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- EMDB-12237: Subtomogram average reconstruction of humanVPS34 complex II bound... -

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Basic information

Entry
Database: EMDB / ID: EMD-12237
TitleSubtomogram average reconstruction of humanVPS34 complex II bound to Rab5a on a lipid membrane
Map dataA reconstruction of the VPS34 complex II bound to Rab5a on lipid vesicles created by subtomogram averaging. The map was sharpened with LAFTER and resampled onto the map from EMD-12214
Sample
  • Complex: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a on lipid vesicles
    • 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-1BECN1
    • Protein or peptide: Ras-related protein Rab-5A
Function / homology
Function and homology information


lytic vacuole / maintenance of Golgi location / regulation of protein serine/threonine kinase activity / autophagy of peroxisome / regulation of endosome size / postsynaptic early endosome / nucleus-vacuole junction / cellular response to aluminum ion / cytoplasmic side of early endosome membrane / Toll Like Receptor 9 (TLR9) Cascade ...lytic vacuole / maintenance of Golgi location / regulation of protein serine/threonine kinase activity / autophagy of peroxisome / regulation of endosome size / postsynaptic early endosome / nucleus-vacuole junction / 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 / Synthesis of PIPs at the early endosome membrane / phosphatidylinositol 3-kinase complex, class III / synaptic vesicle recycling / cellular response to oxygen-glucose deprivation / phosphatidylinositol 3-kinase complex, class III, type II / phosphatidylinositol 3-kinase complex, class III, type I / positive regulation by host of viral genome replication / response to mitochondrial depolarisation / amyloid-beta clearance by transcytosis / positive regulation of attachment of mitotic spindle microtubules to kinetochore / cytoplasmic side of mitochondrial outer membrane / negative regulation of lysosome organization / modulation by host of viral process / Synthesis of PIPs at the Golgi membrane / positive regulation of autophagosome assembly / engulfment of apoptotic cell / regulation of autophagosome assembly / negative regulation of autophagosome assembly / receptor catabolic process / phosphatidylinositol kinase activity / protein localization to phagophore assembly site / protein targeting to vacuole / protein targeting to lysosome / suppression by virus of host autophagy / regulation of filopodium assembly / RAB geranylgeranylation / multivesicular body sorting pathway / early endosome to late endosome transport / cellular response to nitrogen starvation / late endosome to vacuole transport / RAB GEFs exchange GTP for GDP on RABs / double-strand break repair via classical nonhomologous end joining / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / early phagosome / SMAD protein signal transduction / phosphatidylinositol-mediated signaling / phagophore assembly site / Translation of Replicase and Assembly of the Replication Transcription Complex / response to iron(II) ion / negative regulation of programmed cell death / TBC/RABGAPs / centrosome cycle / SNARE complex assembly / positive regulation of autophagosome maturation / autolysosome / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / regulation of synaptic vesicle exocytosis / 1-phosphatidylinositol-3-kinase activity / Macroautophagy / spindle organization / mitotic metaphase chromosome alignment / lysosome organization / cytoplasmic pattern recognition receptor signaling pathway / positive regulation of cardiac muscle hypertrophy / positive regulation of exocytosis / p38MAPK cascade / axoneme / autophagosome maturation / autophagosome membrane / Synthesis of PIPs at the plasma membrane / phosphatidylinositol phosphate biosynthetic process / mitophagy / autophagosome assembly / chromosome, centromeric region / RHO GTPases Activate NADPH Oxidases / PI3K Cascade / neuron development / autophagosome / canonical Wnt signaling pathway / negative regulation of reactive oxygen species metabolic process / response to vitamin E / regulation of macroautophagy / endomembrane system / cellular response to glucose starvation / cellular defense response / phosphatidylinositol 3-kinase binding / phagocytic vesicle / amyloid-beta metabolic process / axon terminus / phagocytosis / positive regulation of autophagy / positive regulation of intrinsic apoptotic signaling pathway / JNK cascade / somatodendritic compartment / Prevention of phagosomal-lysosomal fusion / cellular response to epidermal growth factor stimulus / ruffle
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. / 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 / 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: 13.3 Å
AuthorsTremel S / Ohashi Y / Morado DR / Bertram J / Perisic O / Brandt LTL / von Wrisberg M-K / Chen ZA / Maslen SL / Kovtun O ...Tremel S / Ohashi Y / Morado DR / Bertram J / Perisic O / Brandt LTL / von Wrisberg M-K / Chen ZA / Maslen SL / Kovtun O / Skehel M / Rappsilber J / Lang K / Munro S / Briggs JAG / Williams RL
Funding support United Kingdom, Germany, 9 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 United Kingdom
European Research Council (ERC)ERC-CoG-648432 United Kingdom
German Research Foundation (DFG)Sonderforschungsbereich 1035, project number 201302640, project B10 Germany
German Research Foundation (DFG)392923329 Germany
Wellcome Trust103139 United Kingdom
Wellcome Trust203149 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 23, 2021-
Header (metadata) releaseJun 16, 2021-
Map releaseJun 16, 2021-
UpdateJun 16, 2021-
Current statusJun 16, 2021Processing 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
  • Download
  • Surface view colored by height
  • Surface level: 0.008
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12237.png

