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- EMDB-12214: human complex II-BATS bound to membrane-attached Rab5a-GTP -

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Basic information

Entry
Database: EMDB / ID: EMD-12214
Titlehuman complex II-BATS bound to membrane-attached Rab5a-GTP
Map dataSubtomogram averaging reconstruction of human complex II-BATS bound to membrane-attached Rab5a-GTP at 9.8A with the membrane masked out after local-denoising with the LAFTER algorithm
Sample
  • Complex: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a
    • Complex: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG)
      • Protein or peptide: UV radiation resistance-associated gene protein
      • Protein or peptide: Phosphatidylinositol 3-kinase catalytic subunit type 3
      • Protein or peptide: Phosphoinositide 3-kinase regulatory subunit 4
      • Protein or peptide: Beclin-1
      • Protein or peptide: unknown peptide
    • Complex: Ras-related protein Rab-5A
      • Protein or peptide: Ras-related protein Rab-5A
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsRab GTPase / Phosphatidylinositol 3-kinase / ENDOCYTOSIS
Function / homology
Function and homology information


lytic vacuole / maintenance of Golgi location / regulation of protein serine/threonine kinase activity / regulation of endosome size / nucleus-vacuole junction / postsynaptic early endosome / cellular response to aluminum ion / cytoplasmic side of early endosome membrane / Toll Like Receptor 9 (TLR9) Cascade / protein lipidation ...lytic vacuole / maintenance of Golgi location / regulation of protein serine/threonine kinase activity / regulation of endosome size / nucleus-vacuole junction / postsynaptic early endosome / cellular response to aluminum ion / cytoplasmic side of early endosome membrane / Toll Like Receptor 9 (TLR9) Cascade / protein lipidation / Synthesis of PIPs at the late endosome membrane / phosphatidylinositol 3-kinase complex, class III / Synthesis of PIPs at the early endosome membrane / synaptic vesicle recycling / phosphatidylinositol 3-kinase complex, class III, type II / positive regulation of stress granule assembly / cellular response to oxygen-glucose deprivation / phosphatidylinositol 3-kinase complex, class III, type I / response to mitochondrial depolarisation / positive regulation of attachment of mitotic spindle microtubules to kinetochore / amyloid-beta clearance by transcytosis / cytoplasmic side of mitochondrial outer membrane / negative regulation of lysosome organization / positive regulation by host of viral genome replication / modulation by host of viral process / Synthesis of PIPs at the Golgi membrane / regulation of autophagosome assembly / positive regulation of autophagosome assembly / negative regulation of autophagosome assembly / receptor catabolic process / engulfment of apoptotic cell / phosphatidylinositol kinase activity / protein localization to phagophore assembly site / regulation of filopodium assembly / suppression by virus of host autophagy / multivesicular body sorting pathway / RAB geranylgeranylation / protein targeting to lysosome / protein targeting to vacuole / early endosome to late endosome transport / cellular response to nitrogen starvation / late endosome to vacuole transport / SMAD protein signal transduction / double-strand break repair via classical nonhomologous end joining / RAB GEFs exchange GTP for GDP on RABs / early phagosome / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / phagophore assembly site / Translation of Replicase and Assembly of the Replication Transcription Complex / negative regulation of programmed cell death / response to iron(II) ion / TBC/RABGAPs / positive regulation of autophagosome maturation / centrosome cycle / SNARE complex assembly / autolysosome / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / spindle organization / mitotic metaphase chromosome alignment / regulation of synaptic vesicle exocytosis / Macroautophagy / cytoplasmic pattern recognition receptor signaling pathway / 1-phosphatidylinositol-3-kinase activity / lysosome organization / RSV-host interactions / positive regulation of cardiac muscle hypertrophy / positive regulation of exocytosis / p38MAPK cascade / axoneme / Synthesis of PIPs at the plasma