been directly

visualized.

Concomitant with the occupation of the 40S decoding center

by an ASL and the stabilization of the codon-anticodon duplex

by the flipped-out 18S rRNA bases A1824/A1825, a progressive

tightening of the interaction surface between the top of 18S rRNA

h44 and the shoulder region (h18 and uS12) can be observed

(Figure 6A). These more local changes appear embedded in an

overall tightening of the 40S subunit around the ASL, which in-

volves a subtle movement of the shoulder region, in particular

h16, and the 40S head (Movie S3). These changes are reminis-

cent of the bacterial domain closure movement of the 30S sub-

unit (Ogle et al., 2002). However, the mammalian conformational

Figure 6. Codon Recognition and 40S

Domain Closure at the Decoding Center

(A) Models for the codon recognition / GTPase

activation state complex. The decoding center is

shown from the 60S side, with h44 in light blue,

h18 containing the 626 (530, E. coli numbering)

loop in dark blue, A/T-tRNA in pink,mRNA in green

and uS12 in gold. Bases 1824 and 1825 (1492 and

1493, E. coli numbering) of h44 are highlighted in

red and are shown in the flipped out positions

modeled to the codon recognition state.

(B–F) Density maps for the (B) POST state, (C) the

codon sampling state, (D) the codon recognition /

GTPase activation state, (E) the PRE classical-2

and (F) the PRE classical-1 state. The models are

kept fixed in the recognition state in all panels

for better comparison. Electron density maps

are aligned to h44 and 40S platform. For clarity,

the density around h44 and uS12 has been

segmented to show only the surface-most layer.

See also Movie S3.

change is not completed at the decoding step and still pro-

gresses from the two decoding subpopulations to the classical

PRE states (Figure 6, Movie S3).

Differences in Conformation and Interaction Pattern ofthe Ternary Complexes with the RibosomeWhen the X-ray structures of the 70S,tRNA,EF-Tu complexes

stalled by kirromycin (Schmeing et al., 2009) or GDPCP

(Voorhees et al., 2010) or the cryo-EM map of the

70S,tRNA,EF-Tu,GDP,kirromycin complex (Schuette et al.,

2009) are compared to the present 80S,Val-tRNA,eEF1A,GMPPNP cryo-EM maps by aligning the large ribosomal sub-

units, the bacterial 30S subunit is found to be rotated by �2�

and in-between the positions of the mammalian 40S subunit

of the 80S,Val-tRNA,eEF1A,GMPPNP and PRE complexes,

respectively. This is smaller than the full subunit rolling between

the mammalian POST and PRE complexes but leads to a more

open-factor-binding site of the mammalian ribosome and signif-

icant differences in the relative positions of ribosomal elements

that interact with the ternary complex. The difference in position

of the tip of the functionally important SRL, for example, is in the

range of �5 A when the 80S and 70S complexes are compared

in a common alignment of the small ribosomal subunits. From

geometric consideration some corresponding adjustment in

the ternary complex can be expected.

Indeed, when the ternary complexes are compared from the

perspective of the 40S subunit, differences in the positioning

can be detected between the bacterial codon-recognition com-

plex and the mammalian codon sampling state (Figure 4D), or

codon recognition states (Figure 4E), respectively. Positional

differences between bacterial and eukaryotic complexes mani-

fest mainly at the tRNA elbow, whereas the positions and inter-

actions of the two ends of the A/T-tRNA, i.e. the ASL and the

30-CCA-end are similar (Figures 4D and 4E). The interaction of

the 30-CCA-end with the 40S subunit occurs in the context of

an interaction with domain II of eEF1A with the shoulder region

of the 40S subunit. Consistent with their functional importance

for decoding (Voorhees and Ramakrishnan, 2013), these interac-

tions appear highly conserved from bacteria to mammals. Thus,

similar to the changes between the codon sampling and codon

recognition states of the 80S,Val-tRNA,eEF1A,GMPPNP com-

plex, the interaction of domain II with the shoulder of the 40S

subunit serves as anchor point (Figure 5A).

However, the presented mammalian model of the ternary

complex shows a subtle change in the relative orientations of

eEF1A and tRNA. Compared to the ribosome-bound Thermus

thermophilus ternary complex (Voorhees et al., 2010) the T-arm

ismoved away from the core of the protein by 4–5 A (Figure S5A).

In light of the limited resolution of our study this could be re-

garded as not meaningful. However, a movement in this range

can be predicted from a structural comparison of EF-Tu (Voo-

rhees et al., 2010) and aEF1A (Kobayashi et al., 2012). Due

to sequence variation the interaction surface of domain III of

aEF1A is grown out andwould clash with the bacterial tRNA (Fig-

ure S5A). Tentative interactions of the mammalian ternary com-

plex are formed by the loops around His349/Pro350 (human

numbering) and Asp428/Met429 (human numbering) with the

tRNA around positions 52 and 63/64, respectively (Figure S5A).

Thus, our structural findings are corroborated by evolutionary

changes of EF1A, which apparently adapt the ternary complex

to a changed ribosomal environment.

Moreover, the different position of the ternary complex in the

two states of the mammalian decoding complex and in the bac-

terial complex leads to a differential interaction pattern with the

ribosome. For the mammalian codon sampling state there is a

contact between between uS12 of the 40S shoulder and domain

III of eEF1A nearby Pro409 (human numbering) (Figure 5D) facil-

itated by a eukaryotic-specific rearrangement of the C-terminal

tail of uS12 (rpS23). In the mammalian codon recognition state,

this specific contact and also the nearby evolutionary conserved

contact between nucleotide 68 of the A/T-tRNA and protein

uS12 is less obvious than for the mammalian codon sampling

Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc. 127

state (Figure 5D, right) and can be observed only at lower con-

tour level.

