Molecular Cell, Volume 27

Supplemental Data

Structure of TOR

and Its Complex with KOG1 Alessandra Adami, Begoña García-Álvarez, Ernesto Arias-Palomo, David Barford, and Oscar Llorca

SUPPLEMENTARY EXPERIMENTAL PROCEDURES

N-terminal TAP tagging of TOR1 and C-terminal TAP tagging of KOG1

PCR products were integrated into the yeast genome by homologous

recombination, and positive integrations were selected for on appropriate antibiotic

plates. Because endogenous N-terminal tagging of a gene causes disruption of the

native promoter region, the vector used for TOR1 tagging was designed to encode for

the native promoter. The N-terminal tagging pFA-6A-kanMX6-TOR1p-NTAP vector

was derived from the pFA6a-kanMX6-PGAL-GST vector through several modification

steps. The NTAP tag is a TAP tag in which the order of the two tags has been inverted:

first the calmodulin binding peptide (CBP) separated from the protein A tag by two

TEV cleavage sites. The final product was a vector that codes for the yeast TOR1

native promoter and an inverted TAP tag (NTAP). Subsequently, two yeast codon-

optimised linker sequences were introduced 3’ to the NTAP in order to allow more

accessibility to the CBP tag portion, as initial purification trials with the yeast strain

lacking the linker sequences showed failure of efficient binding to the calmodulin resin.

The linker sequences were designed to flank a PreScission Protease site to allow

removal of the tag after purification if desired. The yeast strain expressing NTAP-

TOR1 carried also a CBP (Calmodulin Binding Protein tag) at the C-terminus of KOG1.

For purification of the TOR-KOG1 complex, a TAP tag was incorporated into the C-

terminus of KOG1 using the pFA-6a-TAP-KanMX6 PCR-based cloning vector and in

vivo genetic recombination (Passmore et al., 2003). To incorporate a His-tag for nano-

gold labeling of the KOG1 C-terminus, the pFA-6a-TAP-KanMX6 vector was modified

by the insertion of a His10 sequence N-terminal to the CBP.

Large scale TAP-tagged purification of TOR1 and TOR-KOG1 complex

The yeast colony of interest was grown with shaking (200 rpm) at 30°C until an

OD600 was 0.6-1.0. Cells were harvested at 3500 rpm for 12 minutes, pellets rinsed with

50 ml of cold water to eliminate any extracellular proteases and again spun down and

flash-frozen in dry ice/ethanol bath. Cell pellets were resuspended in one volume of

cell lysis buffer (Table S1) with the addition of one EDTA-free protease inhibitor tablet

and 1/100 volume DNaseI. Cells were lysed with four passes through the Emulsiflex

C5 homogeniser at 20 000 psi on ice. Following centrifugation at 20 000 rpm, the

lysate was loaded onto a IgG sepharose column. For purification of free TOR1 from the

N-TAP-TOR1 strain, 0.01% Tween20 was added to disrupt the association with KOG1.

IgG columns were prepared by adding 0.5 ml of IgG sepharose for every 6 litres of

culture (about 30 g of cell pellet). For yeast cultures up to 20 litres, the resin was

packed into Amicon Vantage L column - 10 ml capacity and equilibrated according to

the manufacturer’s instructions at 4°C. TEV cleavage was then performed by adding 10

units of TEV protease (Invitrogen) (about 15 µg) for every 100 µl of IgG resin.

Cleavage reactions were incubated at 16°C for 2 hours with gentle shaking. The resin

was then centrifugated 1 min at 4°C in order to separate the supernatant where the

cleaved protein should be found, which was then loaded on a calmodulin affinity resin

(0.05 ml of resin for every litre of cell culture). The resin packed in an Amicon Vantage

L column (10 ml capacity) that had been previously equilibrated with calmodulin

binding buffer (Table S1). 1M CaCl2 to a final concentration of 5mM (1/200 volume)

was also added to the protein. The column was sealed and incubated at 4°C for 1 hour

on rollers. After binding, the column was washed with 70 bed volumes of calmodulin

binding buffer. TORC1 was eluted in calmodulin elution buffer in 14 fractions. Each

fraction was 1 bed volume. Samples were analysed by SDS-PAGE on 8%

polyacrylamide large gel and silver stained. Fractions were then pooled and

concentrated using Centricon YM-50 (Millipore). Protein concentration was measured

at 280 nm. The concentrated protein was aliquoted, flash frozen in liquid nitrogen and

stored at -80°C.

