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- EMDB-10133: cryo-EM structure of mTORC1 bound to active RagA/C GTPases -

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Basic information

Entry
Database: EMDB / ID: EMD-10133
Titlecryo-EM structure of mTORC1 bound to active RagA/C GTPases
Map dataCryo-EM structure of mTORC1 bound to active RagA/C GTPases. The Final map for the coordinates.
Sample
  • Complex: cryo-EM structure of mTORC1 bound to active RagA/C complex
    • Complex: mTORC1
      • Protein or peptide: mTOR,Serine/threonine-protein kinase mTOR,mTOR,Serine/threonine-protein kinase mTOR
      • Protein or peptide: Target of rapamycin complex subunit LST8MTOR
    • Complex: RagA/C
      • Protein or peptide: Ras-related GTP-binding protein A
      • Protein or peptide: Ras-related GTP-binding protein C
      • Protein or peptide: Regulatory-associated protein of mTOR
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
Function / homology
Function and homology information


Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / RNA polymerase III type 2 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of pentose-phosphate shunt / T-helper 1 cell lineage commitment / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / regulation of TORC1 signaling ...Gtr1-Gtr2 GTPase complex / FNIP-folliculin RagC/D GAP / RNA polymerase III type 2 promoter sequence-specific DNA binding / positive regulation of cytoplasmic translational initiation / RNA polymerase III type 1 promoter sequence-specific DNA binding / positive regulation of pentose-phosphate shunt / T-helper 1 cell lineage commitment / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / regulation of TORC1 signaling / cellular response to leucine starvation / TFIIIC-class transcription factor complex binding / TORC2 complex / heart valve morphogenesis / regulation of membrane permeability / negative regulation of lysosome organization / RNA polymerase III type 3 promoter sequence-specific DNA binding / TORC1 complex / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / protein localization to lysosome / calcineurin-NFAT signaling cascade / regulation of autophagosome assembly / TORC1 signaling / regulation of TOR signaling / positive regulation of odontoblast differentiation / voluntary musculoskeletal movement / regulation of osteoclast differentiation / positive regulation of keratinocyte migration / cellular response to L-leucine / MTOR signalling / Amino acids regulate mTORC1 / cellular response to nutrient / energy reserve metabolic process / Energy dependent regulation of mTOR by LKB1-AMPK / nucleus localization / ruffle organization / protein serine/threonine kinase inhibitor activity / negative regulation of cell size / cellular response to osmotic stress / positive regulation of osteoclast differentiation / protein localization to membrane / enzyme-substrate adaptor activity / anoikis / cardiac muscle cell development / positive regulation of transcription by RNA polymerase III / negative regulation of protein localization to nucleus / regulation of myelination / negative regulation of calcineurin-NFAT signaling cascade / Macroautophagy / regulation of cell size / negative regulation of macroautophagy / lysosome organization / positive regulation of oligodendrocyte differentiation / small GTPase-mediated signal transduction / positive regulation of actin filament polymerization / protein kinase activator activity / positive regulation of myotube differentiation / behavioral response to pain / TOR signaling / oligodendrocyte differentiation / mTORC1-mediated signalling / germ cell development / Constitutive Signaling by AKT1 E17K in Cancer / social behavior / cellular response to nutrient levels / CD28 dependent PI3K/Akt signaling / positive regulation of phosphoprotein phosphatase activity / positive regulation of translational initiation / neuronal action potential / HSF1-dependent transactivation / positive regulation of TOR signaling / positive regulation of G1/S transition of mitotic cell cycle / positive regulation of epithelial to mesenchymal transition / regulation of macroautophagy / endomembrane system / 'de novo' pyrimidine nucleobase biosynthetic process / response to amino acid / positive regulation of lipid biosynthetic process / phagocytic vesicle / positive regulation of lamellipodium assembly / heart morphogenesis / regulation of cellular response to heat / protein-membrane adaptor activity / cytoskeleton organization / cardiac muscle contraction / positive regulation of stress fiber assembly / positive regulation of TORC1 signaling / tumor necrosis factor-mediated signaling pathway / cellular response to amino acid starvation / T cell costimulation / cellular response to starvation / positive regulation of endothelial cell proliferation / protein serine/threonine kinase activator activity / positive regulation of glycolytic process / negative regulation of autophagy / response to nutrient levels / post-embryonic development / response to nutrient / RNA splicing / 14-3-3 protein binding
Similarity search - Function
Raptor, N-terminal CASPase-like domain / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / RagA/B / Gtr1/RagA G protein / RagC/D / Gtr1/RagA G protein conserved region / Target of rapamycin complex subunit LST8 / Domain of unknown function DUF3385, target of rapamycin protein ...Raptor, N-terminal CASPase-like domain / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / RagA/B / Gtr1/RagA G protein / RagC/D / Gtr1/RagA G protein conserved region / Target of rapamycin complex subunit LST8 / Domain of unknown function DUF3385, target of rapamycin protein / Domain of unknown function (DUF3385) / Domain of unknown function / FKBP12-rapamycin binding domain / Serine/threonine-protein kinase TOR / FKBP12-rapamycin binding domain superfamily / FKBP12-rapamycin binding domain / HEAT repeat / HEAT repeat / Rapamycin binding domain / PIK-related kinase, FAT / FAT domain / FATC domain / FATC / FATC domain / PIK-related kinase / FAT domain profile. / FATC domain profile. / Quinoprotein alcohol dehydrogenase-like superfamily / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Serine/threonine-protein kinase mTOR / Ras-related GTP-binding protein A / Regulatory-associated protein of mTOR / Target of rapamycin complex subunit LST8 / Ras-related GTP-binding protein C
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.2 Å
AuthorsAnandapadamanaban M / Berndt A / Masson GR / Perisic O / Williams RL
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_U105184308 United Kingdom
European Molecular Biology Organizationlong-term fellowship United Kingdom
Cancer Research UKC14801/A21211 United Kingdom
CitationJournal: Science / Year: 2019
Title: Architecture of human Rag GTPase heterodimers and their complex with mTORC1.
Authors: Madhanagopal Anandapadamanaban / Glenn R Masson / Olga Perisic / Alex Berndt / Jonathan Kaufman / Chris M Johnson / Balaji Santhanam / Kacper B Rogala / David M Sabatini / Roger L Williams /
Abstract: The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag ...The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagA/RagC nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes.
History
DepositionJul 18, 2019-
Header (metadata) releaseOct 16, 2019-
Map releaseOct 16, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6sb2
  • Surface level: 0.018
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10133.png