Most interestingly, the changed position of the elbow of the

eukaryotic A/T-tRNA is stabilized by specific interactions with

the 60S subunit (Figure 5C). In the mammalian codon sampling

state the T-loop (near position 62/63) and D-loop (near position

17) appear to interact with the prominent SRL and helix H89,

respectively (Figure 5C, left). Direct interaction of the A/T-tRNA

with 28S rRNA (outside of the stalk base) constitutes a surprising

difference between bacterial and eukaryotic systems. These

unique eukaryotic-specific contacts could stabilize binding of

the A/T-tRNA and make the reconstruction of the initial codon

sampling state possible. For the mammalian codon recogni-

tion/GTPase activation state there is a complex interaction

pattern between the SRL and eEF1A (Figure 5B, right). It is not

entirely clear whether the contact between the apical loop of

the SRL and the ternary complex directly involves the T-loop of

the A/T-tRNA as well or occurs indirectly via the eEF1A region

around Gln431, which is adjacent to the A/T-tRNA. In addition,

the cryo-EM density shows several contact regions between

the SRL and eEF1A. Onemay involve the P loop of eEF1A around

Asp17 and/or His95 from the switch II region (Figure 5B, right, d)

and a second one Arg69 of the switch I region (Figure 5B, right,

c). Interestingly, also eukaryotic-specific elements participate in

the interaction network, i.e. the helical insertion of eEF1A near

Gly121 and Gly131, respectively (Figure 5B, right, e and f).

The conformation of the switch I region differs between the

bacterial and the eukaryotic/archaeal systems. We note a helical

insertion next to the switch I region that is specific for eukaryotes

and archae. When the G domains of yeast eEF1A in complex

with the catalytic C terminus of its nucleotide exchange factor

eEF1Ba (Andersen et al., 2000) and aEF1A,GTP (Kobayashi

et al., 2012) are compared, this helical insertion is found to

be rotated by �90�, suggesting that it is part of an extended

switch I subdomain (Figure S5B). Interestingly, density for this

expanded sequence suggests an interaction with h14 of 18S

rRNA in the mammalian codon sampling state (Figure 5A,

left, d). In bacteria, h14 has been proposed as a contact site

for the switch I region of the translational GTPases EF-G (Connell

et al., 2007), EF-Tu (Schuette et al., 2009; Villa et al., 2009), and

EF4 (Connell et al., 2008). In X-ray maps of ribosome-bound

EF-Tu, this interaction has not been observed directly, but the

importance of the h8/h14 region for decoding has been demon-

strated (Fagan et al., 2013).

DISCUSSION

Conformational Modes of theMammalian 80SRibosomeThe ribosome is a dynamic macromolecular machine, in which

the elongation steps of translation are facilitated by large-scale

conformational changes of the ribosome and its ligands. As out-

lined by the metastable energy landscape view (Munro et al.,

2009), the ribosome is rather a stochastic Brownian machine

than a mechanical one. The spontaneous nature of confor-

mational modes causes intrinsic conformational heterogeneity,

even for compositionally and functionally defined ribosomal

complexes. Thus, rather than displaying a single, unique struc-

ture, dynamic heterogeneous ensembles are exhibited that

128 Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc.

reflect an exchange between distinct, functionally relevant struc-

tural configurations. A prominent example is the PRE state of the

bacterial 70S ribosome (for review seeMunro et al., 2009), where

spontaneous intersubunit rotation and the fluctuation between

classical and hybrid tRNA configurations have been well

documented. Bacterial and eukaryotic ribosomes differ in their

preferential PRE state: the bacterial 70S ribosome is found pre-

dominantly in the classical nonrotated conformation. In contrast,

vacant yeast 80S ribosomes (Ben-Shem et al., 2011; Spahn

et al., 2004a) or the mammalian 80S PRE complex have been

found to prefer the rotated intersubunit state, unless an excess

of deacylated tRNA or the antibiotic cycloheximide shifts the

landscape toward the classical, nonrotated state (Budkevich

et al., 2011). It follows that functional differences between the

translational apparatus from different kingdoms or species are

not necessarily caused by gross differences in binding sites or

conformation. More subtle changes to the energy landscape

may lead to differences in the distributions of states.

Here, we have determined the cryo-EMmap of themammalian

POST state (Figure 1A). Whereas the mammalian PRE complex

coexists in at least four structurally distinct and interchangeable

substates (Budkevich et al., 2011), intrinsic large-scale con-

formational changes such as the intersubunit rotation within

the POST complex have not been found. Thus, the POST state

may be distinguished by a deep minimum of energy/enthalpy

in the energy landscape of the elongating ribosome to compen-

sate for the relatively low entropy.

Surprisingly, the POST state deviates with respect to overall

conformation not only from the rotated PRE states but also

from the classical PRE states because a novel conformational

mode, termed subunit rolling, exists for mammalian 80S ribo-

somes (Figures 3 and S3 and Movie S1). Rolling converts the

POST to the classical PRE state subunit configurations and oc-

curs largely during the accommodation step of A-site occupation

(Figure 7). Back-rolling occurs in the opposite direction during

translocation from the PRE to the POST state; however, as trans-

location involves large ratchet-like intersubunit rearrangements

including the rotation of 40S subunit relative to 60S subunit

and swivel-like rotation of the small subunit head, back-rolling

may happen not as a stand-alone step, but in combination

with intersubunit rotation/back-rotation movements (Figure 7).

