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- EMDB-13097: human DEPTOR in a complex with mutant human mTORC1 A1459P -

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Basic information

Entry
Database: EMDB / ID: EMD-13097
Titlehuman DEPTOR in a complex with mutant human mTORC1 A1459P
Map datamTORC1 with mTOR A1459P mutant in a complex with DEPTOR
Sample
  • Complex: COMPLEX OF HUMAN MUTANT MTORC1 A1459P WITH THE INHIBITORY PROTEIN DEPTOR
    • Complex: mTORC1 with mutant A1459P mTOR
      • Protein or peptide: Serine/threonine-protein kinase mTOR
      • Protein or peptide: Target of rapamycin complex subunit LST8
      • Protein or peptide: Regulatory-associated protein of mTOR
    • Complex: DEPTOR
      • Protein or peptide: DEP domain-containing mTOR-interacting protein
Function / homology
Function and homology information


negative regulation of TORC2 signaling / regulation of extrinsic apoptotic signaling pathway / positive regulation of cytoplasmic translational initiation / positive regulation of pentose-phosphate shunt / RNA polymerase III type 1 promoter sequence-specific DNA binding / RNA polymerase III type 2 promoter sequence-specific DNA binding / T-helper 1 cell lineage commitment / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / cellular response to leucine starvation ...negative regulation of TORC2 signaling / regulation of extrinsic apoptotic signaling pathway / positive regulation of cytoplasmic translational initiation / positive regulation of pentose-phosphate shunt / RNA polymerase III type 1 promoter sequence-specific DNA binding / RNA polymerase III type 2 promoter sequence-specific DNA binding / T-helper 1 cell lineage commitment / regulation of locomotor rhythm / positive regulation of wound healing, spreading of epidermal cells / cellular response to leucine starvation / regulation of membrane permeability / heart valve morphogenesis / TFIIIC-class transcription factor complex binding / negative regulation of lysosome organization / RNA polymerase III type 3 promoter sequence-specific DNA binding / TORC2 complex / TORC1 complex / positive regulation of transcription of nucleolar large rRNA by RNA polymerase I / regulation of autophagosome assembly / calcineurin-NFAT signaling cascade / nucleus localization / TORC1 signaling / voluntary musculoskeletal movement / positive regulation of odontoblast differentiation / 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 / phosphatidic acid binding / negative regulation of cell size / ruffle organization / protein serine/threonine kinase inhibitor activity / positive regulation of osteoclast differentiation / cellular response to osmotic stress / enzyme-substrate adaptor activity / negative regulation of protein localization to nucleus / anoikis / cardiac muscle cell development / negative regulation of TOR signaling / positive regulation of transcription by RNA polymerase III / negative regulation of calcineurin-NFAT signaling cascade / regulation of myelination / regulation of cell size / Macroautophagy / positive regulation of oligodendrocyte differentiation / negative regulation of macroautophagy / positive regulation of actin filament polymerization / lysosome organization / positive regulation of myotube differentiation / protein kinase inhibitor activity / protein kinase activator activity / behavioral response to pain / oligodendrocyte differentiation / mTORC1-mediated signalling / Constitutive Signaling by AKT1 E17K in Cancer / germ cell development / CD28 dependent PI3K/Akt signaling / cellular response to nutrient levels / positive regulation of phosphoprotein phosphatase activity / social behavior / HSF1-dependent transactivation / TOR signaling / positive regulation of TOR signaling / neuronal action potential / positive regulation of translational initiation / response to amino acid / positive regulation of G1/S transition of mitotic cell cycle / regulation of macroautophagy / endomembrane system / 'de novo' pyrimidine nucleobase biosynthetic process / positive regulation of autophagy / positive regulation of epithelial to mesenchymal transition / positive regulation of lamellipodium assembly / positive regulation of lipid biosynthetic process / heart morphogenesis / cardiac muscle contraction / regulation of cellular response to heat / positive regulation of stress fiber assembly / negative regulation of TORC1 signaling / 14-3-3 protein binding / cytoskeleton organization / positive regulation of endothelial cell proliferation / T cell costimulation / cellular response to amino acid starvation / positive regulation of glycolytic process / cellular response to starvation / phagocytic vesicle / negative regulation of autophagy / protein serine/threonine kinase activator activity / response to nutrient levels / response to nutrient / post-embryonic development / VEGFR2 mediated vascular permeability / positive regulation of peptidyl-threonine phosphorylation / regulation of signal transduction by p53 class mediator / Regulation of PTEN gene transcription
