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- EMDB-54359: VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202 mutant) bound to RA... -

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Basic information

Entry
Database: EMDB / ID: EMD-54359
TitleVPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202 mutant) bound to RAB5A (Q79L) on the VPS15 subunit, composite map processed with CryoTEN.
Map dataVPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202 mutant) bound to RAB5A (Q79L) on the VPS15 subunit
Sample
  • Complex: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/BECLIN1/UVRAG) bound to RAB5A-GTP (C19S, C63S, Q79L) on the VPS15 subunit
    • Protein or peptide: Ras-related protein Rab-5A
    • Protein or peptide: Beclin-1
    • Protein or peptide: UV radiation resistance associated protein
    • Protein or peptide: Phosphatidylinositol 3-kinase catalytic subunit type 3
  • Protein or peptide: Phosphoinositide 3-kinase regulatory subunit 4
  • Ligand: MYRISTIC ACID
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
KeywordsLipid kinase / GTPase / kinase / endocytic sorting / endosome maturation / SIGNALING PROTEIN / ENDOCYTOSIS
Function / homology
Function and homology information


protein-containing complex organization / regulation of endosome size / nucleus-vacuole junction / cytoplasmic side of early endosome membrane / cellular response to aluminum ion / positive regulation of protein lipidation / protein localization to early endosome / postsynaptic endosome / Toll Like Receptor 9 (TLR9) Cascade / positive regulation of stress granule assembly ...protein-containing complex organization / regulation of endosome size / nucleus-vacuole junction / cytoplasmic side of early endosome membrane / cellular response to aluminum ion / positive regulation of protein lipidation / protein localization to early endosome / postsynaptic endosome / Toll Like Receptor 9 (TLR9) Cascade / positive regulation of stress granule assembly / Synthesis of PIPs at the late endosome membrane / phosphatidylinositol 3-kinase complex, class III / cellular response to oxygen-glucose deprivation / Synthesis of PIPs at the early endosome membrane / phosphatidylinositol 3-kinase complex, class III, type II / phosphatidylinositol 3-kinase complex, class III, type I / amyloid-beta clearance by transcytosis / response to mitochondrial depolarisation / presynaptic endosome / positive regulation of attachment of mitotic spindle microtubules to kinetochore / host-mediated activation of viral genome replication / synaptic vesicle recycling / plasma membrane to endosome transport / Dengue virus modulates apoptosis / engulfment of apoptotic cell / negative regulation of lysosome organization / host-mediated perturbation of viral process / phosphatidylinositol kinase activity / SMAD protein signal transduction / positive regulation of autophagosome assembly / regulation of filopodium assembly / Synthesis of PIPs at the Golgi membrane / cytoplasmic side of mitochondrial outer membrane / early endosome to late endosome transport / receptor catabolic process / RAB geranylgeranylation / response to L-leucine / protein targeting to vacuole / late endosome to vacuole transport / regulation of autophagosome assembly / protein targeting to lysosome / endosome organization / pexophagy / RAB GEFs exchange GTP for GDP on RABs / early phagosome / positive regulation of natural killer cell mediated cytotoxicity / phagophore assembly site / Translation of Replicase and Assembly of the Replication Transcription Complex / TBC/RABGAPs / cellular response to nitrogen starvation / centrosome cycle / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / endosomal transport / 1-phosphatidylinositol-3-kinase activity / negative regulation of programmed cell death / response to vitamin E / Macroautophagy / response to iron(II) ion / regulation of synaptic vesicle exocytosis / RSV-host interactions / p38MAPK cascade / cytoplasmic pattern recognition receptor signaling pathway / phosphatidylinositol phosphate biosynthetic process / Synthesis of PIPs at the plasma membrane / mitotic metaphase chromosome alignment / phosphatidylinositol-mediated signaling / autolysosome / positive regulation of exocytosis / Respiratory syncytial virus (RSV) attachment and entry / canonical Wnt signaling pathway / PI3K Cascade / chromosome, centromeric region / RHO GTPases Activate NADPH Oxidases / regulation of macroautophagy / autophagosome maturation / endocytic vesicle / axoneme / synaptic vesicle endocytosis / cellular defense response / autophagosome assembly / phagocytosis / phosphatidylinositol 3-kinase binding / cellular response to glucose starvation / intercellular bridge / mitophagy / JNK cascade / phagocytic vesicle / ruffle / positive regulation of intrinsic apoptotic signaling pathway / vesicle-mediated transport / somatodendritic compartment / endomembrane system / positive regulation of autophagy / Prevention of phagosomal-lysosomal fusion / autophagosome / cellular response to epidermal growth factor stimulus / axon terminus / cellular response to copper ion / cellular response to amino acid starvation