It follows that the conformational landscape of the mammalian

80S ribosome is more complex than the prokaryotic one with

three overall types of subunit configuration: the classical PRE,

the rotated PRE and the POST conformations. The underlying

basis for this new conformational degree of freedom may relate

to the presence of additional, dynamic intersubunit bridges in the

eukaryotic system (Figure S3D). Moreover, bound ligands can

influence the preferred conformation and can lead to additional

intermediate conformational states. As a consequence of sub-

unit rolling, the distinction between PRE and POST states is

much more pronounced in the mammalian system, which also

has implications for the exact nature of classical tRNA-binding

sites (Figure S2).

The Mammalian Decoding ComplexWe have shown here that the mammalian decoding complex

with the ternary complex being trapped in the A/T-state by

Figure 7. The Mammalian Elongation Circle Features Unique

Motions of the 40S Subunit

Cartoon representation of the eukaryotic elongation circle highlighting the

individual subunit motions necessary to convert one functional intermediate

state to the next. Movements of the small ribosomal subunit shared with the

prokaryotic system are noted in black, while eukaryotic-specific intersubunit

movements, i.e., rolling and back-rolling, are noted in red.

GMPPNP also exhibits subtle intrinsic structural dynamics as it

coexists in (at least) two substates. Because of the local config-

uration of the ribosomal decoding center (Figure 6) and the differ-

ential interaction pattern of eEF1A with the SRL (Figures 5B and

5C), we interpret them as states representing codon sampling

and codon recognition /GTPase activation, respectively. Both

substates are present essentially in a nonrotated, nonrolled

POST-like configuration. As the A-site is more open than in the

bacterial 70S complex, the binding site for the ternary complex

is altered between the bacterial and eukaryotic domains of life.

Indeed, we can observe conserved as well as divergent features

in the conformation/structure of the ternary complex itself and in

the interaction patterns of ribosomal ligandswith the ribosome to

account for this difference.

The interactions of the shoulder region of the 40S subunit with

two evolutionary conserved loops of domain II of eEF1A and

the 30-CCA-end of the aa-tRNA acts as an evolutionarily pre-

served anchor (Figures 4D, 4E, and 5A). On the other hand, local

changes in the ribosome exist in the elbow position of tRNA and

the exact location of eEF1A (Figure S5A). For example, the di-

vergent structure of the C-terminal end of uS12 (rpS23) is

seen to interact with eEF1A predominantly in the mammalian

codon sampling state (Figure 5D). Also, an insertion sequence

in eEF1A domain III dictates a change in relative orientation to

the T-loop/stem of the aa-tRNA (Figure S5A). Apparently, the

ternary complex evolved to interact with the altered configura-

tion of factor-binding sites between bacteria and mammals.

Most unexpected is the observed interaction of the A/T-tRNA

elbow with the apical loop of the highly conserved SRL and helix

H89 in the mammalian complex (Figure 5C). In the bacterial

system the tRNA elbow interacts only with the mobile stalk

base region of 23S rRNA. This indicates a more rigid and tighter

binding state for the tRNA elbow in the mammalian system.

The Mammalian Decoding PathwaySelection of the cognate tRNA has to proceed with optimal

speed and accuracy. This is achieved by a complex, multistep

pathway involving an initial selection step and a kinetic proof-

reading step (Rodnina and Wintermeyer, 2001; Geggier et al.,

2010). Crucial for tRNA selection is the codon recognition step

in the decoding center, and in particular the stabilization

of codon-anticodon interaction by A-minor interactions with

A1492/A1493 of 16S rRNA (A1824/A1825 in human) in the flip-

ped-out conformation (Ogle et al., 2002). Therefore, enforcing

Watson-Crick geometry of the codon-anticodon duplex pro-

vides discrimination energy between the cognate and near-

cognate interactions. Conformational changes in the ribosomes

decoding center in turn provide the signal for activation of GTP

hydrolysis by EF-Tu/eEF1A about 80 A away. For the bacterial

system, cryo-EM (Schuette et al., 2009; Villa et al., 2009) and

finally X-ray crystallography (Schmeing et al., 2009; Voorhees

et al., 2010) have revealed a series of coupled conformational

changes that start at the decoding center and are transferred

via domain closure of the 30S subunit and a distortion of the

tRNA to EF-Tu, inducing the activated conformation. Less clear

is the preceding step, because direct structural information

about the codon-sampling state is lacking.

For the mammalian sytem, however, we have now obtained

the cryo-EMmap of a substate of the GMPPNP-stalled mamma-

lian decoding complex that shows all hallmark features of a

codon sampling state (Figures 4 and 5). The ASL of the ternary

complex is located in the decoding center allowing the formation

of the codon-anticodon duplex. However, the appearance of the

ribosomal decoding center indicates that the stabilizing A-minor

interactions with A1824/A1825 of 18S rRNA do not yet occur, in

contrast to the codon recognition state (Figures 6C and 6D).

Interestingly, the conformation of the A/T-tRNA is in a bent

conformation also for the codon sampling state, likely facilitating

efficient monitoring of the codon. In the mammalian system,

codon recognition is apparently not essential for keeping the

tRNA in the distorted conformation.

Comparison of both subpopulations of the GMPPNP-stalled

decoding complex suggests a model for the mammalian decod-

ing pathway with similarities, but also surprising distinctions to

the generally accepted model for the bacterial system (Voorhees

and Ramakrishnan, 2013). As in the bacterial system, stabiliza-

tion of the codon-anticodon duplex by A-minor interactions

with A1824/A1825 of 18S rRNA and long-range signaling of

this event to the GTPase center of eEF1A are key for decoding.