Downloads & links

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Map

FileDownload / File: emd_12237.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationA reconstruction of the VPS34 complex II bound to Rab5a on lipid vesicles created by subtomogram averaging. The map was sharpened with LAFTER and resampled onto the map from EMD-12214
Voxel sizeX=Y=Z: 1.0665 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.008
Minimum - Maximum-0.08853249 - 0.12369968
Average (Standard dev.)2.3871984e-05 (±0.005715127)
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
start NX/NY/NZ696888
NX/NY/NZ828048
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0890.1240.000

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Supplemental data

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Half map: A reconstruction of the VPS34 complex II bound...

Fileemd_12237_half_map_1.map
AnnotationA reconstruction of the VPS34 complex II bound to Rab5a on lipid vesicles created by subtomogram averaging. The even half map, before LAFTER sharpening.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: A reconstruction of the VPS34 complex II bound...

Fileemd_12237_half_map_2.map
AnnotationA reconstruction of the VPS34 complex II bound to Rab5a on lipid vesicles created by subtomogram averaging. The odd half map, before LAFTER sharpening.
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 on lipid vesicles
Components
  • Complex: human VPS34 complex II (VPS34-VPS15-Beclin1-UVRAG) bound to human Rab5a on lipid vesicles
    • 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-1BECN1
    • Protein or peptide: Ras-related protein Rab-5A

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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 on lipid vesicles
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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)
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 392.16 KDa

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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 / Details: Human UVRAG / Enantiomer: LEVO / EC number: phosphatidylinositol 3-kinase
Source (natural)Organism: Homo sapiens (human)
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

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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 / Details: human vps34 / Enantiomer: LEVO / EC number: phosphatidylinositol 3-kinase
Source (natural)Organism: Homo sapiens (human)
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

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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 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
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

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Macromolecule #4: Beclin-1

MacromoleculeName: Beclin-1 / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
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

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

MacromoleculeName: Ras-related protein Rab-5A / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
NKISQFKLVL LGESAVGKSS LVLRFVKGQF HEFQESTIGA AFLTQTVSLD DTTVKFEIWD TAGLERYHSL APMYYRGAQA AIVVYDITN EESFARAKNW VKELQRQASP NIVIALSGNK ADLANKRAVD FQEAQSYADD NSLLFMETSA KTSMNVNEIF M AIAKKLPK

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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 mMNaClSodium chlorideNaClSodium chloride
0.5 mMTCEPTCEP
GridModel: Quantifoil / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: Quorum SC7620
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 316 K / Instrument: FEI VITROBOT MARK I / Details: blot force was 20, with a blot time of 6 s.

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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 / 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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 6000 pixel / Digitization - Dimensions - Height: 4000 pixel / Digitization - Sampling interval: 5.0 µm / Number grids imaged: 1 / Average exposure time: 0.55 sec. / Average electron dose: 2.99 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 105 / Number images used: 191196
Reference model: two gaussian-filtered elipsoids forming a V shape
Details: To identify particles, subtomograms were aligned to an initial reference consisting of two gaussian-filtered ellipsoids forming a V-shape (Supplementary Fig. 6a, reference). After aligning ...Details: To identify particles, subtomograms were aligned to an initial reference consisting of two gaussian-filtered ellipsoids forming a V-shape (Supplementary Fig. 6a, reference). After aligning against the V-shape, some subtomograms converged and formed clusters, which indicated the presence of a particle The subtomogram coordinates were cleaned by a minimal distance threshold (distance cut off 8 px, cluster size 2, cluster distance 2 px) and cross correlation cut-off so that 191,169 particles remained.
CTF correctionSoftware - Name: NOVACTF (ver. 1.0.0)
Final 3D classificationNumber classes: 20 / Avg.num./class: 9600 / Software - Name: MATLAB (ver. R2016b)
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Merit function: Correlation coefficient
Software - Name: MATLAB (ver. R2016b) / Software - details: Local MATLAB program SUBTOM
Final reconstructionNumber classes used: 6 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 13.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number subtomograms used: 26979
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7bl1

chain_id: A

7bl1

chain_id: B

7bl1

chain_id: C

7bl1

chain_id: D

7bl1

chain_id: E

7bl1

chain_id: F
DetailsThe model in PDB 7BL1 was built and refined into the subtomogram averaged density map in EMD-12214. This map had the membrane portion of the reconstruction masked out. The reconstruction described in the final reconstruction described above includes the membrane that was masked out in EMD-12214. The final reconstruction was sharpened in LAFTER then resampled onto the density of EMD-12214, for ease of fitting the 7BL1 model.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 320 / Target criteria: Correlation coefficient

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