membrane / autophagosome membrane / phosphatidylinositol-mediated signaling / Respiratory syncytial virus (RSV) attachment and entry / phosphatidylinositol phosphate biosynthetic process / mitophagy / chromosome, centromeric region / autophagosome maturation / autophagosome assembly / PI3K Cascade / RHO GTPases Activate NADPH Oxidases / response to vitamin E / autophagosome / neuron development / negative regulation of reactive oxygen species metabolic process / regulation of macroautophagy / canonical Wnt signaling pathway / amyloid-beta metabolic process / endomembrane system / cellular defense response / cellular response to glucose starvation / positive regulation of autophagy / phosphatidylinositol 3-kinase binding / phagocytosis / axon terminus / positive regulation of intrinsic apoptotic signaling pathway / JNK cascade / Prevention of phagosomal-lysosomal fusion / somatodendritic compartment / cellular response to epidermal growth factor stimulus
Similarity search - Function
UV radiation resistance protein/autophagy-related protein 14 / Vacuolar sorting 38 and autophagy-related subunit 14 / Serine/threonine-protein kinase Vps15-like / Beclin-1, BH3 domain / Beclin-1 BH3 domain, Bcl-2-interacting / Atg6/Beclin / Atg6/Beclin C-terminal domain superfamily / Atg6, BARA domain / Atg6/beclin, coiled-coil domain / Apg6 BARA domain ...UV radiation resistance protein/autophagy-related protein 14 / Vacuolar sorting 38 and autophagy-related subunit 14 / Serine/threonine-protein kinase Vps15-like / Beclin-1, BH3 domain / Beclin-1 BH3 domain, Bcl-2-interacting / Atg6/Beclin / Atg6/Beclin C-terminal domain superfamily / Atg6, BARA domain / Atg6/beclin, coiled-coil domain / Apg6 BARA domain / Apg6 coiled-coil region / Phosphatidylinositol 3-kinase, Vps34 type / HEAT repeat profile. / HEAT, type 2 / small GTPase Rab1 family profile. / Phosphoinositide 3-kinase C2 / Phosphoinositide 3-kinase, region postulated to contain C2 domain / C2 phosphatidylinositol 3-kinase-type domain / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase, accessory (PIK) domain / Phosphatidylinositol kinase / PIK helical domain profile. / Protein kinase C conserved region 2 (CalB) / C2 domain / C2 domain profile. / 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 / Ran (Ras-related nuclear proteins) /TC4 subfamily of small GTPases / C2 domain superfamily / Rho (Ras homology) subfamily of Ras-like small GTPases / Ras subfamily of RAS small GTPases / Small GTPase / Ras family / Rab subfamily of small GTPases / Armadillo-like helical / Small GTP-binding protein domain / Armadillo-type fold / 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 / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Ras-related protein Rab-5A / Beclin-1 / Phosphatidylinositol 3-kinase catalytic subunit type 3 / Phosphoinositide 3-kinase regulatory subunit 4 / UV radiation resistance-associated gene protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 9.8 Å
AuthorsTremel S / Morado DR
Funding support United Kingdom, 7 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_U105184308 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_U10517878 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/16 United Kingdom
Cancer Research UKC14801/A21211
European Research Council (ERC)ERC-CoG-648432 MEMBRANEFUSION United Kingdom
Wellcome TrustNo. 103139 United Kingdom
Wellcome TrustNo. 203149 United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes.
Authors: Shirley Tremel / Yohei Ohashi / Dustin R Morado / Jessie Bertram / Olga Perisic / Laura T L Brandt / Marie-Kristin von Wrisberg / Zhuo A Chen / Sarah L Maslen / Oleksiy Kovtun / Mark Skehel ...Authors: Shirley Tremel / Yohei Ohashi / Dustin R Morado / Jessie Bertram / Olga Perisic / Laura T L Brandt / Marie-Kristin von Wrisberg / Zhuo A Chen / Sarah L Maslen / Oleksiy Kovtun / Mark Skehel / Juri Rappsilber / Kathrin Lang / Sean Munro / John A G Briggs / Roger L Williams /
Abstract: The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal ...The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.
History
DepositionJan 17, 2021-
Header (metadata) releaseMar 3, 2021-
Map releaseMar 3, 2021-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7bl1
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12214.png