However, in eukaryotes codon recognition results in a subtle

movement of the ASL deeper into the decoding cleft (Movie

S2). With the interaction between the two evolutionary

conserved 258GIGTV and 289VKS/312VKN loops of eEF1A domain

II and the shoulder region of the 40S subunit serving as an an-

chor, pulling on the ASL leads in first approximation to a rotation

of the ternary complex as a whole. Due to a lever arm effect,

movements are largest at the elbow region of the tRNA and the

Gdomain of eEF1A.We do not exclude the presence of concom-

itant conformational changes in the ternary complex such as a

Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc. 129

smaller rotation of the G domain of the factor relative to domains

II and III that has been observed in the bacterial system (Voo-

rhees et al., 2010). As a result of the rotational movement of

the ternary complex, the G domain of eEF1A becomes posi-

tioned at the SRL by a seesaw-like mechanism. The SRL has

been suggested to play a prominent role by facilitating a rear-

rangement of a catalytically important His84 (E. coli nomencla-

ture) within the switch II regions of translational GTPase factors

(Connell et al., 2007; Schuette et al., 2009; Voorhees et al.,

2010). Thus, the codon-recognition-dependent movement of

eEF1A may lead to further conformational changes within the

G-nucleotide-binding site resulting in the GTPase activated

state.

CONCLUSIONS

We have presented here cryo-EM maps of the mammalian 80S

ribosome POST and decoding complexes at subnanometer res-

olution. The maps reveal mechanistic differences between the

mammalian and the bacterial elongation cycle. Most surp-

risingly, a unique rolling of the ribosomal 40S subunit by �6�

around the upper region of the h44 occurs during the transition

from the POST to the PRE state. Accordingly, there must be

changes during the tRNA selection step and indeed we can

describe some prominent differences in the conformation of

the ribosome-bound aa-tRNA,eEF1A,GMPPNP ternary com-

plex as well as its interaction pattern with the ribosome. Evolu-

tionary changes in key steps of elongation demonstrate that in

structural terms the bacterial and the mammalian elongation cy-

cle are less similar than previously thought. Further studies will

be needed to understand whether and how these structural

changes translate into changes of mechanisms or functions in

order to achieve optimal translation or translational control in

the eukaryotic environment.

EXPERIMENTAL PROCEDURES

Sample Preparation

Reassociated 80S ribosomes from rabbit liver, free of endogenous tRNAs and

mRNAs, were prepared according to (Bommer et al., 1997). The POST 80S

complex bearing deacylated tRNAPhe in the E site and N-acetyl-Lys-tRNALys3

in the P-site was prepared by addition of elongation factor 2 (eEF2) in the

presence of 200 mM GTP to PRE complex (0.8 mM) programmed with MFK-

mRNA (Budkevich et al., 2008). The occupancy of N-acylated Lys-tRNALys3

was approximately 0.66 per 80S ribosome and more than 90% of the bound

tRNA was reactive with puromycin, indicating a nearly quantitative transloca-

tion and P-site location. In order to prepare the decoding complex, 80S ribo-

somes programmed with MFV-mRNA and occupied by Ac[3H]Phe-tRNAPhe

were incubated with preformed ternary complex eEF1A,GMPPNP,[14C]Val-

tRNAVal in the presence of 400 mM GMPPNP (Budkevich et al., 2008).

Electron Microscopy and Image Processing

The 80S complexes were diluted to a final concentration of 30 nM and flash-

frozen in liquid ethane and images were recorded under low-dose conditions

using an FEI Tecnai G2 Polara operating at 300 kV and a nominal magnification

of 39,000. The resulting micrographs were digitized on a drum-scanner (Hei-

delberg) with a pixel size of 1.26 A on the object scale. Multiparticle refinement

using SPIDER (Frank et al., 1996) was carried out as described previously

(Budkevich et al., 2011; Loerke et al., 2010; Penczek et al., 2006) in order to

overcome sample heterogeneity caused by substochiometric binding of the

ligands and inhomogeneity in the preparation of themammalian 80S ribosome.

130 Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc.

Final reconstructions were calculated with SPARX (Hohn et al., 2007). The final

resolution for all maps was estimated using the 0.5 cutoff criteria from the

Fourier shell correlation curves. For further details see Extended Experimental

Procedures.

Modeling

A secondary structure map for rabbit 80S ribosomes is not available, so

the human ribosome as target for the modeling was chosen. Initial structural

models of the human ribosomal RNAswere built following a homologymodeling

approach (Tung and Sanbonmatsu, 2004). For homology modeling of the ribo-

somal proteins we used Prime (from the Schrodinger suite) (Jacobson et al.,

2002; Jacobson et al., 2004). Initial structural models of the 80S ribosome

were fit to the cryo-EM map of the 80S POST complex using MDfit (Ratje

etal., 2010)whichallowsflexibledockingwhilemaintainingstereochemistrypre-

sent in the initialmodel. Simulationswereperformedatstructure-basedpotential

temperature of 20. EMweightwas adjusted to the number of atoms in themodel.

