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Entry
Database: EMDB / ID: EMD-13595
TitleHelical reconstruction of TOROID (TORC1 Organized in Inhibited Domains) filaments.
Map dataHelical reconstruction of TOROID (TORC1 Organized in Inhibited Domains) filaments.
Sample
  • Complex: TORC1 Organized in Inhibited Domains (TOROID)
    • Protein or peptide: Target of rapamycin complex 1 subunit KOG1
    • Protein or peptide: Serine/threonine-protein kinase TOR2
    • Protein or peptide: Target of rapamycin complex subunit LST8MTOR
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodhelical reconstruction / cryo EM / Resolution: 9.1 Å
AuthorsFelix J / Prouteau M / Bourgoint C / Bonnadei L / Desfosses A / Gabus C / Sadian Y / Savvides SN / Gutsche I / Loewith R
Funding support Switzerland, France, 4 items
OrganizationGrant numberCountry
European Research Council (ERC)834394 Switzerland
Swiss National Science Foundation179517 Switzerland
H2020 Marie Curie Actions of the European Commission789385 France
European Research Council (ERC)64778 France
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: EGOC inhibits TOROID polymerization by structurally activating TORC1.
Authors: Manoël Prouteau / Clélia Bourgoint / Jan Felix / Lenny Bonadei / Yashar Sadian / Caroline Gabus / Savvas N Savvides / Irina Gutsche / Ambroise Desfosses / Robbie Loewith /
Abstract: Target of rapamycin complex 1 (TORC1) is a protein kinase controlling cell homeostasis and growth in response to nutrients and stresses. In Saccharomyces cerevisiae, glucose depletion triggers a ...Target of rapamycin complex 1 (TORC1) is a protein kinase controlling cell homeostasis and growth in response to nutrients and stresses. In Saccharomyces cerevisiae, glucose depletion triggers a redistribution of TORC1 from a dispersed localization over the vacuole surface into a large, inactive condensate called TOROID (TORC1 organized in inhibited domains). However, the mechanisms governing this transition have been unclear. Here, we show that acute depletion and repletion of EGO complex (EGOC) activity is sufficient to control TOROID distribution, independently of other nutrient-signaling pathways. The 3.9-Å-resolution structure of TORC1 from TOROID cryo-EM data together with interrogation of key interactions in vivo provide structural insights into TORC1-TORC1' and TORC1-EGOC interaction interfaces. These data support a model in which glucose-dependent activation of EGOC triggers binding to TORC1 at an interface required for TOROID assembly, preventing TORC1 polymerization and promoting release of active TORC1.
History
DepositionSep 17, 2021-
Header (metadata) releaseJan 18, 2023-
Map releaseJan 18, 2023-
UpdateMar 29, 2023-
Current statusMar 29, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13595.png

Downloads & links

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Map

FileDownload / File: emd_13595.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHelical reconstruction of TOROID (TORC1 Organized in Inhibited Domains) filaments.
Voxel sizeX=Y=Z: 2.7 Å
Density
Contour LevelBy AUTHOR: 0.028
Minimum - Maximum-0.040822297 - 0.086129285
Average (Standard dev.)0.0020309312 (±0.0068145893)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 810.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Helical reconstruction of TOROID (TORC1 Organized in Inhibited...

Fileemd_13595_half_map_1.map
AnnotationHelical reconstruction of TOROID (TORC1 Organized in Inhibited Domains) filaments, half map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Helical reconstruction of TOROID (TORC1 Organized in Inhibited...

Fileemd_13595_half_map_2.map
AnnotationHelical reconstruction of TOROID (TORC1 Organized in Inhibited Domains) filaments, half map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : TORC1 Organized in Inhibited Domains (TOROID)

EntireName: TORC1 Organized in Inhibited Domains (TOROID)
Components
  • Complex: TORC1 Organized in Inhibited Domains (TOROID)
    • Protein or peptide: Target of rapamycin complex 1 subunit KOG1
    • Protein or peptide: Serine/threonine-protein kinase TOR2
    • Protein or peptide: Target of rapamycin complex subunit LST8MTOR

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Supramolecule #1: TORC1 Organized in Inhibited Domains (TOROID)

SupramoleculeName: TORC1 Organized in Inhibited Domains (TOROID) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Target of rapamycin complex 1 subunit KOG1

