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- EMDB-43645: Cryo-EM structure of human core Rab3GAP1/2 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-43645
TitleCryo-EM structure of human core Rab3GAP1/2 complex
Map data
Sample
  • Complex: Core Rab3GAP1/2 complex
    • Complex: Rab3GAP1
      • Protein or peptide: Rab3GAP1
    • Complex: Rab3GAP2
      • Protein or peptide: Rab3GAP2
KeywordsComplex / GAP / GEF / Rab3GAP / Lipid droplet / ER / Rab regulator / membrane trafficking / CYTOSOLIC PROTEIN
Function / homology
Function and homology information


positive regulation of glutamate neurotransmitter secretion in response to membrane depolarization / positive regulation of endoplasmic reticulum tubular network organization / establishment of protein localization to endoplasmic reticulum membrane / regulation of calcium ion-dependent exocytosis of neurotransmitter / synaptic signaling / endoplasmic reticulum tubular network / positive regulation of protein lipidation / positive regulation of autophagosome assembly / lipid droplet organization / RAB GEFs exchange GTP for GDP on RABs ...positive regulation of glutamate neurotransmitter secretion in response to membrane depolarization / positive regulation of endoplasmic reticulum tubular network organization / establishment of protein localization to endoplasmic reticulum membrane / regulation of calcium ion-dependent exocytosis of neurotransmitter / synaptic signaling / endoplasmic reticulum tubular network / positive regulation of protein lipidation / positive regulation of autophagosome assembly / lipid droplet organization / RAB GEFs exchange GTP for GDP on RABs / cis-Golgi network / camera-type eye development / regulation of short-term neuronal synaptic plasticity / COPI-independent Golgi-to-ER retrograde traffic / face morphogenesis / regulation of GTPase activity / hypothalamus development / enzyme regulator activity / lipid droplet / positive regulation of GTPase activity / GTPase activator activity / autophagosome / enzyme activator activity / guanyl-nucleotide exchange factor activity / excitatory postsynaptic potential / intracellular protein transport / macroautophagy / brain development / small GTPase binding / presynaptic membrane / postsynapse / endoplasmic reticulum membrane / Golgi apparatus / protein-containing complex / extracellular exosome / plasma membrane / cytosol
Similarity search - Function
Rab3GAP regulatory subunit / Rab3GAP catalytic subunit, conserved domain / Rab3GAP regulatory subunit, C-terminal / Rab3-GAP regulatory subunit, N-terminal / Rab3GAP catalytic subunit, C-terminal / Rab3GAP catalytic subunit / Rab3 GTPase-activating protein catalytic subunit / Rab3 GTPase-activating protein regulatory subunit N-terminus / Rab3 GTPase-activating protein regulatory subunit C-terminus / Rab3 GTPase-activating protein catalytic subunit C-terminal
Similarity search - Domain/homology
Rab3 GTPase-activating protein catalytic subunit / Rab3 GTPase-activating protein non-catalytic subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsNguyen KM / Yip CK
Funding support Canada, 2 items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-168907 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2018-03951 Canada
CitationJournal: Nat Commun / Year: 2025
Title: Biochemical and structural characterization of Rab3GAP reveals insights into Rab18 nucleotide exchange activity.
Authors: Gage M J Fairlie / Kha M Nguyen / Sung-Eun Nam / Alexandria L Shaw / Matthew A H Parson / Hannah R Shariati / Xinyin Wang / Meredith L Jenkins / Michael Gong / John E Burke / Calvin K Yip /
Abstract: The heterodimeric Rab3GAP complex is a guanine nucleotide exchange factor (GEF) for the Rab18 GTPase that regulates lipid droplet metabolism, ER-to-Golgi trafficking, secretion, and autophagy. Why ...The heterodimeric Rab3GAP complex is a guanine nucleotide exchange factor (GEF) for the Rab18 GTPase that regulates lipid droplet metabolism, ER-to-Golgi trafficking, secretion, and autophagy. Why both subunits of Rab3GAP are required for Rab18 GEF activity and the molecular basis of how Rab3GAP engages and activates its cognate substrate are unknown. Here we show that human Rab3GAP is conformationally flexible and potentially autoinhibited by the C-terminal domain of its Rab3GAP2 subunit. Our high-resolution structure of the catalytic core of Rab3GAP, determined by cryo-EM, shows that the Rab3GAP2 N-terminal domain binds Rab3GAP1 via an extensive interface. AlphaFold3 modelling analysis together with targeted mutagenesis and in vitro activity assay reveal that Rab3GAP likely engages its substrate Rab18 through an interface away from the switch and interswitch regions. Lastly, we find that three Warburg Micro Syndrome-associated missense mutations do not affect the overall architecture of Rab3GAP but instead likely interfere with substrate binding.
History
DepositionFeb 6, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43645.png

Downloads & links

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Map

FileDownload / File: emd_43645.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.59 Å/pix.
x 640 pix.
= 377.6 Å		0.59 Å/pix.
x 640 pix.
= 377.6 Å		0.59 Å/pix.
x 640 pix.
= 377.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.59 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.62163204 - 1.2874148
Average (Standard dev.)0.00043749632 (±0.016117629)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions640640640
Spacing640640640
CellA=B=C: 377.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43645_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43645_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Core Rab3GAP1/2 complex

EntireName: Core Rab3GAP1/2 complex
Components
  • Complex: Core Rab3GAP1/2 complex
    • Complex: Rab3GAP1
      • Protein or peptide: Rab3GAP1
    • Complex: Rab3GAP2
      • Protein or peptide: Rab3GAP2

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Supramolecule #1: Core Rab3GAP1/2 complex