ACCESSION NUMBERS

The European Molecular Biology Laboratory-European Bioinformatics Insti-

tute (Cambridge, UK) 3D-EM data base accession numbers for the electron

density maps of the mammalian POST, PRE (classical-1/2), and decoding

(codon sampling and codon recognition/ GTPase activation) complexes re-

ported in this paper are EMD-2620, EMD-2621, EMD-2622, EMD-2623 and

EMD-2624, respectively. The Protein Data Bank accession numbers for the

human models for 40S and 60S subunits are 4cxc (40S), 4cxd (60S ribosomal

proteins), 4cxe (60S rRNA), and 4cxb (ligands) and for the ternary complexes

are 4cxg (initial codon sampling state) and 4cxh (codon recognition state).

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures, five

figures, one table, and three movies and can be found with this article online

at http://dx.doi.org/10.1016/j.cell.2014.04.044.

AUTHOR CONTRIBUTION

T.V.B. prepared the 80S-POST and decoding complexes. T.V.B., J.G. and

T.M. collected the cryo-EM data. T.V.B., J.G., J.L., and C.M.T.S. carried out

the image processing. E.B., J.L., D.J.F.R., J.I., P.W.H., C.-S.T., K.Y.S. and

C.M.T.S. carried out the modeling of the human 80S ribosomes. T.V.B.,

E.B., J.L., K.H.N. and C.M.T.S. discussed the results and wrote the paper.

ACKNOWLEDGMENTS

We thankDr. Scott Blanchard for helpful discussion. Thepresentworkwas sup-

ported by grants from the Deutsche Forschungsgemeinschaft DFG (SFB 740

to C.M.T.S., P.W.H., and T.M.; 436 UKR 113/64/1-1 to T.B. and HI 1502 to

P.W.H.), HSFP and Senatsverwaltung fur Wissenschaft, Forschung und Kultur

Berlin (UltraStructureNetwork, Anwenderzentrum). K.Y.S. was supported by

HFSP, NIH Grant R01-GM072686 and Los Alamos Institutional Computing.

We acknowledge the use of computational resources supplied by the North-

German Supercomputing Alliance (HLRN; project beb00001 to C.M.T.S and

bec00085 to P.W.H.).

Received: October 1, 2013

Revised: February 24, 2014

Accepted: April 18, 2014

Published: July 3, 2014

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Supplemental Information

EXTENDED EXPERIMENTAL PROCEDURES

Sample PreparationReassociated 80S ribosomes from rabbit liver, free of endogenous tRNAs and mRNAs, were prepared according to (Bommer et al.,

1997) with slight modifications. In order to wash endogenous RNases from polysomes, the postmitochondrial supernatant was

centrifuged through a 1M sucrose cushion, which contained 0.5 M KCl. Reassociation of 40S and 60S subunits was confirmed by

analytical centrifugation in SW40 rotor (18,000 rpm, 17 hr). The ribosome concentrations were calculated assuming the following

ratios: 60 pmol/A260 for 40S subunits, 30 pmol/A260 for 60S subunits and 20 pmol/A260 for 80S ribosomes. 80S particles were pro-

grammedwith the heteropolymeric MFK-mRNA for POST, andMFV-mRNA for decoding complex preparedwith run-off transcription

according to (Triana-Alonso et al., 1995). Bovine liver tRNAPhe, tRNALys3 and tRNAVal were purified from tRNAbulk preparations by us-

ing ion-exchange DEAE and reverse phase Hypersil 5 C4 HPLC chromatography. N-acetyl-[14C]Lys-tRNALys3 , N-acetyl-[3H]Phe-

tRNAPhe, and [14C]Val-tRNAVal were purified by reverse-phase HPLC on a Nucleosil 300-5 C4 column using a methanol gradient

as described (Cayama et al., 2000).

Elongation factors eEF1A and eEF2 from rabbit liver were isolated using a combination of gel-filtration and ion-exchange chroma-

tography as described (Kemper and Merrick, 1979; Shalak et al., 1997).

The extent of tRNA bindingwas determined by nitrocellulose filtration and the specificity of tRNA binding by the puromycin reaction

(0.8 mM). All incubations were performed in polyamine buffer containing 20 mM HEPES–KOH (pH 7.5 at 4�C), 5 mMMgCl2, 100 mM

KCl, 0.6 mM spermine, 0.8 mM spermidine and 6 mM 2-mercaptoethanol (El’skaya et al., 1997).

Electron Microscopy and Image ProcessingReassociated 80S complexes were diluted to a final concentration of 30 nM and flash-frozen in liquid ethane on carbon-coated

Quantifoil grids (Quantifoil, Germany) using a Vitrobot (FEI) device. Images were recorded under low-dose conditions on film (Kodak

SO-163) using an FEI Tecnai G2 Polara operating at 300 kV and a nominal magnification of 39,000. The resulting micrographs were

digitized on a drum-scanner (Heidelberg) with a pixel size of 1.26 A on the object scale. After visual inspection and evaluation of

micrographs according to their power spectra, particles were preselected using Signature (Chen and Grigorieff, 2007), boxed out

and decimated. This and all further image processing steps were carried out using SPIDER (Frank et al., 1996), final reconstructions

were calculated with SPARX (Hohn et al., 2007). In order to overcome sample heterogeneity caused by substochiometric binding of

the ligands and inhomogeneity in the preparation of the mammalian 80S ribosome, multiparticle refinement was carried out as

described previously (Budkevich et al., 2011; Loerke et al., 2010; Penczek et al., 2006). A reconstruction of the vacant 80S ribosome

from rabbit was introduced as a seed structure to increase the number of references until stable subpopulations were obtained.