Downloads & links

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Map

FileDownload / File: emd_10133.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of mTORC1 bound to active RagA/C GTPases. The Final map for the coordinates.
Voxel sizeX=Y=Z: 1.43 Å
Density
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.018
Minimum - Maximum-0.06333706 - 0.1044751
Average (Standard dev.)0.00029986416 (±0.00356351)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 457.59998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.431.431.43
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z457.600457.600457.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0630.1040.000

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Supplemental data

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Mask #1

Fileemd_10133_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: The experimental EM map for mTORC1-RagA/C monomer after...

Fileemd_10133_additional.map
AnnotationThe experimental EM map for mTORC1-RagA/C monomer after focussed classification with signal subtraction.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half1 map from the Refine 3D reconstruction (RELION-3.0.6)

Fileemd_10133_half_map_1.map
AnnotationHalf1 map from the Refine 3D reconstruction (RELION-3.0.6)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half2 map from the Refine 3D reconstruction (RELION-3.0.6)

Fileemd_10133_half_map_2.map
AnnotationHalf2 map from the Refine 3D reconstruction (RELION-3.0.6)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : cryo-EM structure of mTORC1 bound to active RagA/C complex

EntireName: cryo-EM structure of mTORC1 bound to active RagA/C complex
Components
  • Complex: cryo-EM structure of mTORC1 bound to active RagA/C complex
    • Complex: mTORC1
      • Protein or peptide: mTOR,Serine/threonine-protein kinase mTOR,mTOR,Serine/threonine-protein kinase mTOR
      • Protein or peptide: Target of rapamycin complex subunit LST8MTOR
    • Complex: RagA/C
      • Protein or peptide: Ras-related GTP-binding protein A
      • Protein or peptide: Ras-related GTP-binding protein C
      • Protein or peptide: Regulatory-associated protein of mTOR
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: cryo-EM structure of mTORC1 bound to active RagA/C complex

SupramoleculeName: cryo-EM structure of mTORC1 bound to active RagA/C complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Molecular weightExperimental: 1.09 MDa

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Supramolecule #2: mTORC1

SupramoleculeName: mTORC1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: RagA/C

SupramoleculeName: RagA/C / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#5
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: mTOR,Serine/threonine-protein kinase mTOR,mTOR,Serine/threonine-p...

