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- EMDB-56458: Cryo-EM structure of human VPS34-CI with ADP:MgF3 - composite map -

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Basic information

Entry
Database: EMDB / ID: EMD-56458
TitleCryo-EM structure of human VPS34-CI with ADP:MgF3 - composite map
Map data
Sample
  • Complex: Protein complex VPS34-CI with NRBF2 MIT and ADP:MgF3
    • Complex: Human VPS34-CI
      • 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: Beclin 1-associated autophagy-related key regulator
    • Complex: NRBF2 MIT domain
      • Protein or peptide: Nuclear receptor-binding factor 2
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: MYRISTIC ACID
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION
KeywordsLipid kinase / Complex / Autophagy / LIPID BINDING PROTEIN
Function / homology
Function and homology information


extrinsic component of omegasome membrane / phosphatidylinositol 3-kinase inhibitor activity / extrinsic component of phagophore assembly site membrane / nucleus-vacuole junction / cellular response to aluminum ion / positive regulation of protein lipidation / postsynaptic endosome / Toll Like Receptor 9 (TLR9) Cascade / Synthesis of PIPs at the late endosome membrane / cellular response to oxygen-glucose deprivation ...extrinsic component of omegasome membrane / phosphatidylinositol 3-kinase inhibitor activity / extrinsic component of phagophore assembly site membrane / nucleus-vacuole junction / cellular response to aluminum ion / positive regulation of protein lipidation / postsynaptic endosome / Toll Like Receptor 9 (TLR9) Cascade / Synthesis of PIPs at the late endosome membrane / cellular response to oxygen-glucose deprivation / Synthesis of PIPs at the early endosome membrane / positive regulation of stress granule assembly / phosphatidylinositol 3-kinase complex, class III, type II / phosphatidylinositol 3-kinase complex, class III, type I / response to mitochondrial depolarisation / presynaptic endosome / phosphatidylinositol 3-kinase complex, class III / positive regulation of attachment of mitotic spindle microtubules to kinetochore / host-mediated activation of viral genome replication / engulfment of apoptotic cell / negative regulation of lysosome organization / regulation of protein complex stability / phosphatidylinositol kinase activity / SMAD protein signal transduction / Synthesis of PIPs at the Golgi membrane / phosphatidylinositol 3-kinase regulator activity / phagophore assembly site membrane / early endosome to late endosome transport / receptor catabolic process / cytoplasmic side of mitochondrial outer membrane / response to L-leucine / protein targeting to vacuole / protein targeting to lysosome / mitochondria-associated endoplasmic reticulum membrane contact site / late endosome to vacuole transport / endosome organization / Dengue virus modulates apoptosis / positive regulation of natural killer cell mediated cytotoxicity / pexophagy / positive regulation of autophagosome assembly / phagophore assembly site / Translation of Replicase and Assembly of the Replication Transcription Complex / cellular response to nitrogen starvation / phosphatidylinositol 3-kinase / phosphatidylinositol-3-phosphate biosynthetic process / 1-phosphatidylinositol-3-kinase activity / post-transcriptional regulation of gene expression / response to vitamin E / Macroautophagy / endosome to lysosome transport / p38MAPK cascade / autophagosome membrane docking / RSV-host interactions / response to iron(II) ion / cytoplasmic pattern recognition receptor signaling pathway / phosphatidylinositol phosphate biosynthetic process / negative regulation of protein phosphorylation / phosphatidylinositol-mediated signaling / protein-membrane adaptor activity / autolysosome / autophagosome membrane / positive regulation of intrinsic apoptotic signaling pathway / PI3K Cascade / autophagosome maturation / RHO GTPases Activate NADPH Oxidases / JNK cascade / mitotic metaphase chromosome alignment / autophagosome assembly / axoneme / mitophagy / negative regulation of programmed cell death / synaptic vesicle endocytosis / cellular defense response / cellular response to glucose starvation / phosphatidylinositol 3-kinase binding / regulation of macroautophagy / phagocytic vesicle / positive regulation of autophagy / response to endoplasmic reticulum stress / cellular response to epidermal growth factor stimulus / autophagosome / cellular response to copper ion / cellular response to amino acid starvation / cellular response to starvation / phosphatidylinositol 3-kinase/protein kinase B signal transduction / macroautophagy / regulation of cytokinesis / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / trans-Golgi network / circadian rhythm / autophagy / protein processing / Nuclear Receptor transcription pathway / GABA-ergic synapse / regulation of autophagy / positive regulation of protein phosphorylation / response to lead ion / ISG15 antiviral mechanism / phagocytic vesicle membrane / mitochondrial membrane
Similarity search - Function
Nuclear receptor-binding factor 2, C-terminal / Nuclear receptor-binding factor 2, MIT domain / Nuclear receptor-binding factor 2 / Nuclear receptor-binding factor 2, autophagy regulator / MIT domain of nuclear receptor-binding factor 2 / 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 ...Nuclear receptor-binding factor 2, C-terminal / Nuclear receptor-binding factor 2, MIT domain / Nuclear receptor-binding factor 2 / Nuclear receptor-binding factor 2, autophagy regulator / MIT domain of nuclear receptor-binding factor 2 / 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. / 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. / 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 / C2 domain superfamily / Armadillo-like helical / WD domain, G-beta repeat / Armadillo-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / Serine/Threonine protein kinases, catalytic domain / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Beclin-1 / Beclin 1-associated autophagy-related key regulator / Phosphatidylinositol 3-kinase catalytic subunit type 3 / Nuclear receptor-binding factor 2 / Phosphoinositide 3-kinase regulatory subunit 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.66 Å
AuthorsDessus AN / 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
CitationJournal: Nat Commun / Year: 2026
Title: A GABARAP-PtdIns3K-C1 positive feedback loop at the heart of the phagophore nucleation.
Authors: Antoine N Dessus / Yohei Ohashi / Maxime Bourguet / Tomos E Morgan / Anastasia Nunez / Maria Manifava / Nicholas T Ktistakis / Roger L Williams /
Abstract: In mammalian cells, autophagosomes can reach diameters of over 1000 nm within 30 min after triggering starvation, but how such substantial amounts of membranes can be synthesized remains elusive. ...In mammalian cells, autophagosomes can reach diameters of over 1000 nm within 30 min after triggering starvation, but how such substantial amounts of membranes can be synthesized remains elusive. The phagophore initiation needs the lipid kinase PIK3C3-Complex 1 (PtdIns3K-C1), which produces phosphatidylinositol-3-phosphate (PtdIns3P). PtdIns3P recruits WIPI2 that facilitates lipidation of mammalian ATG8 (mATG8) family proteins on phagophores. Here we show that recombinant membrane-coupled GABARAP binds to and potently activates PtdIns3K-C1. By a combination of cryo-electron microscopy, structural mass spectrometry, activity assays and mutagenesis, we show that GABARAP activates PtdIns3K-C1 through two binding sites. We propose that once GABARAP is indirectly recruited by PtdIns3P generated by basal activity of PtdIns3K-C1, a positive feedback loop is formed where PtdIns3K-C1 interacts with GABARAP and becomes activated to produce more PtdIns3P, thereby further stimulating GABARAP lipidation. This mechanism would be central for autophagosome biogenesis, where enlarged membranes need to be rapidly synthesized.
History
DepositionJan 22, 2026-
Header (metadata) releaseAug 5, 2026-
Map releaseAug 5, 2026-
UpdateSep 16, 2026-
Current statusSep 16, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_56458.png

