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- EMDB-10132: cryo-EM structure of mTORC1 bound to PRAS40-fused active RagA/C G... -

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Basic information

Entry
Database: EMDB / ID: EMD-10132
Titlecryo-EM structure of mTORC1 bound to PRAS40-fused active RagA/C GTPases
Map dataCryoEM structure of RagA/C heterodimer GTPases in complex with mTORC1
Sample
  • Complex: cryoEM structure of mTORC1 bound to 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
      • Protein or peptide: Regulatory-associated protein of mTOR
    • Complex: RagA/C
      • Protein or peptide: Ras-related GTP-binding protein A
      • Protein or peptide: Ras-related GTP-binding protein C
      • Protein or peptide: Proline-rich AKT1 substrate 1
  • 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 / negative regulation of TOR signaling / 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 / AKT phosphorylates targets in the cytosol / regulation of myelination / negative regulation of calcineurin-NFAT signaling cascade / Macroautophagy / regulation of cell size / negative regulation of macroautophagy / lysosome organization / small GTPase-mediated signal transduction / positive regulation of oligodendrocyte differentiation / positive regulation of actin filament polymerization / protein kinase activator activity / protein kinase inhibitor 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 / neurotrophin TRK receptor signaling pathway / 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 neuron apoptotic process / regulation of cellular response to heat / protein-membrane adaptor activity / cytoskeleton organization / cardiac muscle contraction / negative regulation of TORC1 signaling / 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 / positive regulation of glycolytic process / protein serine/threonine kinase activator activity
Similarity search - Function
Proline-rich AKT1 substrate 1 protein / Proline-rich AKT1 substrate 1 / 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 ...Proline-rich AKT1 substrate 1 protein / Proline-rich AKT1 substrate 1 / 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 / Proline-rich AKT1 substrate 1 / 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: 5.5 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
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  • Surface view with fitted model
  • Atomic models: PDB-6sb0
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10132.png

Downloads & links

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Map

FileDownload / File: emd_10132.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM structure of RagA/C heterodimer GTPases in complex with mTORC1
Voxel sizeX=Y=Z: 1.43 Å
Density
Contour LevelBy AUTHOR: 0.018 / Movie #1: 0.018
Minimum - Maximum-0.06416719 - 0.117651746
Average (Standard dev.)0.00024526447 (±0.0035791127)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 500.49997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.431.431.43
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z500.500500.500500.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS350350350
D min/max/mean-0.0640.1180.000

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Supplemental data

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Mask #1

Fileemd_10132_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 of 3D reconstruction of mTORC1-RagA/C monomer

Fileemd_10132_additional.map
AnnotationThe experimental EM map of 3D reconstruction of mTORC1-RagA/C monomer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half1 map from 3D reconstruction RELION-3.0.6

Fileemd_10132_half_map_1.map
AnnotationHalf1 map from 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 3D reconstruction RELION-3.0.6

Fileemd_10132_half_map_2.map
AnnotationHalf2 map from 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 : cryoEM structure of mTORC1 bound to RagA/C complex

EntireName: cryoEM structure of mTORC1 bound to RagA/C complex
Components
  • Complex: cryoEM structure of mTORC1 bound to 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
      • Protein or peptide: Regulatory-associated protein of mTOR
    • Complex: RagA/C
      • Protein or peptide: Ras-related GTP-binding protein A
      • Protein or peptide: Ras-related GTP-binding protein C
      • Protein or peptide: Proline-rich AKT1 substrate 1
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: cryoEM structure of mTORC1 bound to RagA/C complex

SupramoleculeName: cryoEM structure of mTORC1 bound to RagA/C complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Molecular weightExperimental: 1.09 MDa