MacromoleculeName: mTOR,Serine/threonine-protein kinase mTOR,mTOR,Serine/threonine-protein kinase mTOR
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 287.235188 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)EMSQ EESTRFYDQL NHHIFELVSS SDANERKGGI LAIASLIGVE GGNATRIGRF A NYLRNLLP SNDPVVMEMA SKAIGRLAMA GDTFTAEYVE FEVKRALEWL GADRNEGRRH AAVLVLRELA ISVPTFFFQQ VQ PFFDNIF VAVWDPKQAI REGAVAALRA CLILTTQREP KEMQKPQWYR HTFEEAEKGF DETLAKEKGM NRDDRIHGAL LIL NELVRI SSMEGERLRE EMEEITQQQL VHDKYCKDLM GFGTKPRHIT PFTSFQAVQP QQSNALVGLL GYSSHQGLMG FGTS PSPAK ST(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)RN SKNSLIQMTI LNLLPRLAAF RPS AFTDTQ YLQDTMNHVL SCVKKEKERT AAFQALGLLS VAVRSEFKVY LPRVLDIIRA ALPPKDFAHK RQKAMQVDAT VFTC ISMLA RAMGPGIQQD IKELLEPMLA VGLSPALTAV LYDLSRQIPQ LKKDIQDGLL KMLSLVLMHK PLRHPGMPKG LAHQL ASPG LTTLPEASDV GSITLALRTL GSFEFEGHSL TQFVRHCADH FLNSEHKEIR MEAARTCSRL LTPSIHLISG HAHVVS QTA VQVVADVLSK LLVVGITDPD PDIRYCVLAS LDERFDAHLA QAENLQALFV ALNDQVFEIR ELAICTVGRL SSMNPAF VM PFLRKMLIQI LTELEHSGIG RIKEQSARML GHLVSNAPRL IRPYMEPILK ALILKLKDPD PDPNPGVINN VLATIGEL A QVSGLEMRKW VDELFIIIMD MLQDSSLLAK RQVALWTLGQ LVASTGYVVE PYRKYPTLLE VLLNFLKTEQ NQGTRREAI RVLGLLGALD PYKHKVNIGM IDQSRDASAV SLSESKSSQD SSDYSTSEML VNMGNLPLDE FYPAVSMVAL MRIFRDQSLS HHHTMVVQA ITFIFKSLGL KCVQFLPQVM PTFLNVIRVC DGAIREFLFQ QLGMLVSFVK SHIRPYMDEI VTLMREFWVM N TSIQSTII LLIEQIVVAL GGEFKLYLPQ LIPHMLRVFM HDNSPGRIVS IKLLAAIQLF GANLDDYLHL LLPPIVKLFD AP EAPLPSR KAALETVDRL TESLDFTDYA SRIIHPIVRT LDQSPELRST AMDTLSSLVF QLGKKYQIFI PMVNKVLVRH RIN HQRYDV LICRIVKGYT LADEEEDPLI YQHRMLRSGQ GDALASGPVE TGPMKKLHVS TINLQKAWGA ARRVSKDDWL EWLR RLSLE LLKDSSSPSL RSCWALAQAY NPMARDLFNA AFVSCWSELN EDQQDELIRS IELALTSQDI AEVTQTLLNL AEFME HSDK GPLPLRDDNG IVLLGERAAK CRAYAKALHY KELEFQKGPT PAILESLISI NNKLQQPEAA AGVLEYAMKH FGELEI QAT WYEKLHEWED ALVAYDKKMD TNKDDPELML GRMRCLEALG EWGQLHQQCC EKWTLVNDET QAKMARMAAA AAWGLGQ WD SMEEYTCMIP RDTHDGAFYR AVLALHQDLF SLAQQCIDKA RDLLDAELTA MAGESYSRAY GAMVSCHMLS ELEEVIQY K LVPERREIIR QIWWERLQGC QRIVEDWQKI LMVRSLVVSP HEDMRTWLKY ASLCGKSGRL ALAHKTLVLL LGVDPSRQL DHPLPTVHPQ VTYAYMKNMW KSARKIDAFQ HMQHFVQTMQ QQAQHAIATE DQQHKQELHK LMARCFLKLG EWQLNLQGIN ESTIPKVLQ YYSAATEHDR SWYKAWHAWA VMNFEAVLHY KHQNQARDEK KKLRHASGAN ITNATTAATT AATATTTAST E GSNSESEA ESTENSPTPS PLQKKVTEDL SKTLLMYTVP AVQGFFRSIS LSRGNNLQDT LRVLTLWFDY GHWPDVNEAL VE GVKAIQI DTWLQVIPQL IARIDTPRPL VGRLIHQLLT DIGRYHPQAL IYPLTVASKS TTTARHNAAN KILKNMCEHS NTL VQQAMM VSEELIRVAI LWHEMWHEGL EEASRLYFGE RNVKGMFEVL EPLHAMMERG PQTLKETSFN QAYGRDLMEA QEWC RKYMK SGNVKDLTQA WDLYYHVFRR ISKQLPQLTS LELQYVSPKL LMCRDLELAV PGTYDPNQPI IRIQSIAPSL QVITS KQRP RKLTLMGSNG HEFVFLLKGH EDLRQDERVM QLFGLVNTLL ANDPTSLRKN LSIQRYAVIP LSTNSGLIGW VPHCDT LHA LIRDYREKKK ILLNIEHRIM LRMAPDYDHL TLMQKVEVFE HAVNNTAGDD LAKLLWLKSP SSEVWFDRRT NYTRSLA VM SMVGYILGLG DRHPSNLMLD RLSGKILHID FGDCFEVAMT REKFPEKIPF RLTRMLTNAM EVTGLDGNYR ITCHTVME V LREHKDSVMA VLEAFVYDPL LNWRLMDTNT KGNKRSRTRT DSYSAGQSVE ILDGVELGEP AHKKTGTTVP ESIHSFIGD GLVKPEALNK KAIQIINRVR DKLTGRDFSH DDTLDVPTQV ELLIKQATSH ENLCQCYIGW CPFW