Cloning of full length KOG1 and full length LST8 and TOR1 fragments

The open reading frame sequences of full length KOG1 and LST8 were obtained

by PCR from S. cerevisiae BJ2168 colonies. Primers were designed so that the KOG1-

PCR product was flanked by the SacI and XhoI restriction sites, whereas the LST8-

PCR product was flanked by the BamHI and NotI restriction sites. The purified PCR

products were then ligated with the pET28a vector (previously digested with the

appropriate restriction enzymes) and then transformed in BL21-Gold (DE3) cells.

Clones were verified by restriction enzyme digestion and sequencing.

For TOR fragments, PCR products whose sequences partially overlapped one

another and that spanned the entire length of TOR1 were amplified using the primers

that were also flanked by PstI and SacI restriction sites 5’ and 3’ respectively. Colony

PCR was carried out from S. cerevisiae strain BJ2168 and inserted into a pSP64

Poly(A) vector (Promega).

C-terminal TAP tagging of LST8

Tagging of the C-terminus of LST8 with a hexa-His tag using the pFA6a-His6-

nourseothricin vector (in the yeast strain carrying KOG1-TAP) was attempted with the

aim of detecting LST8 in the complex by Western blot or nano-gold-labelling.

However, tagging resulted in lethality, most likely because it interfered with LST8

protein stability (data not shown).

In vitro coupled transcription and translation

TOR1 fragments cloned in pSP64 Poly(A) vector and full-length LST8 and full-

length KOG1 cloned in pET28a vectors were translated using the TNT Quick Coupled

in vitro Transcription and Translation (IVT) System (Promega). TNT reactions for

TOR1 fragments were additionally radioactively-labelled by including Redivue L-

[35S]methionine (Amersham) in order to detect binding to the interacting partner (LST8

or KOG1) following a MagZ (Promega) pull-down. Control reactions to verify the

correct translation of LST8 and KOG1 were also set up with the addition of

[35S]methionine. Following the TNT reaction, hexa-His tagged-fusion proteins (LST8

and KOG1) were immobilized on 40 µl of MagZ binding particles with the addition of

100 µl MagZ binding buffer + 1% BSA. The substrates were left incubating for 15

minutes at room temperature with mixing to prevent particles from settling. Unbound

protein was removed by placing the tubes on a magnetic stand. Washing with the

binding buffer was carried out before adding the TNT reaction of the TOR1 fragments

with 100 µl of MagZ binding buffer +1% BSA. The binding reaction was left to

incubate for 1 hour at room temperature with mixing. After washing, unbound TOR1

fragments were removed by placing the tubes on a magnetic stand, and the remaining

MagZ particles were resuspended in 20 µl SDS-PAGE sample buffer. Reactions were

analysed on 10% SDS-PAGE small gels. Gels were fixed and stained with Coomassie

blue, dried at 80°C for 1 hour and exposed to BioMax MR-1 film (Kodak).

Electron microscopy and image processing of TOR1 and TOR-KOG1

The purified samples were applied to carbon-coated grids, negatively stained

with 1% uranyl acetate and observed in a JEOL 1230 electron microscope operating at

100kV. Micrographs were recorded at a magnification of 50,000 under low dose

conditions and scanned using a MINOLTA Dimage Scan Multi Pro scanner at 2400 dpi

and averaged to a final 4.2 Å/pixel at the specimen. The contrast transfer function of

the microscope (CTF) for each micrograph was estimated using CTFIND3 (Mindell and

Grigorieff, 2003). Individual molecules were boxed, processed and refined using

EMAN (Ludtke et al., 1999). To avoid model bias the particles were processed using

several models as starting references for refinement as performed before (Spagnolo et

al., 2006). Notably we never used information from any previous works and the 3D

reconstructions for TOR1 and TOR-KOG1 were processed completely independently.

Some models were constructed by randomly generated noisy Gaussian blobs using

EMAN (Ludtke et al., 1999) and XMIPP (Sorzano et al., 2004), while others were built

after reference-free image classification, averaging and using common lines as

implemented in EMAN. Resolution was estimated by Fourier Shell Correlation using

the eotest command in EMAN and a correlation of 0.5 which was found to correspond

to 25 Å and 26 Å for TOR1 and TOR-KOG1 respectively. The handedness of the 3D

reconstruction of TOR1 was defined by fitting and comparison with the reconstruction

of DNA-PKcs since the absolute hand for DNA-PKcs has been previously determined

using 2D crystals and electron crystallography (Leuther et al., 1999). In order to select

the correct hand for the TOR-KOG1 complex, the two possible hands of this

reconstruction were aligned in 3D using the align3d command in EMAN (Ludtke et al.,

1999) and the ADP platform. Only one possible hand of the complex was the output for

all the fitting experiments performed. All other alignments and fittings described were

performed using the COLORES (Chacon and Wriggers, 2002) in SITUS (Wriggers et

al., 1999), EMAN (Ludtke et al., 1999) and ADP_EM platforms (Garzon et al., 2006).