MacromoleculeName: Target of rapamycin complex 1 subunit KOG1 / type: protein_or_peptide / ID: 1
Details: S. cerevisiae TOROIDs (TORC1 Organized in Inhibited Domains) in the sample are composed of Kog1 (P38873), Lst8 (P41318) and Tor2 (P32600).
Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MPEIYGPQPL KPLNTVMRHG FEEQYQSDQL LQSLANDFIF YFDDKRHKTN GNPIPEEDKQ RDVNRYYQP ITDWKIMKDR QKTVSAALLL CLNLGVDPPD VMKTHPCARV EAWVDPLNFQ D SKKAIEQI GKNLQAQYET LSLRTRYKQS LDPCVEDVKR FCNSLRRTSK ...String:
MPEIYGPQPL KPLNTVMRHG FEEQYQSDQL LQSLANDFIF YFDDKRHKTN GNPIPEEDKQ RDVNRYYQP ITDWKIMKDR QKTVSAALLL CLNLGVDPPD VMKTHPCARV EAWVDPLNFQ D SKKAIEQI GKNLQAQYET LSLRTRYKQS LDPCVEDVKR FCNSLRRTSK EDRILFHYNG HG VPKPTKS GEIWVFNRGY TQYIPVSLYD LQTWLGAPCI FVYDCNSAEN ILINFQKFVQ KRI KDDEEG NHDVAAPSPT SAYQDCFQLA SCTSDELLLM SPELPADLFS CCLTCPIEIS IRIF LMQSP LKDSKYKIFF ENSTSNQPFG DSKNSFKSKI PNVNIPGMLS DRRTPLGELN WIFTA ITDT IAWTSLPRPL FKKLFRHDLM IAALFRNFLL AKRIMPWYNC HPVSDPELPD SITTHP MWK SWDLAMDEVL TKIVIDLKNA PPATALESQM ILQQQETLQN GGSSKSNAQD TKAGSIQ TQ SRFAVANLST MSLVNNPALQ SRKSISLQSS QQQLQQQQQQ QQQFTGFFEQ NLTAFELW L KYASNVRHPP EQLPIVLQVL LSQVHRIRAL VLLSRFLDLG PWAVYLSLSI GIFPYVLKL LQSPAPELKP ILVFIWARIM SIDYKNTQSE LIKEKGYMYF VTVLVPDWGV NGMSATNGSA MINSGNPLT MTASQNINGP SSRYYERQQG NRTSNLGHNN LPFYHSNDTT DEQKAMAVFV L ASFVRNFP LGQKNCFSLE LVNKLCFYID NSEIPLLRQW CVILLGLLFA DNPLNRFVCM NT GAVEILL KSLKDPVPEV RTASIFALKH FISGFQDAEV ILRLQQEFEE QYQQLHSQLQ HLQ NQSHLQ QQQSQQQQQH LEQQQMKIEK QIRHCQVMQN QLEVIDLRKL KRQEIGNLIS ILPL INDGS SLVRKELVVY FSHIVSRYSN FFIVVVFNDL LEEIKLLEKS DINTRNTSDK YSVSQ GSIF YTVWKSLLIL AEDPFLENKE LSKQVIDYIL LELSAHKELG GPFAVMEKFL LKRSSK AHQ TGKFGFNSSQ VQFVKSSLRS FSPNERVDNN AFKKEQQQHD PKISHPMRTS LAKLFQS LG FSESNSDSDT QSSNTSMKSH TSKKGPSGLY LLNGNNNIYP TAETPRFRKH TEPLQLPL N SSFLDYSREY FQEPQMKKQE ADEPGSVEYN ARLWRRNRNE TIIQETQGEK KLSIYGNWS KKLISLNNKS QPKLMKFAQF EDQLITADDR STITVFDWEK GKTLSKFSNG TPFGTKVTDL KLINEDDSA LLLTGSSDGV IKIYRDYQDV DTFKIVSAWR GLTDMLLTPR STGLLTEWLQ I RGSLLTTG DVKVIRVWDA HTETVEVDIP AKTSSLITSL TADQLAGNIF VAGFADGSLR VY DRRLDPR DSMIRRWRAG NDKQGVWINN VHLQRGGYRE LVSGATNGVV ELWDIRSEDP VES FVDQNV TSQYGSQQKP TTMTCMQVHE HAPIIATGTK QIKIWTTSGD LLNSFKNSHN NGVT STLAA TGIPKSLSYS STSDAFLSSM AFHPHRMMIA ATNSHDSIVN IYKCEDERID YF

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Macromolecule #2: Serine/threonine-protein kinase TOR2