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Macromolecule #2: Target of rapamycin complex subunit LST8

MacromoleculeName: Target of rapamycin complex subunit LST8 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.91009 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MNTSPGTVGS DPVILATAGY DHTVRFWQAH SGICTRTVQH QDSQVNALEV TPDRSMIAAA GYQHIRMYDL NSNNPNPIIS YDGVNKNIA SVGFHEDGRW MYTGGEDCTA RIWDLRSRNL QCQRIFQVNA PINCVCLHPN QAELIVGDQS GAIHIWDLKT D HNEQLIPE ...String:
MNTSPGTVGS DPVILATAGY DHTVRFWQAH SGICTRTVQH QDSQVNALEV TPDRSMIAAA GYQHIRMYDL NSNNPNPIIS YDGVNKNIA SVGFHEDGRW MYTGGEDCTA RIWDLRSRNL QCQRIFQVNA PINCVCLHPN QAELIVGDQS GAIHIWDLKT D HNEQLIPE PEVSITSAHI DPDASYMAAV NSTGNCYVWN LTGGIGDEVT QLIPKTKIPA HTRYALQCRF SPDSTLLATC SA DQTCKIW RTSNFSLMTE LSIKSGNPGE SSRGWMWGCA FSGDSQYIVT ASSDNLARLW CVETGEIKRE YGGHQKAVVC LAF NDSVLG

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Macromolecule #3: Ras-related GTP-binding protein A

MacromoleculeName: Ras-related GTP-binding protein A / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 36.600195 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPNTAMKKKV LLMGKSGSGK TSMRSIIFAN YIARDTRRLG ATIDVEHSHV RFLGNLVLNL WDCGGLDTFM ENYFTSQRDN IFRNVEVLI YVFDVESREL EKDMHYYQSC LEAILQNSPD AKIFCLVHKM DLVQEDQRDL IFKEREEDLR RLSRPLECAC F RTSIWDET ...String:
MPNTAMKKKV LLMGKSGSGK TSMRSIIFAN YIARDTRRLG ATIDVEHSHV RFLGNLVLNL WDCGGLDTFM ENYFTSQRDN IFRNVEVLI YVFDVESREL EKDMHYYQSC LEAILQNSPD AKIFCLVHKM DLVQEDQRDL IFKEREEDLR RLSRPLECAC F RTSIWDET LYKAWSSIVY QLIPNVQQLE MNLRNFAQII EADEVLLFER ATFLVISHYQ CKEQRDVHRF EKISNIIKQF KL SCSKLAA SFQSMEVRNS NFAAFIDIFT SNTYVMVVMS DPSIPSAATL INIRNARKHF EKLERVDGPK HSLLMR