The volumes were rendered to show 100 % of the protein mass assuming an average

density of 1.35 g/ml and visualised using UCSF Chimera (Pettersen et al., 2004). Rough

estimation of the protein mass accounted by different parts of the reconstructions were

performed by segmenting and using the volume command in EMAN at a threshold

equivalent to the one used in the complete protein and an average density of 1.35 g/ml.

To calculate the difference map between the TOR1 and TOR-KOG1 structures

the reconstructions were initially aligned. Then, the head and arm domains of the fitted

TOR1 were segmented to allow a further refinement of the fitting of each segment.

After this fitting the difference between the two structures was calculated with the

voldiff command in SITUS.

Gold-labelling was performed by incubating TOR-KOG1 complexes His-tagged

at the C-terminus of KOG1 with a 1.8 gold derivate (Nanogold). To allow binding of

the gold label, the TOR-KOG1 complex was previously dialyzed to remove any

remaining of the elution buffer (from the purification of the His-tagged product). The

conjugates were formed by incubation with a 10 molar excess of nanogold for 30 min at

4ºC. These were observed in a JEOL 1230 electron microscope after negative staining.

339 particles that seemed to have some putative gold density were extracted and refined

with EMAN (Ludtke et al., 1999) using the TOR-KOG1 complex as a reference. Some

averages displayed a putative gold density not present in the reference projection used

for classification and alignment. The location of the gold on the 3D reconstruction was

inferred by the orientation of the volume projection used as reference for alignment of

each class. Because negative staining can produce a dark background around averages

we only considered as possible positives those averages containing a black spot present

in the class averages within the actual density of the protein and which were not present

in the reference projection (corroborated by difference mapping). Several 2D averages

complied with these requirements and importantly all of them were found to correspond

to several different orientations of the 3D reconstruction but where the putative label

was detected in the same area, supporting the result.

CryoEM of the TOR-KOG1 complex was performed after vitrification of the

sample by plunging into liquid ethane. Samples were observed in a Tecnai G2 FEG 200

operated at 200 kV and equipped with a Gatan liquid nitrogen specimen holder. Images

were recorded at 50,000 and different defocus on Kodak SO-163 film and digitized with

a Zeiss scanner and a step of 7 µm. Particles were extracted and averaged to a final 2.8

Å/pixel. Despite considerable effort, only a few hundreds of single molecules were

collected, mainly due to the low amount and concentration of the purified material.

Several commands in EMAN (Ludtke et al., 1999) were used to obtain reference-free

averages of the cryoEM data.

Maximum Likelihood analysis of the heterogeneity of the data set

The consistency of the reconstructions of TOR1 and TOR-KOG1 complex with

more that one 3D solution was tested using the multirefine command in EMAN (Ludtke

et al., 1999) and a new maximum likelihood method (Scheres et al., 2007). We applied

the method as described in the XMIPP home page (Sorzano et al., 2004) and with the

help of Sorjs Scheres (CNB, Madrid).

Table S1. Buffers used during purifications

Buffer Composition Cell Lysis Buffer

50 mM HEPES-KOH pH7.1, 10 mM B-glycerophosphate, 50 mM NaF, 0.5 mM EGTA, 0.5 mM EDTA, 10% glycerol, 0.25 % Tween 20 for TOR-KOG1 or 0.01% Tween 20 for free TOR1, 150 mM KCl

IgG Wash Buffer I

50 mM HEPES-KOH pH7.1, 10 mM B-glycerophosphate, 50 mM NaF, 0.2 mM PMSF, 0.5 mM EGTA, 0.5 mM EDTA, 10% glycerol, 0.25 % Tween 20 for TOR-KOG1 or 0.01% Tween 20 for free TOR1, 150 mM KCl

IgG Wash Buffer II

3 mM DTT, 50 mM HEPES-KOH pH7.1, 10 mM B-glycerophosphate, 50 mM NaF, 0.5 mM EDTA, 10% glycerol, 0.25 % Tween 20 for TOR-KOG1 or 0.01% Tween 20 for free TOR1, 150 mM KCl

CaM binding buffer

50mM HEPES-KOH pH7.1, 3mM DTT, 10% glycerol, 0.25% Tween 20 for TOR-KOG1 or 0.01% Tween 20 for free TOR1, 150 mM KCl, 1 mM Mg acetate, 2 mM CaCl2

CaM elution buffer

50mM HEPES-KOH pH7.1, 3mM DTT, 10% glycerol, 0.25% Tween 20 for TOR-KOG1 or 0.01% Tween 20 for free TOR1, 150 mM KCl, 1 mM Mg acetate, 2 mM EGTA

Supplementary References

Chacon, P., and Wriggers, W. (2002). Multi-resolution contour-based fitting of macromolecular structures. J Mol Biol 317, 375-384.