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Supramolecule #2: mTORC1

SupramoleculeName: mTORC1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2, #5
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-#4, #6
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: 287.012031 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)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)STRFYDQL NHHIFELVSS SDANERK GG ILAIASLIGV EGGNATRIGR FANYLRNLLP SNDPVVMEMA SKAIGRLAMA GDTFTAEYVE FEVKRALEWL GADRNEGR R HAAVLVLREL AISVPTFFFQ QVQPFFDNIF VAVWDPKQAI REGAVAALRA CLILTTQREP KEMQKPQWYR HTFEEAEKG FDETLAKEKG MNRDDRIHGA LLILNELVRI SSMEGERLRE EMEEITQQQL VHDKYCKDLM GFGTKPRHIT PFTSFQAVQP QQSNALVGL LGYSSHQGLM GFGTSPSPAK 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) R NSKNSLIQMT ILNLLPRLAA FRPSAFTDTQ YLQDTMNHVL SCVKKEKERT AAFQALGLLS VAVRSEFKVY LPRVLDIIR AALPPKDFAH KRQKAMQVDA TVFTCISMLA RAMGPGIQQD IKELLEPMLA VGLSPALTAV LYDLSRQIPQ LKKDIQDGLL KMLSLVLMH KPLRHPGMPK GLAHQLASPG LTTLPEASDV GSITLALRTL GSFEFEGHSL TQFVRHCADH FLNSEHKEIR M EAARTCSR LLTPSIHLIS GHAHVVSQTA VQVVADVLSK LLVVGITDPD PDIRYCVLAS LDERFDAHLA QAENLQALFV AL NDQVFEI RELAICTVGR LSSMNPAFVM PFLRKMLIQI LTELEHSGIG RIKEQSARML GHLVSNAPRL IRPYMEPILK ALI LKLKDP DPDPNPGVIN NVLATIGELA QVSGLEMRKW VDELFIIIMD MLQDSSLLAK RQVALWTLGQ LVASTGYVVE PYRK YPTLL EVLLNFLKTE QNQGTRREAI RVLGLLGALD PYKHKVNIGM IDQSRDASAV SLSESKSSQD SSDYSTSEML VNMGN LPLD EFYPAVSMVA LMRIFRDQSL SHHHTMVVQA ITFIFKSLGL KCVQFLPQVM PTFLNVIRVC DGAIREFLFQ QLGMLV SFV KSHIRPYMDE IVTLMREFWV MNTSIQSTII LLIEQIVVAL GGEFKLYLPQ LIPHMLRVFM HDNSPGRIVS IKLLAAI QL FGANLDDYLH LLLPPIVKLF DAPEAPLPSR KAALETVDRL TESLDFTDYA SRIIHPIVRT LDQSPELRST AMDTLSSL V FQLGKKYQIF IPMVNKVLVR HRINHQRYDV LICRIVKGYT LADEEEDPLI YQHRMLRSGQ GDALASGPVE TGPMKKLHV STINLQKAWG AARRVSKDDW LEWLRRLSLE LLKDSSSPSL RSCWALAQAY NPMARDLFNA AFVSCWSELN EDQQDELIRS IELALTSQD IAEVTQTLLN LAEFMEHSDK GPLPLRDDNG IVLLGERAAK CRAYAKALHY KELEFQKGPT PAILESLISI N NKLQQPEA AAGVLEYAMK HFGELEIQAT WYEKLHEWED ALVAYDKKMD TNKDDPELML GRMRCLEALG EWGQLHQQCC EK WTLVNDE TQAKMARMAA AAAWGLGQWD SMEEYTCMIP RDTHDGAFYR AVLALHQDLF SLAQQCIDKA RDLLDAELTA MAG ESYSRA YGAMVSCHML SELEEVIQYK LVPERREIIR QIWWERLQGC QRIVEDWQKI LMVRSLVVSP HEDMRTWLKY ASLC GKSGR LALAHKTLVL LLGVDPSRQL DHPLPTVHPQ VTYAYMKNMW KSARKIDAFQ HMQHFVQTMQ QQAQHAIATE DQQHK QELH KLMARCFLKL GEWQLNLQGI NESTIPKVLQ YYSAATEHDR SWYKAWHAWA VMNFEAVLHY KHQNQARDEK KKLRHA SGA NITNATTAAT TAATATTTAS TEGSNSESEA ESTENSPTPS PLQKKVTEDL SKTLLMYTVP AVQGFFRSIS LSRGNNL QD TLRVLTLWFD YGHWPDVNEA LVEGVKAIQI DTWLQVIPQL IARIDTPRPL VGRLIHQLLT DIGRYHPQAL IYPLTVAS K STTTARHNAA NKILKNMCEH SNTLVQQAMM VSEELIRVAI LWHEMWHEGL EEASRLYFGE RNVKGMFEVL EPLHAMMER GPQTLKETSF NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR ISKQLPQLTS LELQYVSPKL LMCRDLELAV PGTYDPNQP IIRIQSIAPS LQVITSKQRP RKLTLMGSNG HEFVFLLKGH EDLRQDERVM QLFGLVNTLL ANDPTSLRKN L SIQRYAVI PLSTNSGLIG WVPHCDTLHA LIRDYREKKK ILLNIEHRIM LRMAPDYDHL TLMQKVEVFE HAVNNTAGDD LA KLLWLKS PSSEVWFDRR TNYTRSLAVM SMVGYILGLG DRHPSNLMLD RLSGKILHID FGDCFEVAMT REKFPEKIPF RLT RMLTNA MEVTGLDGNY RITCHTVMEV LREHKDSVMA VLEAFVYDPL LNWRLMDTNT KGNKRSRTRT DSYSAGQSVE ILDG VELGE PAHKKTGTTV PESIHSFIGD GLVKPEALNK KAIQIINRVR DKLTGRDFSH DDTLDVPTQV ELLIKQATSH ENLCQ CYIG WCPFW

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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: Homo sapiens (human)
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: Proline-rich AKT1 substrate 1

MacromoleculeName: Proline-rich AKT1 substrate 1 / type: protein_or_peptide / ID: 6 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 27.412293 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASGRPEELW EAVVGAAERF RARTGTELVL LTAAPPPPPR PGPCAYAAHG RGALAEAARR CLHDIALAHR AATAARPPAP PPAPQPPSP TPSPPRPTLA REDNEEDEDE PTETETSGEQ LGISDNGGLF VMDEDATLQD LPPFCESDPE STDDGSLSEE T PAGPPTCS ...String:
MASGRPEELW EAVVGAAERF RARTGTELVL LTAAPPPPPR PGPCAYAAHG RGALAEAARR CLHDIALAHR AATAARPPAP PPAPQPPSP TPSPPRPTLA REDNEEDEDE PTETETSGEQ LGISDNGGLF VMDEDATLQD LPPFCESDPE STDDGSLSEE T PAGPPTCS VPPASALPTQ QYAKSLPVSV PVWGFKEKRT EARSSDEENG PPSSPDLDRI AASMRALVLR EAEDTQVFGD LP RPRLNTS DFQKLKRKY

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Macromolecule #7: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / 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 #8: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 8 / 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: 50mM HEPES pH 7.0, 100mM NaCl, 2mM MgCl2, 1mM TCEP
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 200 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
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 interacting partner, PRAS40-fused-RagA/C (referred as RagA/C here) forms complex with mTORC1 for its 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 - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / 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: 580707
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 reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.5 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 5.5 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: 90809
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: 283
Output model

PDB-6sb0:
cryo-EM structure of mTORC1 bound to PRAS40-fused active RagA/C GTPases

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