Downloads & links

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Map

FileDownload / File: emd_12214.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram averaging reconstruction of human complex II-BATS bound to membrane-attached Rab5a-GTP at 9.8A with the membrane masked out after local-denoising with the LAFTER algorithm
Voxel sizeX=Y=Z: 1.0665 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.008
Minimum - Maximum-0.045468677 - 0.06802449
Average (Standard dev.)0.00014741789 (±0.002034834)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 341.27997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06651.06651.0665
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z341.280341.280341.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0450.0680.000

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Supplemental data

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Half map: Odd Half-map masked to remove density from the membrane.

Fileemd_12214_half_map_1.map
AnnotationOdd Half-map masked to remove density from the membrane.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Even Half-map masked to remove density from the membrane.

Fileemd_12214_half_map_2.map
AnnotationEven Half-map masked to remove density from the membrane.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human...

EntireName: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a
Components
  • Complex: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a
    • Complex: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG)
      • Protein or peptide: UV radiation resistance-associated gene protein
      • Protein or peptide: Phosphatidylinositol 3-kinase catalytic subunit type 3
      • Protein or peptide: Phosphoinositide 3-kinase regulatory subunit 4
      • Protein or peptide: Beclin-1
      • Protein or peptide: unknown peptide
    • Complex: Ras-related protein Rab-5A
      • Protein or peptide: Ras-related protein Rab-5A
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human...

SupramoleculeName: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Details: The subunits were expressed using transient transfection in HEK293T cells. Cells were transfected with three plasmids: pYO1025 (encoding VPS34 and VPS15 in a pCAG backbone), pYO1124 ...Details: The subunits were expressed using transient transfection in HEK293T cells. Cells were transfected with three plasmids: pYO1025 (encoding VPS34 and VPS15 in a pCAG backbone), pYO1124 (encoding UVGRAG 1-464 fused to the BATS of ATG14, residues 413-492 in pVAG) and pYO1006 (Beclin1 in pCAG)
Molecular weightTheoretical: 392615 kDa/nm

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Supramolecule #2: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG)

SupramoleculeName: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Ras-related protein Rab-5A

SupramoleculeName: Ras-related protein Rab-5A / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #6
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: UV radiation resistance-associated gene protein

MacromoleculeName: UV radiation resistance-associated gene protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 78.258836 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSASASVGGP VPQPPPGPAA ALPPGSAARA LHVELPSQQR RLRHLRNIAA RNIVNRNGHQ LLDTYFTLHL CSTEKIYKEF YRSEVIKNS LNPTWRSLDF GIMPDRLDTS VSCFVVKIWG GKENIYQLLI EWKVCLDGLK YLGQQIHARN QNEIIFGLND G YYGAPFEH ...String:
MSASASVGGP VPQPPPGPAA ALPPGSAARA LHVELPSQQR RLRHLRNIAA RNIVNRNGHQ LLDTYFTLHL CSTEKIYKEF YRSEVIKNS LNPTWRSLDF GIMPDRLDTS VSCFVVKIWG GKENIYQLLI EWKVCLDGLK YLGQQIHARN QNEIIFGLND G YYGAPFEH KGYSNAQKTI LLQVDQNCVR NSYDVFSLLR LHRAQCAIKQ TQVTVQKIGK EIEEKLRLTS TSNELKKKSE CL QLKILVL QNELERQKKA LGREVALLHK QQIALQDKGS AFSAEHLKLQ LQKESLNELR KECTAKRELF LKTNAQLTIR CRQ LLSELS YIYPIDLNEH KDYFVCGVKL PNSEDFQAKD DGSIAVALGY TAHLVSMISF FLQVPLRYPI IHKGSRSTIK DNIN DKLTE KEREFPLYPK GGEKLQFDYG VYLLNKNIAQ LRYQHGLGTP DLRQTLPNLK NFMEHGLMVR CDRHHTSSAI PVPKR QSSI FGGADVGFSG GIPSPDKGHR KRASSENERL QYKTPPPSYN SALAQPVTTV PSMGETERKI TSLSSSLDTS LDFSKE NKK KGEDLVGSLN GGHANVHPSQ EQGEALSGHR ATVNGTLLPS EQAGSASVQL PGEFHPVSEA ELCCTVEQAE EIIGLEA TG FASGDQLEAF NCIPVDSAVA VECDEQVLGE FEEFSRRIYA LNENVSSFRR PRRSSDK