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 / : / : / PIK3R4-like, middle domain / HEAT, type 2 / HEAT repeat profile. / Small GTPase Rab domain profile. / C2 phosphatidylinositol 3-kinase-type domain / Phosphoinositide 3-kinase C2 / C2 phosphatidylinositol 3-kinase (PI3K)-type domain profile. / Phosphoinositide 3-kinase, region postulated to contain C2 domain / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase, accessory (PIK) domain / Phosphatidylinositol kinase / PIK helical domain profile. / Protein kinase C conserved region 2 (CalB) / C2 domain / 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 / WD domain, G-beta repeat / Armadillo-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Trp-Asp (WD) repeats signature. / Protein kinase domain / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / Serine/Threonine protein kinases, catalytic domain / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / 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 / UV radiation resistance associated protein / Phosphatidylinositol 3-kinase catalytic subunit type 3 / Phosphoinositide 3-kinase regulatory subunit 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsSpokaite S / Ohashi Y / Dessus AN / Bourguet M / Williams RL
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_U105184308 United Kingdom
Cancer Research UKDRCPGM 100014 United Kingdom
Citation
Journal: Elife / Year: 2026
Title: A novel RAB5 binding site in human VPS34-CII that is likely the primordial site in eukaryotic evolution.
Authors: Saule Spokaite / Yohei Ohashi / Maxime Bourguet / Antoine Nicolas Dessus / Roger L Williams /
Abstract: RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII by ...RAB5-GTP activation of the multiprotein VPS34 complex II (VPS34-CII) is critical for endosomal sorting and maturation, phagocytosis, and receptor downregulation. RAB5-GTP activates VPS34-CII by binding to a helical insertion in the C2 domain of VPS34 on the BECLIN1/UVRAG-containing adaptor arm of the complex. The autophagy complex, VPS34 complex I (VPS34-CI), features a unique ATG14L subunit in place of the VPS34-CII UVRAG subunit, and we found that this distorts the adaptor arm to alter the VPS34 RAB-GTPase binding pocket so that it preferentially binds RAB1-GTP. Surprisingly, our higher-resolution single-particle cryo-EM structure of VPS34-CII showed a second RAB5-GTP binding site on the VPS15 solenoid region. This site (VPS15-RAB5-site) appears to be the primordial RAB5-binding region. A mutant in the helical insertion of the C2 domain of human VPS34 that mimics the sequence abolishes RAB5 binding to VPS34. Mutation of the VPS15-RAB5-site ortholog in VPS15 resulted in defective CPY sorting, loss of colocalisation with the RAB5 ortholog Vps21, and loss of binding to Vps21 in vitro. Evolutionary expansion from one to two RAB5-orthologue binding sites may have increased membrane binding and VPS34-CII activity to adapt to more complex endocytic systems.
#1: Journal: Elife / Year: 2026
Title: A novel RAB5 binding site in human VPS34-CII that is likely the primordial site in eukaryotic evolution
Authors: Spokaite S / Ohashi Y / Bourguet M / Dessus AN / Williams RL
History
DepositionJul 10, 2025-
Header (metadata) releaseJun 3, 2026-
Map releaseJun 3, 2026-
UpdateJun 10, 2026-
Current statusJun 10, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_54359.png

Downloads & links

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Map

FileDownload / File: emd_54359.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202 mutant) bound to RAB5A (Q79L) on the VPS15 subunit
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 440 pix.
= 365.2 Å		0.83 Å/pix.
x 440 pix.
= 365.2 Å		0.83 Å/pix.
x 440 pix.
= 365.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.062799275 - 1.6630684
Average (Standard dev.)0.001394914 (±0.022883972)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 365.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/...

EntireName: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/BECLIN1/UVRAG) bound to RAB5A-GTP (C19S, C63S, Q79L) on the VPS15 subunit
Components
  • Complex: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/BECLIN1/UVRAG) bound to RAB5A-GTP (C19S, C63S, Q79L) on the VPS15 subunit
    • Protein or peptide: Ras-related protein Rab-5A
    • Protein or peptide: Beclin-1
    • Protein or peptide: UV radiation resistance associated protein
    • Protein or peptide: Phosphatidylinositol 3-kinase catalytic subunit type 3
  • Protein or peptide: Phosphoinositide 3-kinase regulatory subunit 4
  • Ligand: MYRISTIC ACID
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/...