Downloads & links

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Map

FileDownload / File: emd_56458.map.gz / Format: CCP4 / Size: 352.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 452 pix.
= 327.7 Å		0.73 Å/pix.
x 452 pix.
= 327.7 Å		0.73 Å/pix.
x 452 pix.
= 327.7 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.725 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.053
Minimum - Maximum-0.39804405 - 0.6378031
Average (Standard dev.)0.00063854933 (±0.010466664)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions452452452
Spacing452452452
CellA=B=C: 327.7 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Protein complex VPS34-CI with NRBF2 MIT and ADP:MgF3

EntireName: Protein complex VPS34-CI with NRBF2 MIT and ADP:MgF3
Components
  • Complex: Protein complex VPS34-CI with NRBF2 MIT and ADP:MgF3
    • Complex: Human VPS34-CI
      • 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: Beclin 1-associated autophagy-related key regulator
    • Complex: NRBF2 MIT domain
      • Protein or peptide: Nuclear receptor-binding factor 2
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: MYRISTIC ACID
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: ZINC ION

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Supramolecule #1: Protein complex VPS34-CI with NRBF2 MIT and ADP:MgF3

SupramoleculeName: Protein complex VPS34-CI with NRBF2 MIT and ADP:MgF3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 140.86 KDa

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Supramolecule #2: Human VPS34-CI

SupramoleculeName: Human VPS34-CI / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: NRBF2 MIT domain

SupramoleculeName: NRBF2 MIT domain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #5
Source (natural)Organism: Homo sapiens (human)