UniProtKB: UV radiation resistance-associated gene protein

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Macromolecule #2: Phosphatidylinositol 3-kinase catalytic subunit type 3

MacromoleculeName: Phosphatidylinositol 3-kinase catalytic subunit type 3
type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: phosphatidylinositol 3-kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 101.680328 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGEAEKFHYI YSCDLDINVQ LKIGSLEGKR EQKSYKAVLE DPMLKFSGLY QETCSDLYVT CQVFAEGKPL ALPVRTSYKA FSTRWNWNE WLKLPVKYPD LPRNAQVALT IWDVYGPGKA VPVGGTTVSL FGKYGMFRQG MHDLKVWPNV EADGSEPTKT P GRTSSTLS ...String:
MGEAEKFHYI YSCDLDINVQ LKIGSLEGKR EQKSYKAVLE DPMLKFSGLY QETCSDLYVT CQVFAEGKPL ALPVRTSYKA FSTRWNWNE WLKLPVKYPD LPRNAQVALT IWDVYGPGKA VPVGGTTVSL FGKYGMFRQG MHDLKVWPNV EADGSEPTKT P GRTSSTLS EDQMSRLAKL TKAHRQGHMV KVDWLDRLTF REIEMINESE KRSSNFMYLM VEFRCVKCDD KEYGIVYYEK DG DESSPIL TSFELVKVPD PQMSMENLVE SKHHKLARSL RSGPSDHDLK PNAATRDQLN IIVSYPPTKQ LTYEEQDLVW KFR YYLTNQ EKALTKFLKC VNWDLPQEAK QALELLGKWK PMDVEDSLEL LSSHYTNPTV RRYAVARLRQ ADDEDLLMYL LQLV QALKY ENFDDIKNGL EPTKKDSQSS VSENVSNSGI NSAEIDSSQI ITSPLPSVSS PPPASKTKEV PDGENLEQDL CTFLI SRAC KNSTLANYLY WYVIVECEDQ DTQQRDPKTH EMYLNVMRRF SQALLKGDKS VRVMRSLLAA QQTFVDRLVH LMKAVQ RES GNRKKKNERL QALLGDNEKM NLSDVELIPL PLEPQVKIRG IIPETATLFK SALMPAQLFF KTEDGGKYPV IFKHGDD LR QDQLILQIIS LMDKLLRKEN LDLKLTPYKV LATSTKHGFM QFIQSVPVAE VLDTEGSIQN FFRKYAPSEN GPNGISAE V MDTYVKSCAG YCVITYILGV GDRHLDNLLL TKTGKLFHID FGYILGRDPK PLPPPMKLNK EMVEGMGGTQ SEQYQEFRK QCYTAFLHLR RYSNLILNLF SLMVDANIPD IALEPDKTVK KVQDKFRLDL SDEEAVHYMQ SLIDESVHAL FAAVVEQIHK FAQYWRK

UniProtKB: Phosphatidylinositol 3-kinase catalytic subunit type 3

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Macromolecule #3: Phosphoinositide 3-kinase regulatory subunit 4