Similarity search - Function
DEP domain-containing mTOR-interacting protein, DEP domain 1 / DEP domain-containing mTOR-interacting protein, DEP domain 2 / Raptor, N-terminal CASPase-like domain / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / Target of rapamycin complex subunit LST8 / Domain of unknown function DUF3385, target of rapamycin protein / Serine/threonine-protein kinase mTOR domain / Domain of unknown function ...DEP domain-containing mTOR-interacting protein, DEP domain 1 / DEP domain-containing mTOR-interacting protein, DEP domain 2 / Raptor, N-terminal CASPase-like domain / Raptor N-terminal CASPase like domain / Raptor N-terminal CASPase like domain / Regulatory associated protein of TOR / Target of rapamycin complex subunit LST8 / Domain of unknown function DUF3385, target of rapamycin protein / Serine/threonine-protein kinase mTOR domain / 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 / Domain found in Dishevelled, Egl-10, and Pleckstrin (DEP) / DEP domain profile. / Domain found in Dishevelled, Egl-10, and Pleckstrin / DEP 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 / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold / G-protein beta WD-40 repeat / Winged helix DNA-binding domain superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / Winged helix-like DNA-binding domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Serine/threonine-protein kinase mTOR / Regulatory-associated protein of mTOR / DEP domain-containing mTOR-interacting protein / Target of rapamycin complex subunit LST8
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsHeimhalt M / Berndt A / Wagstaff J / Anandapadamanaban M / Perisic O / Maslen S / McLaughlin S / Yu W-H / Masson GR / Boland A ...Heimhalt M / Berndt A / Wagstaff J / Anandapadamanaban M / Perisic O / Maslen S / McLaughlin S / Yu W-H / Masson GR / Boland A / Ni X / Yamashita K / Murshudov GN / Skehel M / Freund SM / Williams RL
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_U105184308 United Kingdom
Cancer Research UKC14801/A21211 United Kingdom
European Molecular Biology Organization (EMBO)EMBO ALTF 603-2019 United Kingdom
CitationJournal: Elife / Year: 2021
Title: Bipartite binding and partial inhibition links DEPTOR and mTOR in a mutually antagonistic embrace.
Authors: Maren Heimhalt / Alex Berndt / Jane Wagstaff / Madhanagopal Anandapadamanaban / Olga Perisic / Sarah Maslen / Stephen McLaughlin / Conny Wing-Heng Yu / Glenn R Masson / Andreas Boland / ...Authors: Maren Heimhalt / Alex Berndt / Jane Wagstaff / Madhanagopal Anandapadamanaban / Olga Perisic / Sarah Maslen / Stephen McLaughlin / Conny Wing-Heng Yu / Glenn R Masson / Andreas Boland / Xiaodan Ni / Keitaro Yamashita / Garib N Murshudov / Mark Skehel / Stefan M Freund / Roger L Williams /
Abstract: The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted ...The mTORC1 kinase complex regulates cell growth, proliferation, and survival. Because mis-regulation of DEPTOR, an endogenous mTORC1 inhibitor, is associated with some cancers, we reconstituted mTORC1 with DEPTOR to understand its function. We find that DEPTOR is a unique mTORC1 inhibitor that may have evolved to preserve feedback inhibition of PI3K. Counterintuitively, mTORC1 activated by RHEB or oncogenic mutation is much more potently inhibited by DEPTOR. Although DEPTOR partially inhibits mTORC1, mTORC1 prevents this inhibition by phosphorylating DEPTOR, a mutual antagonism that requires no exogenous factors. Structural analyses of the mTORC1/DEPTOR complex showed DEPTOR's PDZ domain interacting with the mTOR FAT region, and the unstructured linker preceding the PDZ binding to the mTOR FRB domain. The linker and PDZ form the minimal inhibitory unit, but the N-terminal tandem DEP domains also significantly contribute to inhibition.
History
DepositionJun 18, 2021-
Header (metadata) releaseSep 8, 2021-
Map releaseSep 8, 2021-
UpdateFeb 23, 2022-
Current statusFeb 23, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7owg
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7owg
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13097.png