SupramoleculeName: Core Rab3GAP1/2 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Rab3GAP1

SupramoleculeName: Rab3GAP1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Rab3GAP2

SupramoleculeName: Rab3GAP2 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Rab3GAP1

MacromoleculeName: Rab3GAP1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MWSHPQFEKG GGSGGGSGGG SWSHPQFEKL EVLFQGPRSE ARGIQRPTST MAADSEPESE VFEITDFTTA SEWERFISKV EEVLNDWKLI GNSLGKPLEK GIFTSGTWEE KSDEISFADF KFSVTHHYLV QESTDKEGKD ELLEDVVPQS MQDLLGMNND FPPRAHCLVR ...String:
MWSHPQFEKG GGSGGGSGGG SWSHPQFEKL EVLFQGPRSE ARGIQRPTST MAADSEPESE VFEITDFTTA SEWERFISKV EEVLNDWKLI GNSLGKPLEK GIFTSGTWEE KSDEISFADF KFSVTHHYLV QESTDKEGKD ELLEDVVPQS MQDLLGMNND FPPRAHCLVR WYGLREFVVI APAAHSDAVL SESKCNLLLS SVSIALGNTG CQVPLFVQIH HKWRRMYVGE CQGPGVRTDF EMVHLRKVPN QYTHLSGLLD IFKSKIGCPL TPLPPVSIAI RFTYVLQDWQ QYFWPQQPPD IDALVGGEVG GLEFGKLPFG ACEDPISELH LATTWPHLTE GIIVDNDVYS DLDPIQAPHW SVRVRKAENP QCLLGDFVTE FFKICRRKES TDEILGRSAF EEEGKETADI THALSKLTEP ASVPIHKLSV SNMVHTAKKK IRKHRGVEES PLNNDVLNTI LLFLFPDAVS EKPLDGTTST DNNNPPSESE DYNLYNQFKS APSDSLTYKL ALCLCMINFY HGGLKGVAHL WQEFVLEMRF RWENNFLIPG LASGPPDLRC CLLHQKLQML NCCIERKKAR DEGKKTSASD VTNIYPGDAG KAGDQLVPDN LKETDKEKGE VGKSWDSWSD SEEEFFECLS DTEELKGNGQ ESGKKGGPKE MANLRPEGRL YQHGKLTLLH NGEPLYIPVT QEPAPMTEDL LEEQSEVLAK LGTSAEGAHL RARMQSACLL SDMESFKAAN PGCSLEDFVR WYSPRDYIEE EVIDEKGNVV LKGELSARMK IPSNMWVEAW ETAKPIPARR QRRLFDDTRE AEKVLHYLAI QKPADLARHL LPCVIHAAVL KVKEEESLEN ISSVKKIIKQ IISHSSKVLH FPNPEDKKLE EIIHQITNVE ALIARARSLK AKFGTEKCEQ EEEKEDLERF VSCLLEQPEV LVTGAGRGHA GRIIHKLFVN AQRLTESSDE AAAMTPPEEE LKRMGSPEER RQNSVSDFPP PAGREFILRT TVPRPAPYSK ALPQRMYSVL TKEDFRLAGA FSSDTSFF

UniProtKB: Rab3 GTPase-activating protein catalytic subunit

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Macromolecule #2: Rab3GAP2

MacromoleculeName: Rab3GAP2 / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MACSIVQFCY FQDLQAARDF LFPHLREEIL SGALRRDPSK STDWEDDGWG AWEENEPQEP EEEGNTCKTQ KTSWLQDCVL SLSPTNDLMV IAREQKAVFL VPKWKYSDKG KEEMQFAVGW SGSLNVEEGE CVTSALCIPL ASQKRSSTGR PDWTCIVVGF TSGYVRFYTE ...String:
MACSIVQFCY FQDLQAARDF LFPHLREEIL SGALRRDPSK STDWEDDGWG AWEENEPQEP EEEGNTCKTQ KTSWLQDCVL SLSPTNDLMV IAREQKAVFL VPKWKYSDKG KEEMQFAVGW SGSLNVEEGE CVTSALCIPL ASQKRSSTGR PDWTCIVVGF TSGYVRFYTE NGVLLLAQLL NEDPVLQLKC RTYEIPRHPG VTEQNEELSI LYPAAIVTID GFSLFQSLRA CRNQVAKAAA SGNENIQPPP LAYKKWGLQD IDTIIDHASV GIMTLSPFDQ MKTASNIGGF NAAIKNSPPA MSQYITVGSN PFTGFFYALE GSTQPLLSHV ALAVASKLTS ALFNAASGWL GWKSKHEEEA VQKQKPKVEP ATPLAVRFGL PDSRRHGESI CLSPCNTLAA VTDDFGRVIL LDVARGIAIR MWKGYRDAQI GWIQTVEDLH ERVPEKADFS PFGNSQGPSR VAQFLVIYAP RRGILEVWST QQGPRVGAFN VGKHCRLLYP GYKIMGLNNV TSQSWQPQTY QICLVDPVSG SVKTVNVPFH LALSDKKLRE QKLELGGSGG RQLDYKDHDG DYKDHDIDYK DDDDK

UniProtKB: Rab3 GTPase-activating protein non-catalytic subunit

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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

BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloride
1.0 mMC9H15O6PTCEP
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 2s blot time, 5s blot force.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 215000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 12427326
Startup modelType of model: NONE / Details: CryoSPARC ab initio reconstruction
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 170636
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final 3D classificationNumber classes: 1 / Software - Name: cryoSPARC (ver. 4.2.1)
FSC plot (resolution estimation)

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