MacromoleculeName: Phosphoinositide 3-kinase regulatory subunit 4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 154.790391 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGNQLAGIAP SQILSVESYF SDIHDFEYDK SLGSTRFFKV ARAKHREGLV VVKVFAIQDP TLPLTSYKQE LEELKIRLNS AQNCLPFQK ASEKASEKAA MLFRQYVRDN LYDRISTRPF LNNIEKRWIA FQILTAVDQA HKSGVRHGDI KTENVMVTSW N WVLLTDFA ...String:
MGNQLAGIAP SQILSVESYF SDIHDFEYDK SLGSTRFFKV ARAKHREGLV VVKVFAIQDP TLPLTSYKQE LEELKIRLNS AQNCLPFQK ASEKASEKAA MLFRQYVRDN LYDRISTRPF LNNIEKRWIA FQILTAVDQA HKSGVRHGDI KTENVMVTSW N WVLLTDFA SFKPTYLPED NPADFNYFFD TSRRRTCYIA PERFVDGGMF ATELEYMRDP STPLVDLNSN QRTRGELKRA MD IFSAGCV IAELFTEGVP LFDLSQLLAY RNGHFFPEQV LNKIEDHSIR ELVTQMIHRE PDKRLEAEDY LKQQRGNAFP EIF YTFLQP YMAQFAKETF LSADERILVI RKDLGNIIHN LCGHDLPEKA EGEPKENGLV ILVSVITSCL QTLKYCDSKL AALE LILHL APRLSVEILL DRITPYLLHF SNDSVPRVRA EALRTLTKVL ALVKEVPRND INIYPEYILP GIAHLAQDDA TIVRL AYAE NIALLAETAL RFLELVQLKN LNMENDPNNE EIDEVTHPNG NYDTELQALH EMVQQKVVTL LSDPENIVKQ TLMENG ITR LCVFFGRQKA NDVLLSHMIT FLNDKNDWHL RGAFFDSIVG VAAYVGWQSS SILKPLLQQG LSDAEEFVIV KALYALT CM CQLGLLQKPH VYEFASDIAP FLCHPNLWIR YGAVGFITVV ARQISTADVY CKLMPYLDPY ITQPIIQIER KLVLLSVL K EPVSRSIFDY ALRSKDITSL FRHLHMRQKK RNGSLPDCPP PEDPAIAQLL KKLLSQGMTE EEEDKLLALK DFMMKSNKA KANIVDQSHL HDSSQKGVID LAALGITGRQ VDLVKTKQEP DDKRARKHVK QDSNVNEEWK SMFGSLDPPN MPQALPKGSD QEVIQTGKP PRSESSAGIC VPLSTSSQVP EVTTVQNKKP VIPVLSSTIL PSTYQIRITT CKTELQQLIQ QKREQCNAER I AKQMMENA EWESKPPPPG WRPKGLLVAH LHEHKSAVNR IRVSDEHSLF ATCSNDGTVK IWNSQKMEGK TTTTRSILTY SR IGGRVKT LTFCQGSHYL AIASDNGAVQ LLGIEASKLP KSPKIHPLQS RILDQKEDGC VVDMHHFNSG AQSVLAYATV NGS LVGWDL RSSSNAWTLK HDLKSGLITS FAVDIHQCWL CIGTSSGTMA CWDMRFQLPI SSHCHPSRAR IRRLSMHPLY QSWV IAAVQ GNNEVSMWDM ETGDRRFTLW ASSAPPLSEL QPSPHSVHGI YCSPADGNPI LLTAGSDMKI RFWDLAYPER SYVVA GSTS SPSVSYYRKI IEGTEVVQEI QNKQKVGPSD DTPRRGPESL PVGHHDIITD VATFQTTQGF IVTASRDGIV KVWKSR PTT ASENLYFQ