Downloads & links

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Map

FileDownload / File: emd_13097.map.gz / Format: CCP4 / Size: 80.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationmTORC1 with mTOR A1459P mutant in a complex with DEPTOR
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.013817274 - 0.038300518
Average (Standard dev.)0.00045145763 (±0.0027693997)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions276276276
Spacing276276276
CellA=B=C: 303.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z276276276
origin x/y/z0.0000.0000.000
length x/y/z303.600303.600303.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS276276276
D min/max/mean-0.0140.0380.000

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Supplemental data

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Mask #1

Fileemd_13097_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: mTORC1 with mTOR A1459P mutant in a complex with DEPTOR half map 2

Fileemd_13097_half_map_1.map
AnnotationmTORC1 with mTOR A1459P mutant in a complex with DEPTOR half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: mTORC1 with mTOR A1459P mutant in a complex with DEPTOR half map 1

Fileemd_13097_half_map_2.map
AnnotationmTORC1 with mTOR A1459P mutant in a complex with DEPTOR half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : COMPLEX OF HUMAN MUTANT MTORC1 A1459P WITH THE INHIBITORY PROTEIN...

EntireName: COMPLEX OF HUMAN MUTANT MTORC1 A1459P WITH THE INHIBITORY PROTEIN DEPTOR
Components
  • Complex: COMPLEX OF HUMAN MUTANT MTORC1 A1459P WITH THE INHIBITORY PROTEIN DEPTOR
    • Complex: mTORC1 with mutant A1459P mTOR
      • Protein or peptide: Serine/threonine-protein kinase mTOR
      • Protein or peptide: Target of rapamycin complex subunit LST8
      • Protein or peptide: Regulatory-associated protein of mTOR
    • Complex: DEPTOR
      • Protein or peptide: DEP domain-containing mTOR-interacting protein

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Supramolecule #1: COMPLEX OF HUMAN MUTANT MTORC1 A1459P WITH THE INHIBITORY PROTEIN...

SupramoleculeName: COMPLEX OF HUMAN MUTANT MTORC1 A1459P WITH THE INHIBITORY PROTEIN DEPTOR
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: An mTORC1 complex bound to two copies of DEPTOR. Each mTORC1 complex consists of a dimer of an mTOR/LST8/RAPTOR heterotrimer. The mTOR is an A1459P mutant.
Molecular weightTheoretical: 46.3 MDa

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Supramolecule #2: mTORC1 with mutant A1459P mTOR

SupramoleculeName: mTORC1 with mutant A1459P mTOR / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2, #4 / Details: mTORC1 with mutant A1459P mTOR
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: Expi293F / Recombinant plasmid: pOPL146, pOPL95 and pOPL119

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Supramolecule #3: DEPTOR

SupramoleculeName: DEPTOR / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3 / Details: DEPTOR is a natural inhibitor of mTORC1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: LOBSTR / Recombinant plasmid: pOPL138

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Macromolecule #1: Serine/threonine-protein kinase mTOR

