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- PDB-9qwp: Structure of the human RalGAP2 complex -

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Basic information

Entry
Database: PDB / ID: 9qwp
TitleStructure of the human RalGAP2 complex
Components
  • Ral GTPase-activating protein subunit alpha-2
  • Ral GTPase-activating protein subunit beta
KeywordsSIGNALING PROTEIN / Complex / GTPase activating protein / RAL / Asn thumb GAP / RalGAP
Function / homology
Function and homology information


Ral protein signal transduction / regulation of exocyst localization / activation of GTPase activity / regulation of small GTPase mediated signal transduction / GTPase activator activity / Translocation of SLC2A4 (GLUT4) to the plasma membrane / regulation of protein localization / protein heterodimerization activity / extracellular space / nucleus ...Ral protein signal transduction / regulation of exocyst localization / activation of GTPase activity / regulation of small GTPase mediated signal transduction / GTPase activator activity / Translocation of SLC2A4 (GLUT4) to the plasma membrane / regulation of protein localization / protein heterodimerization activity / extracellular space / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Ral GTPase-activating protein subunit beta / Ral GTPase-activating protein subunit alpha/beta, N-terminal domain / RALGAPB N-terminal domain / Tuberin/Ral GTPase-activating protein subunit alpha / Rap/Ran-GAP domain / Rap/Ran-GAP superfamily / Rap/ran-GAP / Rap GTPase activating proteins domain profile. / Armadillo-type fold
Similarity search - Domain/homology
Ral GTPase-activating protein subunit alpha-2 / Ral GTPase-activating protein subunit beta
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsRasche, R. / Klink, B.U. / Gatsogiannis, C. / Kuemmel, D.
Funding support Germany, 6items
OrganizationGrant numberCountry
German Research Foundation (DFG)KU2531/2 Germany
German Research Foundation (DFG)KU2531/6 Germany
German Research Foundation (DFG)496113311 Germany
German Research Foundation (DFG)CRC 1348 A15 Germany
German Research Foundation (DFG)CRC1430 A04 Germany
German Research Foundation (DFG)INST 211/667-1 Germany
Citation
Journal: Nat Commun / Year: 2025
Title: Structure and mechanism of the RalGAP tumor suppressor complex.
Authors: René Rasche / Björn Udo Klink / Lisa Helene Apken / Esther Michalke / Minghao Chen / Andrea Oeckinghaus / Christos Gatsogiannis / Daniel Kümmel /
Abstract: The RalGAP (GTPase activating protein) complexes are negative regulators of the Ral GTPases and thus crucial components that counteract oncogenic Ras signaling. However, no structural information on ...The RalGAP (GTPase activating protein) complexes are negative regulators of the Ral GTPases and thus crucial components that counteract oncogenic Ras signaling. However, no structural information on the architecture of this tumor suppressor complex is available hampering a mechanistic understanding of its functionality. Here, we present a cryo-EM structure of RalGAP that reveals an extended 58 nm tetrameric architecture comprising two heterodimers of the RalGAPα and RalGAPβ subunits. We show that the catalytic domain of RalGAPα requires stabilization by a unique domain of RalGAPβ, providing the molecular basis for why RalGAP complexes are obligatory heterodimers. Formation of RalGAP tetramers is not required for activity in vitro, but essential for function of the complex in vivo. Structural analysis of RalGAP subunit variants reported in cancer patients suggests effects on complex formation and thus functional relevance, emphasizing the significance of the obtained structural information for medical research.
#1: Journal: J Biol Chem / Year: 2025
Title: The GTPase κB-Ras is an essential subunit of the RalGAP tumor suppressor complex.
Authors: René Rasche / Lisa Helene Apken / Sonja Titze / Esther Michalke / Rohit Kumar Singh / Andrea Oeckinghaus / Daniel Kümmel /
Abstract: κB-Ras1 and κB-Ras2 are small GTPases with noncanonical features that act as tumor suppressors downstream of Ras. Via interaction with the RalGAP (GTPase-activating protein) complex, they limit ...κB-Ras1 and κB-Ras2 are small GTPases with noncanonical features that act as tumor suppressors downstream of Ras. Via interaction with the RalGAP (GTPase-activating protein) complex, they limit activity of Ral GTPases and restrict anchorage-independent proliferation. We here present the crystal structure of κB-Ras1 in complex with the N-terminal domain of RGα2. The structure suggests a mechanism of intrinsic GTP hydrolysis of κB-Ras1 that relies on a scaffolding function of the GTPase rather than on catalytic residues, which we confirm by mutational analysis. The interaction with RGα2 is nucleotide independent and does not involve κB-Ras1 switch regions, which establishes κB-Ras proteins as a constitutive third subunit of RalGAP complexes. Functional studies demonstrate that κB-Ras proteins are not required for RalGAP catalytic activity in vitro but for functionality in vivo. We propose that κB-Ras may thus act as a regulator of RalGAP localization and thereby control the Ras-Ral signaling pathway.
History
DepositionApr 15, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 16, 2025Provider: repository / Type: Initial release
Revision 1.1Aug 6, 2025Group: Data collection / Database references / Category: citation / em_admin / Item: _citation.journal_volume / _em_admin.last_update
Revision 1.2Aug 13, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ral GTPase-activating protein subunit alpha-2
B: Ral GTPase-activating protein subunit beta
C: Ral GTPase-activating protein subunit beta
D: Ral GTPase-activating protein subunit alpha-2


