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- EMDB-53422: Structure of the human RalGAP2 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-53422
TitleStructure of the human RalGAP2 complex
Map dataComposite cryo EM map of the human RalGAP2 complex
Sample
  • Complex: Homodimer of two RalGAPa2-RalGAPb heterodimers
    • Complex: RalGAP subunit alpha2
      • Protein or peptide: Ral GTPase-activating protein subunit alpha-2
    • Complex: RalGAP subunit beta
      • Protein or peptide: Ral GTPase-activating protein subunit beta
KeywordsComplex / GTPase activating protein / RAL / Asn thumb GAP / RalGAP / SIGNALING PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsRasche R / Klink BU / Gatsogiannis C / Kuemmel D
Funding support Germany, 6 items
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
CitationJournal: 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 non-canonical 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 non-canonical 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 regulator of RalGAP localization and thereby control the Ras/Ral signaling pathway.
History
DepositionApr 15, 2025-
Header (metadata) releaseJul 16, 2025-
Map releaseJul 16, 2025-
UpdateJul 16, 2025-
Current statusJul 16, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53422.png

Downloads & links

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Map

FileDownload / File: emd_53422.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite cryo EM map of the human RalGAP2 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.16 Å/pix.
x 360 pix.
= 417.6 Å		1.16 Å/pix.
x 360 pix.
= 417.6 Å		1.16 Å/pix.
x 360 pix.
= 417.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 9.130000000000001
Minimum - Maximum-27.712185000000002 - 56.165095999999998
Average (Standard dev.)0.0019600154 (±1.0653441)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 417.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Homodimer of two RalGAPa2-RalGAPb heterodimers

EntireName: Homodimer of two RalGAPa2-RalGAPb heterodimers
Components
  • Complex: Homodimer of two RalGAPa2-RalGAPb heterodimers
    • Complex: RalGAP subunit alpha2
      • Protein or peptide: Ral GTPase-activating protein subunit alpha-2
    • Complex: RalGAP subunit beta
      • Protein or peptide: Ral GTPase-activating protein subunit beta

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Supramolecule #1: Homodimer of two RalGAPa2-RalGAPb heterodimers

SupramoleculeName: Homodimer of two RalGAPa2-RalGAPb heterodimers / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Co-expression of RalGAP subunit alpha2 and beta
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 170 KDa

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Supramolecule #2: RalGAP subunit alpha2

SupramoleculeName: RalGAP subunit alpha2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: RalGAP subunit beta

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

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Macromolecule #1: Ral GTPase-activating protein subunit alpha-2