The 80S POST complex (initial data set is 826 micrographs with 1,072,632 particle images) was analyzed and sorted according to

multi-particle alignment procedures (Figure S1). During this procedure about 400,000 particle images were discarded. Only about

35% (236,113) of the remaining ribosomal complexes constituted the subpopulation corresponding to the desired 80S POST com-

plex and were used for final reconstruction (Figure S1). In addition, our unsupervised classification procedure revealed the presence

of two subpopulations representing PRE complexes. Themap of the larger PRE subpopulation (29%of the particle images) exhibited

density for tRNAs in classical A-, P- and E-sites, whereas the second PRE complex (15% of the particle images) had tRNAs in hybrid

A/P- and P/E-states. According to our previous analysis of the 80S PRE complex (Budkevich et al., 2011) these two PRE subpop-

ulations correspond to the classical-1 and rotated-2 PRE states, respectively. Further refinement of the PRE classical-1 state yielded

three distinct structures: PRE (classical-1: 66,618 images), PRE (classical-2: 73,951 images) and an unassigned structure with frag-

mented A-site tRNA (51,149 images).

In addition to the POST and PRE-like structures, the initial data set contained a fourth subpopulation of particle images (143,150

images) that exhibited ligand density in the factor-binding site together with density for P/P- and E/E-tRNAs. By its appearance this

density can be identified to correspond to elongation factor eEF2 (Figure S1). It is remarkable that eEF2 is found bound to a subset of

ribosomes without being stalled by an antibiotic or a nonhydrolyzable GTP analog. The significant presence of PRE complexes and

translocation intermediates within the posttranslocational sample cannot be explained by inefficient translocation, because the pu-

romycin reaction indicates nearly quantitative translocation. However, the biochemical analysis and the cryo-EM results can be

reconciled by taking into account the possibility of a reverse translocation reaction. Spontaneous, reverse translocation has been

found in bacterial system and can be driven by an excess of deacylated tRNA in in vitro assays (Shoji et al., 2006). The possibility

of a reverse translocation was also suggested for the eukaryotic system (Nilsson and Nygard, 1992). The presence of eEF2 can

lead to the recurring translocation. It seems that translocation and reverse translocation result in equilibrium of PRE and POST com-

plexes as visualized by cryo-EM. eEF2-dependent translocation can occur also during the incubation with puromycin leading to a

nearly quantitative puromycin reaction.

For the 80S decoding complex, the initial data set (230 micrographs, 626,562 particle images) separated into two major

populations during several rounds of the multi-particle refinement. The first subpopulation, reconstructed from 247,633 particle

images, represents the decoding complex with two classically oriented tRNAs in P- and E-sites and the ternary complex aa-

tRNA,eEF1A,GMPPNP stalled in the A/T-position. The second subpopulation, reconstructed from 210,574 particle images, consists

of PRE 80S ribosomes bearing three tRNA molecules and was described in (Budkevich et al., 2011). For further processing, only the

Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc. S1

first of these two subpopulations was used. After further multi-particle classification and removal of particles images not assigned to

the decoding complex, the remaining 176,675 particles split into three final subpopulations: the codon sampling complex (79,705

particle images), the codon recognition / GTPase activation complex (52,686 particle images), and a third, unassigned population

with 44,284 particle images.

The final resolution for all (POST, classical-1, classical-2, codon sampling and codon recognition / GTPase activation) maps was

estimated using the 0.5 cutoff criteria from the Fourier shell correlation curves (Figure S1B) and the maps were filtered accordingly.

Interpretation and ModelingAs a starting point, models derived from the X-ray structures of S. cerevisiae 80S (Ben-Shem et al., 2010; PDB IDs 3O2Z, 3O58; Ben-

Shem et al., 2011; PDB IDs 3U5B-I), T. thermophila 40S-eIF1 complex (Rabl et al., 2011; PDB ID 2XZN) and T. thermophila 60S-eIF6

(Klinge et al., 2011; PDB IDs 4A17, 4A19) were fit as rigid-bodies into the cryo-EMmaps usingChimera (Pettersen et al., 2004). Crystal

structures of prokaryotic tRNAs (Voorhees et al., 2010; PDB ID 2WDG) were docked into segmented densities corresponding to the

tRNAs. For A/T-tRNA, we created hybrid structures from PDB IDs 2XQD (27-43) and 1TTT (1-26; 44-76) by fitting each fragment inde-

pendently as a rigid-body into the decoding state maps. RNA geometry at the junction sites was then restored using RCrane (Keating

and Pyle, 2012) and CNS 1.3 (Brunger, 2007).

As a secondary structure map for rabbit 80S ribosomes is not available, we chose the human ribosome as target for our modeling.

Importantly, on an overall structural level rabbit and human ribosomes are very similar (Budkevich et al., 2011; Spahn et al., 2004b).

Furthermore, sequence alignment between ribosomal proteins from human and rabbit shows that the sequence identity is generally

high with the majority of the aligned proteins being identical in length and sequence (for example eS6, uS9 (rpS16), uS12 (rpS23),

eS24, eS27, uL15 (L27A), eL19, eL27). Others are identical in length but show minor differences in sequence identity (e.g., eS8

99%, eS26 87%, eL32 94%, eL39 78%). Only few are slightly different in length and sequence identity (e.g., eL6: human 288 residues

versus rabbit 291, 84%).

It is remarkable that mammalian 80S ribosomes contain only one additional protein, eL28 (L28e), compared to ribosomes from

yeast. However, protein eL28 is present in T. thermophila 60S subunits (Klinge et al., 2011). To obtain an all-atommodel highly consis-

tent with the cryo-EM reconstructions, we also employed MDfit (Whitford et al., 2011), which allows for flexible docking while main-

taining stereochemistry present in the initial model. As we did not attempt to model those parts of rRNA expansion elements and

proteins that are apparently too flexible to be observed in the map, our final model of the human 80S ribosome encompasses 33 pro-

teins from the small and 43 proteins from the large subunit and 5,634 out of 7,173 nucleotides for the rRNAs (Figure 1A, Table S1).