Theoretical massNumber of molelcules
Total (without water)769,6864
Polymers769,6864
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Ral GTPase-activating protein subunit alpha-2 / 250 kDa substrate of Akt / AS250 / p220


Mass: 214046.469 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal 3xFLAG tag MDYKDHDGDYKDHDIDYKDDDDKLAAA / Source: (gene. exp.) Homo sapiens (human) / Gene: RALGAPA2, C20orf74, KIAA1272 / Plasmid: pCMV7.1_3xflag_p220 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q2PPJ7
#2: Protein Ral GTPase-activating protein subunit beta / p170


Mass: 170796.406 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal 3xHA tag MYPYDVPDYAGSYPYDVPDYAGSYPYDVPDYAGS
Source: (gene. exp.) Homo sapiens (human) / Gene: RALGAPB, KIAA1219 / Plasmid: pKH3-HA_p170 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q86X10
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Homodimer of two RalGAPa2-RalGAPb heterodimersCOMPLEXCo-expression of RalGAP subunit alpha2 and betaall0RECOMBINANT
2RalGAP subunit alpha2COMPLEX#11RECOMBINANT
3RalGAP subunit betaCOMPLEX#21RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.79 MDaYES
210.22 MDaNO
310.17 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens (human)9606
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
11Homo sapiens (human)9606Expi293F
22Homo sapiens (human)9606Expi293FpCMV7.1-3xFLAG_p220
33Homo sapiens (human)9606Expi293FpKH3-HA_p170
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mmol/lHEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)C8H18N2O4S1
2150 mmol/lsodium chlorideNaCl1
32 mmol/lmagnesium chlorideMgCl21
41 mmol/lTCEP (tris(2-carboxyethyl)phosphine)C9H15O6P1
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Co-expression of RalGAP alpha2 and beta Monodisperse sample from size exclusion chromatography
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 286 K / Details: double blotting

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 215000 X / Nominal defocus max: 4200 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.2 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 64606
Details: Images were collected in EER mode with 793-819 fractions were generated for motion correction. Micrographs were collected at 0, 10 and 30 degree tilt, in 5 subsets of data.
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategoryDetails
1crYOLO1.8.3particle selectionCryolo was used to automatically pick particles using multiple rounds of optimization of the training model.
2EPU3.2.0.4776image acquisitionEPU was used to automatically collect raw movies in EER format
4RELION5CTF correction
7UCSF ChimeraX1.7.dev2023model fitting
9RELION5initial Euler assignment
10RELION5final Euler assignment
11RELION5classification
12RELION53D reconstruction
13PHENIX1.21.1_5286model refinement
14Coot0.9.8.95model refinement
15ISOLDE1.7model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5519589
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 420975 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 129.72 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: cross correlation
Details: A model of the RGalpha2/RGbeta complex was calculated using AlphaFold2 running on a local high performance computing cluster (PALMA II), docked into map from 3D reconstruction with ChimeraX ...Details: A model of the RGalpha2/RGbeta complex was calculated using AlphaFold2 running on a local high performance computing cluster (PALMA II), docked into map from 3D reconstruction with ChimeraX and energy optimized with ISOLDE. In an iterative process, the model was optimized using real space refinement in Phenix Refine against the full density map, and manual model building with Coot using the full density map as well as the resampled multi body refinement maps.
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 129.72 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002327621
ELECTRON MICROSCOPYf_angle_d0.462537523
ELECTRON MICROSCOPYf_chiral_restr0.03824308
ELECTRON MICROSCOPYf_plane_restr0.00374776
ELECTRON MICROSCOPYf_dihedral_angle_d3.42393625

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