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- EMDB-43655: Cryo-EM structure of human core Rab3GAP1/2 complex, local refinement -

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Basic information

Entry
Database: EMDB / ID: EMD-43655
TitleCryo-EM structure of human core Rab3GAP1/2 complex, local refinement
Map dataLocal refinement from CryoSPARC, z-flipped
Sample
  • Complex: Core Rab3GAP1/2 complex
    • Complex: Rab3GAP1
      • Protein or peptide: Isoform 2 of Rab3 GTPase-activating protein catalytic subunit
    • Complex: Rab3GAP2
      • Protein or peptide: Rab3 GTPase-activating protein non-catalytic subunit
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 / positive regulation of protein lipidation / synaptic signaling / endoplasmic reticulum tubular network / 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 / positive regulation of protein lipidation / synaptic signaling / endoplasmic reticulum tubular network / 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 / positive regulation of GTPase activity / enzyme regulator activity / lipid droplet / autophagosome / GTPase activator activity / guanyl-nucleotide exchange factor activity / excitatory postsynaptic potential / macroautophagy / intracellular protein transport / enzyme activator activity / 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: 3.37 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 7, 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_43655.png

Downloads & links

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Map

FileDownload / File: emd_43655.map.gz / Format: CCP4 / Size: 1000 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refinement from CryoSPARC, z-flipped
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: 0.25
Minimum - Maximum-1.1981106 - 1.5430683
Average (Standard dev.)0.00035670164 (±0.018636553)
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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Additional map: Auto-sharpened map from Phenix

Fileemd_43655_additional_1.map
AnnotationAuto-sharpened map from Phenix
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A, z-flipped

Fileemd_43655_half_map_1.map
AnnotationHalf map A, z-flipped
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B, z-flipped

Fileemd_43655_half_map_2.map
AnnotationHalf map B, z-flipped
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: Isoform 2 of Rab3 GTPase-activating protein catalytic subunit
    • Complex: Rab3GAP2
      • Protein or peptide: Rab3 GTPase-activating protein non-catalytic subunit

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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: Isoform 2 of Rab3 GTPase-activating protein catalytic subunit

MacromoleculeName: Isoform 2 of Rab3 GTPase-activating protein catalytic subunit
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 116.747188 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MWSHPQFEKG GGSGGGSGGG SWSHPQFEKL EVLFQGPRSE ARGIQRPTST MAADSEPESE VFEITDFTTA SEWERFISKV EEVLNDWKL IGNSLGKPLE KGIFTSGTWE EKSDEISFAD FKFSVTHHYL VQESTDKEGK DELLEDVVPQ SMQDLLGMNN D FPPRAHCL ...String:
MWSHPQFEKG GGSGGGSGGG SWSHPQFEKL EVLFQGPRSE ARGIQRPTST MAADSEPESE VFEITDFTTA SEWERFISKV EEVLNDWKL IGNSLGKPLE KGIFTSGTWE EKSDEISFAD FKFSVTHHYL VQESTDKEGK DELLEDVVPQ SMQDLLGMNN D FPPRAHCL VRWYGLREFV VIAPAAHSDA VLSESKCNLL LSSVSIALGN TGCQVPLFVQ IHHKWRRMYV GECQGPGVRT DF EMVHLRK VPNQYTHLSG LLDIFKSKIG CPLTPLPPVS IAIRFTYVLQ DWQQYFWPQQ PPDIDALVGG EVGGLEFGKL PFG ACEDPI SELHLATTWP HLTEGIIVDN DVYSDLDPIQ APHWSVRVRK AENPQCLLGD FVTEFFKICR RKESTDEILG RSAF EEEGK ETADITHALS KLTEPASVPI HKLSVSNMVH TAKKKIRKHR GVEESPLNND VLNTILLFLF PDAVSEKPLD GTTST DNNN PPSESEDYNL YNQFKSAPSD SLTYKLALCL CMINFYHGGL KGVAHLWQEF VLEMRFRWEN NFLIPGLASG PPDLRC CLL HQKLQMLNCC IERKKARDEG KKTSASDVTN IYPGDAGKAG DQLVPDNLKE TDKEKGEVGK SWDSWSDSEE EFFECLS DT EELKGNGQES GKKGGPKEMA NLRPEGRLYQ HGKLTLLHNG EPLYIPVTQE PAPMTEDLLE EQSEVLAKLG TSAEGAHL R ARMQSACLLS DMESFKAANP GCSLEDFVRW YSPRDYIEEE VIDEKGNVVL KGELSARMKI PSNMWVEAWE TAKPIPARR QRRLFDDTRE AEKVLHYLAI QKPADLARHL LPCVIHAAVL KVKEEESLEN ISSVKKIIKQ IISHSSKVLH FPNPEDKKLE EIIHQITNV EALIARARSL KAKFGTEKCE QEEEKEDLER FVSCLLEQPE VLVTGAGRGH AGRIIHKLFV NAQRLTESSD E AAAMTPPE EELKRMGSPE ERRQNSVSDF PPPAGREFIL RTTVPRPAPY SKALPQRMYS VLTKEDFRLA GAFSSDTSFF

UniProtKB: Rab3 GTPase-activating protein catalytic subunit

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Macromolecule #2: Rab3 GTPase-activating protein non-catalytic subunit

MacromoleculeName: Rab3 GTPase-activating protein non-catalytic subunit / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 65.121422 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MACSIVQFCY FQDLQAARDF LFPHLREEIL SGALRRDPSK STDWEDDGWG AWEENEPQEP EEEGNTCKTQ KTSWLQDCVL SLSPTNDLM VIAREQKAVF LVPKWKYSDK GKEEMQFAVG WSGSLNVEEG ECVTSALCIP LASQKRSSTG RPDWTCIVVG F TSGYVRFY ...String:
MACSIVQFCY FQDLQAARDF LFPHLREEIL SGALRRDPSK STDWEDDGWG AWEENEPQEP EEEGNTCKTQ KTSWLQDCVL SLSPTNDLM VIAREQKAVF LVPKWKYSDK GKEEMQFAVG WSGSLNVEEG ECVTSALCIP LASQKRSSTG RPDWTCIVVG F TSGYVRFY TENGVLLLAQ LLNEDPVLQL KCRTYEIPRH PGVTEQNEEL SILYPAAIVT IDGFSLFQSL RACRNQVAKA AA SGNENIQ PPPLAYKKWG LQDIDTIIDH ASVGIMTLSP FDQMKTASNI GGFNAAIKNS PPAMSQYITV GSNPFTGFFY ALE GSTQPL LSHVALAVAS KLTSALFNAA SGWLGWKSKH EEEAVQKQKP KVEPATPLAV RFGLPDSRRH GESICLSPCN TLAA VTDDF GRVILLDVAR GIAIRMWKGY RDAQIGWIQT VEDLHERVPE KADFSPFGNS QGPSRVAQFL VIYAPRRGIL EVWST QQGP RVGAFNVGKH CRLLYPGYKI MGLNNVTSQS WQPQTYQICL VDPVSGSVKT VNVPFHLALS DKKLREQKLE LGGSGG RQL DYKDHDGDYK DHDIDYKDDD DK

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: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / 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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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-8vyb:
Cryo-EM structure of human core Rab3GAP1/2 complex

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