The initial structural models of the human ribosomal RNAs were calculated following a protocol developed for E. coli 30S (Tung

et al., 2002) and T. thermophilus 70S (Tung and Sanbonmatsu, 2004) ribosomal RNA modeling. The 4 A resolution crystal structures

(Ben-Shem et al., 2010; PDB IDs 3O2Z, 3O58) of the yeast small and large ribosomal subunits were used as templates for RNA ho-

mologymodeling. The resultingmodels were further improved using the 3 A resolution crystal structures (Ben-Shem et al., 2011; PDB

IDs 3U5B, 3U5D) of the yeast small and large ribosomal subunits. Ourmotif-based approach preserves RNA stereochemistry present

in crystallographic motifs and maintains structural integrity (e.g., bond lengths, van der Waals contacts).

For homology modeling of the ribosomal proteins we used Prime (from the Schrodinger suite) (Jacobson et al., 2002, Jacobson

et al., 2004). The crystal structures from yeast (PDB ID 3U5E) or Tetrahymena thermophila (PDB ID 2XZM) were used as templates.

Appropriate template structures were selected from these two entries based on completeness of the template structure, similarity

between target and template and fitting of the modeled structure to the electron density maps. Loops and insertions were either

modeled with Prime (Schrodinger suite) or with Superlooper (Hildebrand et al., 2009). Superlooper allows visualization and selection

of a loop conformation in the context of the whole molecule. 87% of all residues of these 76 proteins, 4,745 of 5,476 residues of

the small subunit and 6,507 of 7,429 residues of the large subunit, were modeled. The average sequence identity/similarity between

template and target protein structures is 50%/67% for the small subunit and 55%/70% for the large subunit. The sequence identity

between target and template protein structures in the small and the large subunit ranges from 22% (eL28) to 88% (eL41).

Initial structural models of the 80S ribosome were flexibly fit to the cryo-EM reconstruction of the 80S POST configuration using

MDfit (Whitford et al., 2011). Simulations used protocols similar to those in the online tutorial and to our previous studies (Ratje et al.,

2010). Forces associated with EM density were evaluated every 200 time steps. Simulations were performed at structure-based

potential temperature of 20. EMweight was adjusted to the number of atoms in the model. As in previous studies, three successive

simulations were performed: (1) MDfit using the 80S cryo-EM reconstruction with an additional Gaussian filter with a half-width of 4 A,

(2) MDfit using the 80S cryo-EM reconstruction Gaussian with an additional Gaussian filter with a half-width of 2 A, and (3) MDfit using

the 80S cryo-EM reconstruction. Each simulation used between 100,000-300,000 time steps and required �24 hr on a MAC OSX

desktop computer with eight processor cores.

Cryo-EMdensities and the atomicmodels were visualized using the programChimera (Pettersen et al., 2004), which was also used

to create all images and movies.

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Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc. S3

Figure S1. Heterogeneity in the 80S Ribosome POST Complexes from Rabbit Liver, Related to Figure 1

(A) Top: The POST 80S complex (initial data set is 1,072,632 images) was analyzed and sorted into distinct classes according to multi-particle alignment

procedures: POST (236,113 images), PRE-classical-1 (191,718 images), PRE-rotated-2 (100,473 images) and eEF2-containing (143,150 images) (middle).

Subsequent refinement of the POST and PRE (classical-1) classes yielded four distinct structures: POST (236,113 images), PRE (classical-1) (66,618 images),

PRE (classical-2) (73,951 images) and a structure with fragmented A-site tRNA (51,149 images) which is not in the scope of this study (bottom).

(B) Fourier Shell Correlation (FSC) curves of the presented complexes. The inset table shows the final resolutions according to the FSC 0.5 criterion for each

complex.

(C) Cross-resolution curves between each complex and its atomic model indicate resolutions in the same range as the FSC analysis, thus corroborating our

resolution estimation.

S4 Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc.

Figure S2. Eukaryotic-Specific Contacts between P-tRNA and Large Ribosomal Proteins, Related to Figure 2(A) Contact of the P-site tRNA (position 1) with ribosomal protein uL16 (L10e). The same contact of uL16 (L10e) and the peptidyl-tRNA in the A/P-site has been also

observed in the rotated-2 subpopulation of the PRE ribosome (Budkevich et al., 2011). Thus, the uL16 loopmight escort the 50-end of peptidyl-tRNA from the A/P-

hybrid position to the classical P/P-site of the POST state.

(B) Contacts of the P-tRNA elbow with ribosomal proteins eL44 and uL5 (rpL11). The so-called P-site loop (Rhodin and Dinman, 2010), a highly conserved,

basically charged internal loop of uL5 (amino acids 48 to 68) appears to participate in this interaction and also in a nearby interaction with the D-loop of the P-site

tRNA.

White arrows indicate contacts. Themodels for tRNAs and ribosomal proteins are derived from the homology model of the human 80S ribosome presented in this

paper. Ribosome orientations are indicated by the orientation aids (top left).

Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc. S5

Figure S3. 40S Rolling and Its Effect on Integrity of Ribosomal Intersubunit Bridges and on tRNA Positions, Related to Figure 3

(A) Comparison of the 80S ribosome in fully ratcheted state (Spahn et al., 2004b) with the ribosome in classical-1 state (this study). The fully ratcheted state is

characterized by a rotation of the 40S subunit of 5� and a swivel movement of the head of 14�.(B) Comparison of the ribosome in classical-1 state (this study) to the ribosome in rotated-2 state (Budkevich et al., 2011) shows a rotation of the 40S subunit of 8�.(C) Comparison of the 40S subunit positions in classical-1 state to the POST state (both from this study) reveals a new motion of 40S subunit of 6� we term

‘‘subunit rolling.’’ Comparison of the 40S positions in classical-1 and POST states is already shown in the related Figure 3 but color-coded from 0�20 A instead of

0 �12 A.

(D) Intersubunit bridges of the POST complex. In keeping with the previously established nomenclature (Spahn et al., 2001), universally conserved bridges are

numbered B1-B7 (red) and eukaryotic-specific are numbered eB8-eB11 (pink). Furthermore, additional bridges eB12-eB14 that were described for yeast 80S

ribosomes in (Ben-Shem et al., 2011) are also observed. Bridges B6, B7 and eB13 (green) were not observed in the POST state due to subunit rolling. Moreover,

bridge eB8 (eS1(S3A)/H79; green) is absent in the POST state as well because it is a property of the 80S ribosome in the rotated conformation (Ben-Shem et al.,

2011).

(E) Comparison of the positions of the eukaryotic PRE and POST tRNAs near the elbow regions as observed in mammalian system (colored ribbons) with those

observed in T. thermophilus 70S structures (Voorhees et al., 2010), (gray ribbon, PDB ID code 2WDG). Structures of P- and E-site tRNAs along with their paired

mRNA codons were docked independently as rigid bodies to the PRE and POST cryo-EM maps. Color code: P-tRNA in PRE complex (blue), P-tRNA in POST

complex (green), E-tRNA in PRE complex (red), E-tRNA in POST complex (orange). Distances were measured between phosphate backbones at position 56.

(F) Effects of subunit rolling on the tRNA positions. Structures as in (E) but color-coded by distances from 0 to 5 A.

S6 Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc.

(legend on next page)

Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc. S7

Figure S4. Protein Sequence Alignment of EF1A Homologs from Different Species, Related to Figure 4

(A) A protein sequence alignment of EF1A homologs from different species clearly demonstrates that, while EF1A/EF-Tu sequences are largely conserved,

archaeal and eukaryotic homologs feature a conserved helical extension of the switch I region (highlighted yellow in the structural annotation). This insertion is

located between the conserved a-helix 1 and b strand 2 and is not present in EF-Tu. The sequence alignment was calculated using Clustal omega (Sievers et al.,

2011) with sequences obtained from the UniProt database (Apweiler et al., 2004) (IDs Q9YAV0, P68104, P68103, P60338, P53013, P50522, P10126, P0CE47).

Different from the initial E. coli EF-Tu1 structure (PDB ID code 1ETU), we used the UniProt numbering including the leading methionine. This results in a +1

sequence shift, such that e.g.,E. coliH84 is H85 according to our numbering. Visualization of the sequence alignment according to theClustalX-color scheme and

a representation of their phylogenetic relationship were prepared using JalView (Waterhouse et al., 2009). For the consensus sequence, functional regions are

highlighted in color (green: P loop, red: switch I region, blue: switch II region) and the corresponding GTP-binding sequence motifs (Kjeldgaard et al., 1996) in

white.

(B) Location of the eukaryotic-specific insertion (helix 5) between domain I and II. The density corresponded to the helix 5 of rabbit eEF1A is highlighted in pink,

while the corresponding section of the A. pernix X-ray model (PDB ID 3VMF) is shown in dark blue.

S8 Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc.

Figure S5. Reorganization of the Mammalian Ternary Complex to Adopt to a New Ribosomal Environment, Related to Figure 5

(A) Structural and sequence comparison of the interface between domain III of aEF1A/EF-Tu and the acceptor (stem and elbow of A/T-tRNA for archaeal (blue)

and bacterial (gold) X-ray models (bacterial ternary complex: PDB ID 2XQD; archaeal EF1A: PDB ID 3VMF; A/T-tRNA: hybrid structure from PDB IDs 2XQD for the

ASL and 1TTT for the tRNA body). Analysis of atom-atom distances between the bacterial tRNA and the archaeal EF1Amodel reveals multiple clashes (indicated

by red lines in the structure and red-orange bars in the sequence panel). Most likely, several insertions (dark blue) compared to the bacterial EF-Tu extend EF1A

either directly or indirectly (light blue) toward the bacterial tRNA position. As a consequence, the position of the archaeal tRNAmust differ from that of its bacterial

counterpart due to steric hindrance. Interactions of the aEF1A with the A/T-tRNA are formed by the loops around His335/Pro336 (archaeal numbering) and

Asp414/Met415 archaeal numbering) around positions tRNA 52 and 63/64, respectively. Orientation aids between the structure and the sequence are given by

small letters highlighting regions of interest (a, b, c and d). The structure-based sequence comparison was calculated using Chimera (Pettersen et al., 2004).

(B) Superposition of the GTP-binding domains of archaeal EF1A,GTP (Kobayashi et al., 2012; PDB ID code 3VMF; red ribbon) and yeast eEF1A (Andersen et al.,

2000; PDB ID code 1F60; gray ribbon). The helical extension (aminoacids 35-50) is depicted in orange. Conserved switch I helixes (aminoacids 57-61) and

(aminoacids 63-68) are colored yellow while switch II region is depicted blue.

Cell 158, 121–131, July 3, 2014 ª2014 Elsevier Inc. S9