MacromoleculeName: Ral GTPase-activating protein subunit alpha-2 / type: protein_or_peptide / ID: 1 / Details: N-terminal 3xFLAG tag MDYKDHDGDYKDHDIDYKDDDDKLAAA / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 214.046469 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDYKDHDGDY KDHDIDYKDD DDKLAAAFSR RSHGDVKKST QKVLDPKKDV LTRLKHLRAL LDNVDANDLK QFFETNYSQI YFIFYENFI ALENSLKLKG NNKSQREELD SILFLFEKIL QFLPERIFFR WHYQSIGSTL KKLLHTGNSI KIRCEGIRLF L LWLQALQT ...String:
MDYKDHDGDY KDHDIDYKDD DDKLAAAFSR RSHGDVKKST QKVLDPKKDV LTRLKHLRAL LDNVDANDLK QFFETNYSQI YFIFYENFI ALENSLKLKG NNKSQREELD SILFLFEKIL QFLPERIFFR WHYQSIGSTL KKLLHTGNSI KIRCEGIRLF L LWLQALQT NCAEEQVLIF ACLVPGFPAV MSSRGPCTLE TLINPSPSVA DVKIYPEEIT PLLPAISGEK IAEDQTCFFL QI LLKYMVI QAASLEWKNK ENQDTGFKFL FTLFRKYYLP HLFPSFTKLT NIYKPVLDIP HLRPKPVYIT TTRDNENIYS TKI PYMAAR VVFIKWIVTF FLEKKYLTAT QNTKNGVDVL PKIIQTVGGG AVQERAPELD GGGPTEQDKS HSNSSTLSDR RLSN SSLCS IEEEHRMVYE MVQRILLSTR GYVNFVNEVF HQAFLLPSCE IAVTRKVVQV YRKWILQDKP VFMEEPDRKD VAQED AEKL GFSETDSKEA SSESSGHKRS SSWGRTYSFT SAMSRGCVTE EENTNVKAGV QALLQVFLTN SANIFLLEPC AEVPVL LKE QVDACKAVLI IFRRMIMELT MNKKTWEQML QILLRITEAV MQKPKDKQIK DLFAQSLAGL LFRTLMVAWI RANLCVY IS RELWDDFLGV LSSLTEWEEL INEWANIMDS LTAVLARTVY GVEMTNLPLD KLSEQKEKKQ RGKGCVLDPQ KGTTVGRS F SLSWRSHPDV TEPMRFRSAT TSGAPGVEKA RNIVRQKATE VEECQQSENA PAAGSGHLTV GQQQQVLRSS STSDIPEPL CSDSSQGQKA ENTQNSSSSE PQPIQENKGH VKREHEGITI LVRRSSSPAE LDLKDDLQQT QGKCRERQKS ESTNSDTTLG CTNEAELSM GPWQTCEEDP ELNTPTDVVA DADARHWLQL SPTDASNLTD SSECLTDDCS IIAGGSLTGW HPDSAAVLWR R VLGILGDV NNIQSPKIHA RVFCYLYELW YKLAKIRDNL AISLDNQSSP SPPVLIPPLR MFASWLFKAA TLPNEYKEGK LQ AYRLICA MMTRRQDVLP NSDFLVHFYL VMHLGLTSED QDILNTIIRH CPPRFFSLGF PGFSMLVGDF ITAAARVLST DIL TAPRSE AVTVLGSLVC FPNTYQEIPL LQSVPEVNEA ITGTEDVKHY LINILLKNAT EEPNEYARCI AVCSLGVWIC EELA QCTSH PQVKEAINVI GVTLKFPNKI VAQVACDVLQ LLVSYWEKLQ MFETSLPRKM AEILVATVAF LLPSAEYSSV ETDKK FIVS LLLCLLDWCM ALPVSVLLHP VSTAVLEEQH SARAPLLDYI YRVLHCCVCG SSTYTQQSHY ILTLADLSST DYDPFL PLA NVKSSEPVQY HSSAELGNLL TVEEEKKRRS LELIPLTARM VMAHLVNHLG HYPLSGGPAI LHSLVSENHD NAHVEGS EL SFEVFRSPNL QLFVFNDSTL ISYLQTPTEG PVGGSPVGSL SDVRVIVRDI SGKYSWDGKV LYGPLEGCLA PNGRNPSF L ISSWHRDTFG PQKDSSQVEE GDDVLDKLLE NIGHTSPECL LPSQLNLNEP SLTPCGMNYD QEKEIIEVIL RQNAQEDEY IQSHNFDSAM KVTSQGQPSP VEPRGPFYFC RLLLDDLGMN SWDRRKNFHL LKKNSKLLRE LKNLDSRQCR ETHKIAVFYI AEGQEDKCS ILSNERGSQA YEDFVAGLGW EVDLSTHCGF MGGLQRNGST GQTAPYYATS TVEVIFHVST RMPSDSDDSL T KKLRHLGN DEVHIVWSEH SRDYRRGIIP TAFGDVSIII YPMKNHMFFI AITKKPEVPF FGPLFDGAIV SGKLLPSLVC AT CINASRA VKCLIPLYQS FYEERALYLE AIIQNHREVM TFEDFAAQVF SPSPSYSLSG TD

UniProtKB: Ral GTPase-activating protein subunit alpha-2

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Macromolecule #2: Ral GTPase-activating protein subunit beta