MacromoleculeName: Serine/threonine-protein kinase TOR2 / type: protein_or_peptide / ID: 2
Details: S. cerevisiae TOROIDs (TORC1 Organized in Inhibited Domains) in the sample are composed of Kog1 (P38873), Lst8 (P41318) and Tor2 (P32600).
Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MNKYINKYTT PPNLLSLRQR AEGKHRTRKK LTHKSHSHDD EMSTTSNTDS NHNGPNDSGR VITGSAGHI GKISFVDSEL DTTFSTLNLI FDKLKSDVPQ ERASGANELS TTLTSLAREV S AEQFQRFS NSLNNKIFEL IHGFTSSEKI GGILAVDTLI SFYLSTEELP ...String:
MNKYINKYTT PPNLLSLRQR AEGKHRTRKK LTHKSHSHDD EMSTTSNTDS NHNGPNDSGR VITGSAGHI GKISFVDSEL DTTFSTLNLI FDKLKSDVPQ ERASGANELS TTLTSLAREV S AEQFQRFS NSLNNKIFEL IHGFTSSEKI GGILAVDTLI SFYLSTEELP NQTSRLANYL RV LIPSSDI EVMRLAANTL GRLTVPGGTL TSDFVEFEVR TCIDWLTLTA DNNSSSSKLE YRR HAALLI IKALADNSPY LLYPYVNSIL DNIWVPLRDA KLIIRLDAAV ALGKCLTIIQ DRDP ALGKQ WFQRLFQGCT HGLSLNTNDS VHATLLVFRE LLSLKAPYLR DKYDDIYKST MKYKE YKFD VIRREVYAIL PLLAAFDPAI FTKKYLDRIM VHYLRYLKNI DMNAANNSDK PFILVS IGD IAFEVGSSIS PYMTLILDNI REGLRTKFKV RKQFEKDLFY CIGKLACALG PAFAKHL NK DLLNLMLNCP MSDHMQETLM ILNEKIPSLE STVNSRILNL LSISLSGEKF IQSNQYDF N NQFSIEKARK SRNQSFMKKT GESNDDITDA QILIQCFKML QLIHHQYSLT EFVRLITIS YIEHEDSSVR KLAALTSCDL FIKDDICKQT SVHALHSVSE VLSKLLMIAI TDPVAEIRLE ILQHLGSNF DPQLAQPDNL RLLFMALNDE IFGIQLEAIK IIGRLSSVNP AYVVPSLRKT L LELLTQLK FSNMPKKKEE SATLLCTLIN SSDEVAKPYI DPILDVILPK CQDASSAVAS TA LKVLGEL SVVGGKEMTR YLKELMPLII NTFQDQSNSF KRDAALTTLG QLAASSGYVV GPL LDYPEL LGILINILKT ENNPHIRRGT VRLIGILGAL DPYKHREIEV TSNSKSSVEQ NAPS IDIAL LMQGVSPSND EYYPTVVIHN LMKILNDPSL SIHHTAAIQA IMHIFQNLGL RCVSF LDQI IPGIILVMRS CPPSQLDFYF QQLGSLISIV KQHIRPHVEK IYGVIREFFP IIKLQI TII SVIESISKAL EGEFKRFVPE TLTFFLDILE NDQSNKRIVP IRILKSLVTF GPNLEDY SH LIMPIVVRMT EYSAGSLKKI SIITLGRLAK NINLSEMSSR IVQALVRILN NGDRELTK A TMNTLSLLLL QLGTDFVVFV PVINKALLRN RIQHSVYDQL VNKLLNNECL PTNIIFDKE NEVPERKNYE DEMQVTKLPV NQNILKNAWY CSQQKTKEDW QEWIRRLSIQ LLKESPSACL RSCSSLVSV YYPLARELFN ASFSSCWVEL QTSYQEDLIQ ALCKALSSSE NPPEIYQMLL N LVEFMEHD DKPLPIPIHT LGKYAQKCHA FAKALHYKEV EFLEEPKNST IEALISINNQ LH QTDSAIG ILKHAQQHNE LQLKETWYEK LQRWEDALAA YNEKEAAGED SVEVMMGKLR SLY ALGEWE ELSKLASEKW GTAKPEVKKA MAPLAAGAAW GLEQWDEIAQ YTSVMKSQSP DKEF YDAIL CLHRNNFKKA EVHIFNARDL LVTELSALVN ESYNRAYNVV VRAQIIAELE EIIKY KKLP QNSDKRLTMR ETWNTRLLGC QKNIDVWQRI LRVRSLVIKP KEDAQVRIKF ANLCRK SGR MALAKKVLNT LLEETDDPDH PNTAKASPPV VYAQLKYLWA TGLQDEALKQ LINFTSR MA HDLGLDPNNM IAQSVPQQSK RVPRHVEDYT KLLARCFLKQ GEWRVCLQPK WRLSNPDS I LGSYLLATHF DNTWYKAWHN WALANFEVIS MLTSVSKKKQ EGSDASSVTD INEFDNGMI GVNTFDAKEV HYSSNLIHRH VIPAIKGFFH SISLSESSSL QDALRLLTLW FTFGGIPEAT QAMHEGFNL IQIGTWLEVL PQLISRIHQP NQIVSRSLLS LLSDLGKAHP QALVYPLMVA I KSESLSRQ KAALSIIEKM RIHSPVLVDQ AELVSHELIR MAVLWHEQWY EGLDDASRQF FG EHNTEKM FAALEPLYEM LKRGPETLRE ISFQNSFGRD LNDAYEWLMN YKKSKDVSNL NQA WDIYYN VFRKIGKQLP QLQTLELQHV SPKLLSAHDL ELAVPGTRAS GGKPIVKISK FEPV FSVIS SKQRPRKFCI KGSDGKDYKY VLKGHEDIRQ DSLVMQLFGL VNTLLQNDAE CFRRH LDIQ QYPAIPLSPK SGLLGWVPNS DTFHVLIREH REAKKIPLNI EHWVMLQMAP DYDNLT LLQ KVEVFTYALN NTEGQDLYKV LWLKSRSSET WLERRTTYTR SLAVMSMTGY ILGLGDR HP SNLMLDRITG KVIHIDFGDC FEAAILREKF PEKVPFRLTR MLTYAMEVSG IEGSFRIT C ENVMKVLRDN KGSLMAILEA FAFDPLINWG FDLPTKKIEE ETGIQLPVMN ANELLSNGA ITEEEVQRVE NEHKNAIRNA RAMLVLKRIT DKLTGNDIRR FNDLDVPEQV DKLIQQATSV ENLCQHYIG WCPFW