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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 / 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 #2: Phosphoinositide 3-kinase regulatory subunit 4

MacromoleculeName: Phosphoinositide 3-kinase regulatory subunit 4 / type: protein_or_peptide / ID: 2 / Details: C-terminal tag, TEV cleaved / 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 #3: Beclin-1

MacromoleculeName: Beclin-1 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / 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: Beclin 1-associated autophagy-related key regulator

MacromoleculeName: Beclin 1-associated autophagy-related key regulator / type: protein_or_peptide / ID: 4 / Details: Insertion of Threonine at position 2. / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 55.461348 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTASPSGKGA RALEAPGCGP RPLARDLVDS VDDAEGLYVA VERCPLCNTT RRRLTCAKCV QSGDFVYFDG RDRERFIDKK ERLSRLKSK QEEFQKEVLK AMEGKWITDQ LRWKIMSCKM RIEQLKQTIC KGNEEMEKNS EGLLKTKEKN QKLYSRAQRH Q EKKEKIQR ...String:
MTASPSGKGA RALEAPGCGP RPLARDLVDS VDDAEGLYVA VERCPLCNTT RRRLTCAKCV QSGDFVYFDG RDRERFIDKK ERLSRLKSK QEEFQKEVLK AMEGKWITDQ LRWKIMSCKM RIEQLKQTIC KGNEEMEKNS EGLLKTKEKN QKLYSRAQRH Q EKKEKIQR HNRKLGDLVE KKTIDLRSHY ERLANLRRSH ILELTSVIFP IEEVKTGVRD PADVSSESDS AMTSSTVSKL AE ARRTTYL SGRWVCDDHS GDTSISITGP WISLPNNGDY SAYYSWVEEK KTTQGPDMEQ SNPAYTISAA LCYATQLVNI LSH ILDVNL PKKLCNSEFC GENLSKQKFT RAVKKLNANI LYLCFSQHVN LDQLQPLHTL RNLMYLVSPS SEHLGRSGPF EVRA DLEES MEFVDPGVAG ESDESGDERV SDEETDLGTD WENLPSPRFC DIPSQSVEVS QSQSTQASPP IASSSAGGMI SSAAA SVTS WFKAYTGHR

UniProtKB: Beclin 1-associated autophagy-related key regulator

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Macromolecule #5: Nuclear receptor-binding factor 2

MacromoleculeName: Nuclear receptor-binding factor 2 / type: protein_or_peptide / ID: 5 / Details: MIT domain of NRBF2 (1-84), N-terminal tag. / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 9.71619 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
SHMEVMEVME GPLNLAHQQS RRADRLLAAG KYEEAISCHK KAAAYLSEAM KLTQSEQAHL SLELQRDSHM KQLLLIQERW KRAQ

UniProtKB: Nuclear receptor-binding factor 2

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Macromolecule #6: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: MYRISTIC ACID

MacromoleculeName: MYRISTIC ACID / type: ligand / ID: 8 / Number of copies: 1 / Formula: MYR
Molecular weightTheoretical: 228.371 Da
Chemical component information

ChemComp-MYR:
MYRISTIC ACID

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Macromolecule #9: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 9 / 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 #10: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

Concentration1.35 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
25.0 mMHEPESHEPES
100.0 mMNaClsodium chloride
1.0 mMTCEPTris-(2-Carboxyethyl)phosphine
4.0 mMCHAPSOCHAPSO
0.005 % (v/v)Nonidet-P-40Nonidet P-40
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 287.15 K / Instrument: FEI VITROBOT MARK II
Details3.6 uM VPS34-CI was mixed with 3.6 uM NRBF2 MIT, in presence of 13 mM MgCl2, 22.5 mM NaF and 2.5 mM ADP.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 4092 pixel / Digitization - Dimensions - Height: 5760 pixel / Number grids imaged: 1 / Number real images: 8483 / Average exposure time: 1.66 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 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 #1

Image processing ID1
Particle selectionNumber selected: 306134
CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: EMDB MAP
EMDB ID:

56346

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.66 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Number images used: 186859
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.6.2)

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Image processing #2

Image processing ID2
Particle selectionNumber selected: 306134
CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: EMDB MAP
EMDB ID:

56346

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Number images used: 186859
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final 3D classificationSoftware - Name: cryoSPARC (ver. 4.6.2)

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Atomic model buiding 1

Initial modelPDB ID:

9tw2


Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: GABARAP chain F was removed
RefinementSpace: REAL
Output model

PDB-9tz3:
Cryo-EM structure of human VPS34-CI with ADP:MgF3

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