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Macromolecule #4: Ras-related GTP-binding protein C

MacromoleculeName: Ras-related GTP-binding protein C / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.284832 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSLQYGAEET PLAGSYGAAD SFPKDFGYGV EEEEEEAAAA GGGVGAGAGG GCGPGGADSS KPRILLMGLR RSGKSSIQKV VFHKMSPNE NLFLESTNKI YKDDISNSSF VNFQIWDFPG QMDFFDPTFD YEMIFRGTGA LIYVIDAQDD YMEALTRLHI T VSKAYKVN ...String:
MSLQYGAEET PLAGSYGAAD SFPKDFGYGV EEEEEEAAAA GGGVGAGAGG GCGPGGADSS KPRILLMGLR RSGKSSIQKV VFHKMSPNE NLFLESTNKI YKDDISNSSF VNFQIWDFPG QMDFFDPTFD YEMIFRGTGA LIYVIDAQDD YMEALTRLHI T VSKAYKVN PDMNFEVFIH KVDGLSDDHK IETQRDIHQR ANDDLADAGL EKLHLSFYLT SIYDHSIFEA FSKVVQKLIP QL PTLENLL NIFISNSGIE KAFLFDVVSK IYIATDSSPV DMQSYELCCD MIDVVIDVSC IYGLKEDGSG SAYDKESMAI IKL NNTTVL YLKEVTKFLA LVCILREESF ERKGLIDYNF HCFRKAIHEV FEVGVTSHRS CGHQTSASSL KALTHNGTPR NAI

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Macromolecule #5: Regulatory-associated protein of mTOR

MacromoleculeName: Regulatory-associated protein of mTOR / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 149.200016 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MESEMLQSPL LGLGEEDEAD LTDWNLPLAF MKKRHCEKIE GSKSLAQSWR MKDRMKTVSV ALVLCLNVGV DPPDVVKTTP CARLECWID PLSMGPQKAL ETIGANLQKQ YENWQPRARY KQSLDPTVDE VKKLCTSLRR NAKEERVLFH YNGHGVPRPT V NGEVWVFN ...String:
MESEMLQSPL LGLGEEDEAD LTDWNLPLAF MKKRHCEKIE GSKSLAQSWR MKDRMKTVSV ALVLCLNVGV DPPDVVKTTP CARLECWID PLSMGPQKAL ETIGANLQKQ YENWQPRARY KQSLDPTVDE VKKLCTSLRR NAKEERVLFH YNGHGVPRPT V NGEVWVFN KNYTQYIPLS IYDLQTWMGS PSIFVYDCSN AGLIVKSFKQ FALQREQELE VAAINPNHPL AQMPLPPSMK NC IQLAACE ATELLPMIPD LPADLFTSCL TTPIKIALRW FCMQKCVSLV PGVTLDLIEK IPGRLNDRRT PLGELNWIFT AIT DTIAWN VLPRDLFQKL FRQDLLVASL FRNFLLAERI MRSYNCTPVS SPRLPPTYMH AMWQAWDLAV DICLSQLPTI IEEG TAFRH SPFFAEQLTA FQVWLTMGVE NRNPPEQLPI VLQVLLSQVH RLRALDLLGR FLDLGPWAVS LALSVGIFPY VLKLL QSSA RELRPLLVFI WAKILAVDSS CQADLVKDNG HKYFLSVLAD PYMPAEHRTM TAFILAVIVN SYHTGQEACL QGNLIA ICL EQLNDPHPLL RQWVAICLGR IWQNFDSARW CGVRDSAHEK LYSLLSDPIP EVRCAAVFAL GTFVGNSAER TDHSTTI DH NVAMMLAQLV SDGSPMVRKE LVVALSHLVV QYESNFCTVA LQFIEEEKNY ALPSPATTEG GSLTPVRDSP CTPRLRSV S SYGNIRAVAT ARSLNKSLQN LSLTEESGGA VAFSPGNLST SSSASSTLGS PENEEHILSF ETIDKMRRAS SYSSLNSLI GVSFNSVYTQ IWRVLLHLAA DPYPEVSDVA MKVLNSIAYK ATVNARPQRV LDTSSLTQSA PASPTNKGVH IHQAGGSPPA SSTSSSSLT NDVAKQPVSR DLPSGRPGTT GPAGAQYTPH SHQFPRTRKM FDKGPEQTAD DADDAAGHKS FISATVQTGF C DWSARYFA QPVMKIPEEH DLESQIRKER EWRFLRNSRV RRQAQQVIQK GITRLDDQIF LNRNPGVPSV VKFHPFTPCI AV ADKDSIC FWDWEKGEKL DYFHNGNPRY TRVTAMEYLN GQDCSLLLTA TDDGAIRVWK NFADLEKNPE MVTAWQGLSD MLP TTRGAG MVVDWEQETG LLMSSGDVRI VRIWDTDREM KVQDIPTGAD SCVTSLSCDS HRSLIVAGLG DGSIRVYDRR MALS ECRVM TYREHTAWVV KASLQKRPDG HIVSVSVNGD VRIFDPRMPE SVNVLQIVKG LTALDIHPQA DLIACGSVNQ FTAIY NSSG ELINNIKYYD GFMGQRVGAI SCLAFHPHWP HLAVGSNDYY ISVYSVEKRV R