SupramoleculeName: Mutant human VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202/VPS15/BECLIN1/UVRAG) bound to RAB5A-GTP (C19S, C63S, Q79L) on the VPS15 subunit
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #5, #3-#4, #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 409 KDa

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Macromolecule #1: Phosphatidylinositol 3-kinase catalytic subunit type 3

MacromoleculeName: Phosphatidylinositol 3-kinase catalytic subunit type 3
type: protein_or_peptide / ID: 1
Details: Full length VPS34 with a 199-REIE-202 to 199-ERIR-202 mutation
Number of copies: 1 / Enantiomer: LEVO / EC number: phosphatidylinositol 3-kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 101.708406 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 ERIRMINESE 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 #2: Phosphoinositide 3-kinase regulatory subunit 4

MacromoleculeName: Phosphoinositide 3-kinase regulatory subunit 4 / type: protein_or_peptide / ID: 2
Details: Full length VPS15 myristoylated at the G2 residue, bound to GDP, with 13 extra residues at the C-terminus left after TEV protease treatment (VPS15-SRPTTASENLYFQ)
Number of copies: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 154.659188 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GNQLAGIAPS QILSVESYFS DIHDFEYDKS LGSTRFFKVA RAKHREGLVV VKVFAIQDPT LPLTSYKQEL EELKIRLNSA QNCLPFQKA SEKASEKAAM LFRQYVRDNL YDRISTRPFL NNIEKRWIAF QILTAVDQAH KSGVRHGDIK TENVMVTSWN W VLLTDFAS ...String:
GNQLAGIAPS QILSVESYFS DIHDFEYDKS LGSTRFFKVA RAKHREGLVV VKVFAIQDPT LPLTSYKQEL EELKIRLNSA QNCLPFQKA SEKASEKAAM LFRQYVRDNL YDRISTRPFL NNIEKRWIAF QILTAVDQAH KSGVRHGDIK TENVMVTSWN W VLLTDFAS FKPTYLPEDN PADFNYFFDT SRRRTCYIAP ERFVDGGMFA TELEYMRDPS TPLVDLNSNQ RTRGELKRAM DI FSAGCVI AELFTEGVPL FDLSQLLAYR NGHFFPEQVL NKIEDHSIRE LVTQMIHREP DKRLEAEDYL KQQRGNAFPE IFY TFLQPY MAQFAKETFL SADERILVIR KDLGNIIHNL CGHDLPEKAE GEPKENGLVI LVSVITSCLQ TLKYCDSKLA ALEL ILHLA PRLSVEILLD RITPYLLHFS NDSVPRVRAE ALRTLTKVLA LVKEVPRNDI NIYPEYILPG IAHLAQDDAT IVRLA YAEN IALLAETALR FLELVQLKNL NMENDPNNEE IDEVTHPNGN YDTELQALHE MVQQKVVTLL SDPENIVKQT LMENGI TRL CVFFGRQKAN DVLLSHMITF LNDKNDWHLR GAFFDSIVGV AAYVGWQSSS ILKPLLQQGL SDAEEFVIVK ALYALTC MC QLGLLQKPHV YEFASDIAPF LCHPNLWIRY GAVGFITVVA RQISTADVYC KLMPYLDPYI TQPIIQIERK LVLLSVLK E PVSRSIFDYA LRSKDITSLF RHLHMRQKKR NGSLPDCPPP EDPAIAQLLK KLLSQGMTEE EEDKLLALKD FMMKSNKAK ANIVDQSHLH DSSQKGVIDL AALGITGRQV DLVKTKQEPD DKRARKHVKQ DSNVNEEWKS MFGSLDPPNM PQALPKGSDQ EVIQTGKPP RSESSAGICV PLSTSSQVPE VTTVQNKKPV IPVLSSTILP STYQIRITTC KTELQQLIQQ KREQCNAERI A KQMMENAE WESKPPPPGW RPKGLLVAHL HEHKSAVNRI RVSDEHSLFA TCSNDGTVKI WNSQKMEGKT TTTRSILTYS RI GGRVKTL TFCQGSHYLA IASDNGAVQL LGIEASKLPK SPKIHPLQSR ILDQKEDGCV VDMHHFNSGA QSVLAYATVN GSL VGWDLR SSSNAWTLKH DLKSGLITSF AVDIHQCWLC IGTSSGTMAC WDMRFQLPIS SHCHPSRARI RRLSMHPLYQ SWVI AAVQG NNEVSMWDME TGDRRFTLWA SSAPPLSELQ PSPHSVHGIY CSPADGNPIL LTAGSDMKIR FWDLAYPERS YVVAG STSS PSVSYYRKII EGTEVVQEIQ NKQKVGPSDD TPRRGPESLP VGHHDIITDV ATFQTTQGFI VTASRDGIVK VWKSRP TTA SENLYFQ