MacromoleculeName: Ral GTPase-activating protein subunit beta / type: protein_or_peptide / ID: 2
Details: N-terminal 3xHA tag MYPYDVPDYAGSYPYDVPDYAGSYPYDVPDYAGS
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 170.796406 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MYPYDVPDYA GSYPYDVPDY AGSYPYDVPD YAGSMYSEWR SLHLVIQNDQ GHTSVLHSYP ESVGREVANA VVRPLGQVLG TPSVAGSEN LLKTDKEVKW TMEVICYGLT LPLDGETVKY CVDVYTDWIM ALVLPKDSIP LPVIKEPNQY VQTILKHLQN L FVPRQEQG ...String:
MYPYDVPDYA GSYPYDVPDY AGSYPYDVPD YAGSMYSEWR SLHLVIQNDQ GHTSVLHSYP ESVGREVANA VVRPLGQVLG TPSVAGSEN LLKTDKEVKW TMEVICYGLT LPLDGETVKY CVDVYTDWIM ALVLPKDSIP LPVIKEPNQY VQTILKHLQN L FVPRQEQG SSQIRLCLQV LRAIQKLARE SSLMARETWE VLLLFLLQIN DILLAPPTVQ GGIAENLAEK LIGVLFEVWL LA CTRCFPT PPYWKTAKEM VANWRHHPAV VEQWSKVICA LTSRLLRFTY GPSFPAFKVP DEDASLIPPE MDNECVAQTW FRF LHMLSN PVDLSNPAII SSTPKFQEQF LNVSGMPQEL NQYPCLKHLP QIFFRAMRGI SCLVDAFLGI SRPRSDSAPP TPVN RLSMP QSAAVSTTPP HNRRHRAVTV NKATMKTSTV STAHASKVQH QTSSTSPLSS PNQTSSEPRP LPAPRRPKVN SILNL FGSW LFDAAFVHCK LHNGINRDSS MTAITTQASM EFRRKGSQMS TDTMVSNPMF DASEFPDNYE AGRAEACGTL CRIFCS KKT GEEILPAYLS RFYMLLIQGL QINDYVCHPV LASVILNSPP LFCCDLKGID VVVPYFISAL ETILPDRELS KFKSYVN PT ELRRSSINIL LSLLPLPHHF GTVKSEVVLE GKFSNDDSSS YDKPITFLSL KLRLVNILIG ALQTETDPNN TQMILGAM L NIVQDSALLE AIGCQMEMGG GENNLKSHSR TNSGISSASG GSTEPTTPDS ERPAQALLRD YALNTDSAAG LLIRSIHLV TQRLNSQWRQ DMSISLAALE LLSGLAKVKV MVDSGDRKRA ISSVCTYIVY QCSRPAPLHS RDLHSMIVAA FQCLCVWLTE HPDMLDEKD CLKEVLEIVE LGISGSKSKN NEQEVKYKGD KEPNPASMRV KDAAEATLTC IMQLLGAFPS PSGPASPCSL V NETTLIKY SRLPTINKHS FRYFVLDNSV ILAMLEQPLG NEQNDFFPSV TVLVRGMSGR LAWAQQLCLL PRGAKANQKL FV PEPRPVP KNDVGFKYSV KHRPFPEEVD KIPFVKADLS IPDLHEIVTE ELEERHEKLR SGMAQQIAYE IHLEQQSEEE LQK RSFPDP VTDCKPPPPA QEFQTARLFL SHFGFLSLEA LKEPANSRLP PHLIALDSTI PGFFDDIGYL DLLPCRPFDT VFIF YMKPG QKTNQEILKN VESSRTVQPH FLEFLLSLGW SVDVGRHPGW TGHVSTSWSI NCCDDGEGSQ QEVISSEDIG ASIFN GQKK VLYYADALTE IAFVVPSPVE SLTDSLESNI SDQDSDSNMD LMPGILKQPS LTLELFPNHT DNLNSSQRLS PSSRMR KLP QGRPVPPLGP ETRVSVVWVE RYDDIENFPL SELMTEISTG VETTANSSTS LRSTTLEKEV PVIFIHPLNT GLFRIKI QG ATGKFNMVIP LVDGMIVSRR ALGFLVRQTV INICRRKRLE SDSYSPPHVR RKQKITDIVN KYRNKQLEPE FYTSLFQE V GLKNCSS

UniProtKB: Ral GTPase-activating protein subunit beta

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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

Concentration0.7 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mmol/lC8H18N2O4SHEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
150.0 mmol/lNaClsodium chloride
2.0 mmol/lMgCl2magnesium chloride
1.0 mmol/lC9H15O6PTCEP (tris(2-carboxyethyl)phosphine)
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.01 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV / Details: double blotting.
DetailsCo-expression of RalGAP alpha2 and beta Monodisperse sample from size exclusion chromatography

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 4 / Number real images: 64606 / Average exposure time: 3.2 sec. / Average electron dose: 60.0 e/Å2
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.
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 / Cs: 2.7 mm / Nominal defocus max: 4.2 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 215000
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: 5519589
CTF correctionSoftware - Name: RELION (ver. 5) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
In silico model: A model of the RGalpha2/RGbeta complex was calculated using AlphaFold2
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5) / Number images used: 420975
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5)
Final 3D classificationNumber classes: 4 / Avg.num./class: 221930 / Software - Name: RELION (ver. 5)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsA 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.
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 129.72 / Target criteria: cross correlation
Output model

PDB-9qwp:
Structure of the human RalGAP2 complex

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