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Macromolecule #3: Target of rapamycin complex subunit LST8

MacromoleculeName: Target of rapamycin complex subunit LST8 / type: protein_or_peptide / ID: 3
Details: S. cerevisiae TOROIDs (TORC1 Organized in Inhibited Domains) in the sample are composed of Kog1 (P38873), Lst8 (P41318) and Tor2 (P32600).
Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MSVILVSAGY DHTIRFWEAL TGVCSRTIQH SDSQVNRLEI TNDKKLLATA GHQNVRLYDI RTTNPNPVA SFEGHRGNVT SVSFQQDNRW MVTSSEDGTI KVWDVRSPSI PRNYKHNAPV N EVVIHPNQ GELISCDRDG NIRIWDLGEN QCTHQLTPED DTSLQSLSMA ...String:
MSVILVSAGY DHTIRFWEAL TGVCSRTIQH SDSQVNRLEI TNDKKLLATA GHQNVRLYDI RTTNPNPVA SFEGHRGNVT SVSFQQDNRW MVTSSEDGTI KVWDVRSPSI PRNYKHNAPV N EVVIHPNQ GELISCDRDG NIRIWDLGEN QCTHQLTPED DTSLQSLSMA SDGSMLAAAN TK GNCYVWE MPNHTDASHL KPVTKFRAHS TYITRILLSS DVKHLATCSA DHTARVWSID DDF KLETTL DGHQRWVWDC AFSADSAYLV TASSDHYVRL WDLSTREIVR QYGGHHKGAV CVAL NDV

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMMES-NaOH2-(N-morpholino)ethanesulfonic acid-sodium hydroxide
5.0 mMCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
600.0 mMNaClSodium chloridesodium chloride
0.5 mMDTTDithiothreitol
Phosphatase Inhibitor Mix
1.0 1 tablet/50 mlComplete Protease Inhibitor Cocktail
1.0 mMPMSFphenylmethylsulfonyl fluoridePMSF

Details: 50 mM MES-NaOH pH 6.0, 5 mM CHAPS, 600 mM KCl, 0,5 mM DTT, Phosphatase Inhibitor Mix, Complete Protease Inhibitor Cocktail (Roche; 1 tablet/ 50 ml) and 1 mM PMSF)
GridModel: PELCO Ultrathin Carbon with Lacey Carbon / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: OTHER / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 37000
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-16 / Number real images: 4901 / Average exposure time: 8.0 sec. / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 219455 / Software - Name: EMAN2 / Software - details: e2helixboxer module
Details: A total of 219,455 particles (filaments) were extracted in RELION 2.0 using the --helix option, with an extract size of 600 pixels (810 Angstrom), an outer diameter of 620 Angstrom, and a ...Details: A total of 219,455 particles (filaments) were extracted in RELION 2.0 using the --helix option, with an extract size of 600 pixels (810 Angstrom), an outer diameter of 620 Angstrom, and a helical rise of 26.75 Angstrom.
Startup modelType of model: EMDB MAP
EMDB ID:

3814


Details: Map was low-pass filtered to 30 Angstrom.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0) / Software - details: Helical reconstruction
Final reconstructionApplied symmetry - Helical parameters - Δz: 26.7 Å
Applied symmetry - Helical parameters - Δ&Phi: 47.32 °
Applied symmetry - Helical parameters - Axial symmetry: D1 (2x1 fold dihedral)
Resolution.type: BY AUTHOR / Resolution: 9.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Software - details: Helical reconstruction / Number images used: 219455
DetailsMotion correction and dose weighting of the recorded movies were performed using MotionCor2, discarding the first frame.
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT

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