MacromoleculeName: Serine/threonine-protein kinase mTOR / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 287.570312 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLGTGPAAAT TAATTS(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)SRNEET(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)EMSQE ...String:
MLGTGPAAAT TAATTS(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)SRNEET(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)EMSQE E(UNK)TRFYDQLN HHIFELVSSS DANERKGGIL AIASLIGVEG GNATRI GRF ANYLRNLLPS NDPVVMEMAS KAIGRLAMAG DTFTAEYVEF EVKRALEWLG ADRNEGRRHA AVLVLRELAI SVPTFFF QQ VQPFFDNIFV AVWDPKQAIR EGAVAALRAC LILTTQREPK EMQKPQWYRH TFEEAEKGFD ETLAKEKGMN RDDRIHGA L LILNELVRIS SMEGERLREE MEEITQQQLV HDKYCKDLMG FGTKPRHITP FTSFQAVQPQ QSNALVGLLG YSSHQGLMG FGTSPSPAKS T(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)KCRNS KNSLIQMTIL NLLPRLAAFR PSAFT DTQY LQDTMNHVLS CVKKEKERTA AFQALGLLSV AVRSEFKVYL PRVLDIIRAA LPPKDFAHKR QKAMQVDATV FTCISM LAR AMGPGIQQDI KELLEPMLAV GLSPALTAVL YDLSRQIPQL KKDIQDGLLK MLSLVLMHKP LRHPGMPKGL AHQLASP GL TTLPEASDVG SITLALRTLG SFEFEGHSLT QFVRHCADHF LNSEHKEIRM EAARTCSRLL TPSIHLISGH AHVVSQTA V QVVADVLSKL LVVGITDPDP DIRYCVLASL DERFDAHLAQ AENLQALFVA LNDQVFEIRE LAICTVGRLS SMNPAFVMP FLRKMLIQIL TELEHSGIGR IKEQSARMLG HLVSNAPRLI RPYMEPILKA LILKLKDPDP DPNPGVINNV LATIGELAQV SGLEMRKWV DELFIIIMDM LQDSSLLAKR QVALWTLGQL VASTGYVVEP YRKYPTLLEV LLNFLKTEQN QGTRREAIRV L GLLGALDP YKHKVNIGMI DQSRDASAVS LSESKSSQDS SDYSTSEMLV NMGNLPLDEF YPAVSMVALM RIFRDQSLSH HH TMVVQAI TFIFKSLGLK CVQFLPQVMP TFLNVIRVCD GAIREFLFQQ LGMLVSFVKS HIRPYMDEIV TLMREFWVMN TSI QSTIIL LIEQIVVALG GEFKLYLPQL IPHMLRVFMH DNSPGRIVSI KLLAAIQLFG ANLDDYLHLL LPPIVKLFDA PEAP LPSRK AALETVDRLT ESLDFTDYAS RIIHPIVRTL DQSPELRSTA MDTLSSLVFQ LGKKYQIFIP MVNKVLVRHR INHQR YDVL ICRIVKGYTL ADEEEDPLIY QHRMLRSGQG DALASGPVET GPMKKLHVST INLQKAWGAA RRVSKDDWLE WLRRLS LEL LKDSSSPSLR SCWALAQAYN PMARDLFNAA FVSCWSELNE DQQDELIRSI ELALTSQDIA EVTQTLLNLA EFMEHSD KG PLPLRDDNGI VLLGERAAKC RAYAKALHYK ELEFQKGPTP AILESLISIN NKLQQPEAAA GVLEYAMKHF GELEIQAT W YEKLHEWEDP LVAYDKKMDT NKDDPELMLG RMRCLEALGE WGQLHQQCCE KWTLVNDETQ AKMARMAAAA AWGLGQWDS MEEYTCMIPR DTHDGAFYRA VLALHQDLFS LAQQCIDKAR DLLDAELTAM AGESYSRAYG AMVSCHMLSE LEEVIQYKLV PERREIIRQ IWWERLQGCQ RIVEDWQKIL MVRSLVVSPH EDMRTWLKYA SLCGKSGRLA LAHKTLVLLL GVDPSRQLDH P LPTVHPQV TYAYMKNMWK SARKIDAFQH MQHFVQTMQQ QAQHAIATED QQHKQELHKL MARCFLKLGE WQLNLQGINE ST IPKVLQY YSAATEHDRS WYKAWHAWAV MNFEAVLHYK HQNQARDEKK KLRHASGANI TNATTAATTA ATATTTASTE GSN SESEAE STENSPTPSP LQKKVTEDLS KTLLMYTVPA VQGFFRSISL SRGNNLQDTL RVLTLWFDYG HWPDVNEALV EGVK AIQID TWLQVIPQLI ARIDTPRPLV GRLIHQLLTD IGRYHPQALI YPLTVASKST TTARHNAANK ILKNMCEHSN TLVQQ AMMV SEELIRVAIL WHEMWHEGLE EASRLYFGER NVKGMFEVLE PLHAMMERGP QTLKETSFNQ AYGRDLMEAQ EWCRKY MKS GNVKDLTQAW DLYYHVFRRI SKQLPQLTSL ELQYVSPKLL MCRDLELAVP GTYDPNQPII RIQSIAPSLQ VITSKQR PR KLTLMGSNGH EFVFLLKGHE DLRQDERVMQ LFGLVNTLLA NDPTSLRKNL SIQRYAVIPL STNSGLIGWV PHCDTLHA L IRDYREKKKI LLNIEHRIML RMAPDYDHLT LMQKVEVFEH AVNNTAGDDL AKLLWLKSPS SEVWFDRRTN YTRSLAVMS MVGYILGLGD RHPSNLMLDR LSGKILHIDF GDCFEVAMTR EKFPEKIPFR LTRMLTNAME VTGLDGNYRI TCHTVMEVLR EHKDSVMAV LEAFVYDPLL NWRLMDTNTK GNKRSRTRTD SYSAGQSVEI LDGVELGEPA HKKTGTTVPE SIHSFIGDGL V KPEALNKK AIQIINRVRD KLTGRDFSHD DTLDVPTQVE LLIKQATSHE NLCQCYIGWC PFW