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Macromolecule #6: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #7: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 2 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.05 mg/mL
BufferpH: 7
Details: 100mM Tris-HCl pH7.0, 260mM NaCl, 5mM MgCl2, 1mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK III
DetailsmTORC1 (mTOR complex 1) is a dimer consists of three proteins: mTOR, mLST8 and RAPTOR. The small GTPases, RagA/C in its active form bind to mTORC1 for activation. We solved the cryo-EM structure of mTORC1 bound to RagA/C.

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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: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-22 / Average exposure time: 1.8 sec. / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 169971
CTF correctionSoftware - Name: Gctf (ver. 1.18)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6bcx


Details: For building the mTORC1 structure we used the previously published apo-mTORC1 structure 6BCX, and for RagA/C structure we used our high-resolution crystal structure PDB ID 6S6A.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.6)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.6)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.6)
Details: For the final reconstruction of mTORC1-RagA/C structure we used a strategy taking advantage of the relion particle symmetry expand program, and duplicated the C2-refined particles and ...Details: For the final reconstruction of mTORC1-RagA/C structure we used a strategy taking advantage of the relion particle symmetry expand program, and duplicated the C2-refined particles and applied the appropriate rotation and translation to generate a set of monomers. We performed mTORC1-RagA/C 'pseudo-monomer' focussed classification with signal subtraction and obtained a reconstruction of 6.2 A resolution map. This cryo-EM density corresponded to the mTORC1-RagA/C pseudomonomer, where the previously published structure for apo-mTORC1 (PDB ID 6BCX) and our high-resolution crystal structure of RagA/C (6S6A) were fitted with great confidence from our experimental analysis including Pulldown assays, mutational at per-residue level in the binding interface and HDX-Mass Spectrometry.
Number images used: 51902
DetailsThe selected images were processed using MotionCor2 within the RELION-3.0.6 package.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6bcx

,

6s6a

)
DetailsCryo-EM model of mTORC1-RagA/C was refined using REFMAC5 program in CCPEM package, with a composite map of the 3D reconstruction of mTORC1-RagA/C pseudo-monomer (as mentioned in Reconstruction section) of one protomer together with the generated map for the other second protomer of mTORC1-RagA/C. This second protomer of mTORC1-RagA/C map was generated by simply aligning the first 3D reconstructed pseudomonomer map onto the mTORC1 dimer consensus C2 map and then obtained the rotation-translation matrix with CHIMERA and then used Maputils program in CCP4i. From the resulting mTORC1-RagA/C dimer map, the model of mTORC1-RagA/C was built by using previously published structure of apo-mTORC1 (PDB ID 6BCX) and our crystal structure of RagA/C was fitted (PDB ID 6S6A, unreleased). The entire mTORC1-RagA/C final model was refined using REFMAC5 program using the restraints from the crystal structure of RagA/C and previously published mTORC1 structure. Side chains were removed before refinement, since these were not evident in the cryo-EM densities. Separate model refinements were performed against single half-maps, and the resulting models were compared with the other half-maps to confirm the absence of overfitting.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 315
Output model

PDB-6sb2:
cryo-EM structure of mTORC1 bound to active RagA/C GTPases

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