UniProtKB: Phosphoinositide 3-kinase regulatory subunit 4

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Macromolecule #4: Beclin-1

MacromoleculeName: Beclin-1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 51.953102 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MEGSKTSNNS TMQVSFVCQR CSQPLKLDTS FKILDRVTIQ ELTAPLLTTA QAKPGETQEE ETNSGEEPFI ETPRQDGVSR RFIPPARMM STESANSFTL IGEASDGGTM ENLSRRLKVT GDLFDIMSGQ TDVDHPLCEE CTDTLLDQLD TQLNVTENEC Q NYKRCLEI ...String:
MEGSKTSNNS TMQVSFVCQR CSQPLKLDTS FKILDRVTIQ ELTAPLLTTA QAKPGETQEE ETNSGEEPFI ETPRQDGVSR RFIPPARMM STESANSFTL IGEASDGGTM ENLSRRLKVT GDLFDIMSGQ TDVDHPLCEE CTDTLLDQLD TQLNVTENEC Q NYKRCLEI LEQMNEDDSE QLQMELKELA LEEERLIQEL EDVEKNRKIV AENLEKVQAE AERLDQEEAQ YQREYSEFKR QQ LELDDEL KSVENQMRYA QTQLDKLKKT NVFNATFHIW HSGQFGTINN FRLGRLPSVP VEWNEINAAW GQTVLLLHAL ANK MGLKFQ RYRLVPYGNH SYLESLTDKS KELPLYCSGG LRFFWDNKFD HAMVAFLDCV QQFKEEVEKG ETRFCLPYRM DVEK GKIED TGGSGGSYSI KTQFNSEEQW TKALKFMLTN LKWGLAWVSS QFYNK

UniProtKB: Beclin-1

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Macromolecule #5: unknown peptide

MacromoleculeName: unknown peptide / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.581727 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
AAAAAAAAAA AAAAAAAAAA AA

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Macromolecule #6: Ras-related protein Rab-5A

MacromoleculeName: Ras-related protein Rab-5A / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.769314 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. DH10B (bacteria)
SequenceString:
NKISQFKLVL LGESAVGKSS LVLRFVKGQF HEFQESTIGA AFLTQTVSLD DTTVKFEIWD TAGLERYHSL APMYYRGAQA AIVVYDITN EESFARAKNW VKELQRQASP NIVIALSGNK ADLANKRAVD FQEAQSYADD NSLLFMETSA KTSMNVNEIF M AIAKKLPK

UniProtKB: Ras-related protein Rab-5A

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Macromolecule #7: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

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

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state3D array

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Sample preparation

Concentration6.2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMHEPESHEPES
200.0 mMNaClNaCl
0.5 mMTCEPTCEP
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Details: Quorum SC7620
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 316 K / Instrument: FEI VITROBOT MARK I / Details: Blot force 20, blot time 6 s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Average exposure time: 0.55 sec. / Average electron dose: 2.99 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: 5.0 µm / Nominal defocus min: 2.0 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

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 9.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number subtomograms used: 26979
ExtractionNumber tomograms: 105 / Number images used: 191196 / Reference model: two Gaussian filtered elipsoids / Software - Name: MATLAB (ver. R2016b) / Software - details: The locally written subTOM
Final 3D classificationNumber classes: 20 / Avg.num./class: 9600 / Software - Name: MATLAB (ver. R2016b) / Software - details: locally written subTOM
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Merit function: correlation coefficient
Software - Name: MATLAB (ver. R2016b) / Software - details: locally written subTOM
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5dfz

source_name: PDB, initial_model_type: experimental model

3mjh

chain_id: A, source_name: PDB, initial_model_type: experimental model

4ddp

chain_id: A, source_name: PDB, initial_model_type: experimental model

3ihy

chain_id: A, source_name: PDB, initial_model_type: experimental model
DetailsAn initial model was build with SWISSMODEL. using 5DFZ, 4DDP and 3IHY. This was then fit manually to density while running restrained MD with ISOLDE. The model was refined in REFMAC with self-restraints (4.3) and restraints to 3MJH, 4DDP and 3IHY.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 320 / Target criteria: correlation
Output model

PDB-7bl1:
human complex II-BATS bound to membrane-attached Rab5a-GTP

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