Garzon, J. I., Kovacs, J., Abagyan, R., and Chacon, P. (2006). ADP_EM: fast exhaustive multi-resolution docking for high-throughput coverage. Bioinformatics.

Leuther, K. K., Hammarsten, O., Kornberg, R. D., and Chu, G. (1999). Structure of DNA-dependent protein kinase: implications for its regulation by DNA. Embo J 18, 1114-1123.

Ludtke, S. J., Baldwin, P. R., and Chiu, W. (1999). EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128, 82-97.

Mindell, J. A., and Grigorieff, N. (2003). Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol 142, 334-347.

Passmore, L. A., McCormack, E. A., Au, S. W., Paul, A., Willison, K. R., Harper, J. W., and Barford, D. (2003). Doc1 mediates the activity of the anaphase-promoting complex by contributing to substrate recognition. Embo J 22, 786-796.

Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. (2004). UCSF Chimera--a visualization system for exploratory research and analysis. J Comput Chem 25, 1605-1612.

Scheres, S. H., Gao, H., Valle, M., Herman, G. T., Eggermont, P. P., Frank, J., and Carazo, J. M. (2007). Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nat Methods 4, 27-29.

Sorzano, C. O., Marabini, R., Velazquez-Muriel, J., Bilbao-Castro, J. R., Scheres, S. H., Carazo, J. M., and Pascual-Montano, A. (2004). XMIPP: a new generation of an open-source image processing package for electron microscopy. J Struct Biol 148, 194-204.

Spagnolo, L., Rivera-Calzada, A., Pearl, L. H., and Llorca, O. (2006). Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair. Mol Cell 22, 511-519.

Wriggers, W., Milligan, R. A., and McCammon, J. A. (1999). Situs: A package for docking crystal structures into low-resolution maps from electron microscopy. J Struct Biol 125, 185-195.

1 500 1000 1500 2000 2470

HEAT/ARM repeats 1-2025helical1802-2026

kinase2027-2394

FATC2395-2470

FRB1955-2049

FAT 1319-1919

helical/kinase 1802-2394

HEAT (non FAT/helical): 53%FATa: 20%Helical/kinase domain: 24%FATC: 3%

aexcluding overlap with helical domain

Supplementary Figure S1. Schematic of TOR1 domain organization.

N-terminal HEAT/ARM repeats 1-1318

2474

S. cerevisiae TOR2

2470

S. cerevisiae TOR1 (target of rapamycin)

2549

Human mTOR

4128

Human DNA-PKcs (catalytic subunit of DNA dependent protein kinase)

3056

Human ATM (ataxia telangiectasia mutated)

2644

Human ATR (ATM and Rad3 related)

3860

Human TRRAP (transformation/transcription domain associated protein)

3661

Human SMG-1

HEAT repeats

FAT domain

Helical/kinase domain

FATC domain

Supplementary Figure S2. Schematic showing domain organization of PIKK family members, with domain boundary residue numbers shown for TOR1. The FAT and helical/kinase domains overlap.

1331 1919

1802

2395

A

B

Side

view

Bac

k vi

ew

DNA-PKcs/Ku70/Ku80 TOR1 Fitting

Arm Head

Ku70/Ku80 Ku70/Ku80PIKK

PIKK

Arm Head

Head Head

Supplementary Figure S3. The 3D structure of the DNA-PKcs/Ku70/Ku80 complex obtained using negative staining (pink colour) Spagnolo et al., 2006) was fitted with the structure of free TOR1 (green colour). Both structures reveal similarities in the organization of the head domain as revealed in their side (A) and back (B) views.

Spagnolo, L., Rivera-Calzada, A., Pearl, L. H., and Llorca, O. (2006). Three-dimensional structure of the human DNA-PKcs/Ku70/Ku80 complex assembled on DNA and its implications for DNA DSB repair. Mol Cell 22, 511-519.

A B3D

reco

nstr

uctio

nM

odel

C

Fitted PI3K(catalytic and helical)

PI3K catalyticdomain

FATC

DNA-PKcs front view DNA-PKcs back view

N-terminal Repeats PI3K FATFATC

Supplementary Figure S4. (A) 3D cryo-EM structure and atomic model of DNA-PKcstaken from Rivera-Calzada et al. (2005). Cryo-EM density is shown on top and fitted atomic models are shown in the bottom. Colour codes are similar to those used for TOR1. (C) Fitting and proposed organization of the head domain in DNA-PKcs, modified from Rivera-Calzada et al. (2005). (D) The helical and catalytic domains of the PI3K fitted into the cryo-EM map of DNA-PKcs are coloured differently.

PI3K helicaldomain

D