UniProtKB: Phosphoinositide 3-kinase regulatory subunit 4

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Macromolecule #3: Beclin-1

MacromoleculeName: Beclin-1 / type: protein_or_peptide / ID: 3 / 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 #4: UV radiation resistance associated protein

MacromoleculeName: UV radiation resistance associated protein / type: protein_or_peptide / ID: 4 / Details: Full length UVRAG with a P10Q mutation / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 78.289852 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSASASVGGQ VPQPPPGPAA ALPPGSAARA LHVELPSQQR RLRHLRNIAA RNIVNRNGHQ LLDTYFTLHL CSTEKIYKEF YRSEVIKNS LNPTWRSLDF GIMPDRLDTS VSCFVVKIWG GKENIYQLLI EWKVCLDGLK YLGQQIHARN QNEIIFGLND G YYGAPFEH ...String:
MSASASVGGQ 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 protein

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

MacromoleculeName: Ras-related protein Rab-5A / type: protein_or_peptide / ID: 5 / Details: RAB5A-GTP (1-212) C19S C63S Q79L GTP-locked mutant / Number of copies: 1 / Enantiomer: LEVO / EC number: small monomeric GTPase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.333225 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MASRGATRPN GPNTGNKISQ FKLVLLGESA VGKSSLVLRF VKGQFHEFQE STIGAAFLTQ TVSLDDTTVK FEIWDTAGLE RYHSLAPMY YRGAQAAIVV YDITNEESFA RAKNWVKELQ RQASPNIVIA LSGNKADLAN KRAVDFQEAQ SYADDNSLLF M ETSAKTSM ...String:
MASRGATRPN GPNTGNKISQ FKLVLLGESA VGKSSLVLRF VKGQFHEFQE STIGAAFLTQ TVSLDDTTVK FEIWDTAGLE RYHSLAPMY YRGAQAAIVV YDITNEESFA RAKNWVKELQ RQASPNIVIA LSGNKADLAN KRAVDFQEAQ SYADDNSLLF M ETSAKTSM NVNEIFMAIA KKLPKNEPQN PGANSARGRG VDLTEPTQPT RNQC

UniProtKB: Ras-related protein Rab-5A

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Macromolecule #6: MYRISTIC ACID

MacromoleculeName: MYRISTIC ACID / type: ligand / ID: 6 / Number of copies: 1 / Formula: MYR
Molecular weightTheoretical: 228.371 Da
Chemical component information

ChemComp-MYR:
MYRISTIC ACID

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Macromolecule #7: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

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

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Macromolecule #8: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #9: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #10: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 10 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.45 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
200.0 mMNaClSodium chloride
1.0 mMC9H15O6PTCEP
5.0 mMMgCl2Magnesium chloride
1.0 mMC10H17N6O12P3AMPPNP
4.0 mMC32H58N2O8SCHAPSO
0.005 % v/v(C2H4O)nC14H22ONonidet P40 substitute
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 287 K / Instrument: FEI VITROBOT MARK III
Details: 3.5 uL sample, 0 second wait, 3.5 second blot, blot force +8.
DetailsSample was crosslinked with 30 uM BS3 for 20 minutes on ice, then quenched with 100 mM TRIS pH 8.0 prior to vitrification

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 13301 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
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: 370889
CTF correctionType: NONE
Startup modelType of model: OTHER
Details: Density from VPS34-CI bound to NRBF2 MIT domain, determined earlier
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 84296
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9rx6:
VPS34-CII (VPS34 199-REIE-202 to 199-ERIR-202 mutant) bound to RAB5A (Q79L) on the VPS15 subunit

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