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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: 1 / 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: DEP domain-containing mTOR-interacting protein

MacromoleculeName: DEP domain-containing mTOR-interacting protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.365832 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEEGGSTGSA GSDSSTSGSG GAQQRELERM AEVLVTGEQL RLRLHEEKVI KDRRHHLKTY PNCFVAKELI DWLIEHKEAS DRETAIKLM QKLADRGIIH HVCDEHKEFK DVKLFYRFRK DDGTFPLDNE VKAFMRGQRL YEKLMSPENT LLQPREEEGV K YERTFMAS ...String:
MEEGGSTGSA GSDSSTSGSG GAQQRELERM AEVLVTGEQL RLRLHEEKVI KDRRHHLKTY PNCFVAKELI DWLIEHKEAS DRETAIKLM QKLADRGIIH HVCDEHKEFK DVKLFYRFRK DDGTFPLDNE VKAFMRGQRL YEKLMSPENT LLQPREEEGV K YERTFMAS EFLDWLVQEG EATTRKEAEQ LCHRLMEHGI IQHVSSKHPF VDSNLLYQFR MNFRRRRRLM ELLNEKSPSS QE THDSPFC LRKQSHDNRK STSFMSVSPS KEIKIVSAVR RSSMSSCGSS GYFSSSPTLS SSPPVLCNPK SVLKRPVTSE ELL TPGAPY ARKTFTIVGD AVGWGFVVRG SKPCHIQAVD PSGPAAAAGM KVCQFVVSVN GLNVLHVDYR TVNNLILTGP RTIV MEVME ELEC

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Macromolecule #4: Regulatory-associated protein of mTOR

MacromoleculeName: Regulatory-associated protein of mTOR / type: protein_or_peptide / ID: 4 / Number of copies: 1 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 7.5
Details: 50 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM TCEP, 1 mM MgCl2, 500 uM AMP-PNP
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 287 K / Instrument: FEI VITROBOT MARK I / Details: blotting time of 2 s and a force of -15..
DetailsPurified mutant mTORC1 A1459P and DEPTOR were preincubated in the presence of MgCl2 and AMP-PNP for 20 min and used for cryo-EM grid preparation.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 100.0 K / Max: 100.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 2 / Number real images: 4759 / Average exposure time: 2.5 sec. / Average electron dose: 56.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: -3.0 µm / Nominal defocus min: -1.4000000000000001 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 376784
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 500000
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 1 / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6bcx


Chain - Chain ID: B / Chain - Residue range: 1-2549
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 145.7 / Target criteria: Correlation
Output model

PDB-7owg:
human DEPTOR in a complex with mutant human mTORC1 A1459P

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