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- EMDB-53420: Focussed cryo EM map from multibody refinement of human RalGAP2 c... -

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Basic information

Entry
Database: EMDB / ID: EMD-53420
TitleFocussed cryo EM map from multibody refinement of human RalGAP2 complex (body2: alpha2 N-terminus)
Map dataFocussed cryo EM map from multibody refinement of RalGAP2 complex (body2: alpha2 N-terminus)
Sample
  • Complex: Homodimer of two RalGAPa2-RalGAPb heterodimers
    • Complex: RalGAP subunit alpha2
      • Protein or peptide: RalGAP subunit alpha 2
    • Complex: RalGAP subunit beta
      • Protein or peptide: RalGAP 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: 3.93 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 1430 A04 Germany
German Research Foundation (DFG)CRC 1348 A15 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_53420.png

Downloads & links

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Map

FileDownload / File: emd_53420.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocussed cryo EM map from multibody refinement of RalGAP2 complex (body2: alpha2 N-terminus)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (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: 0.00718
Minimum - Maximum-0.0072071506 - 0.018840713
Average (Standard dev.)0.00000030903703 (±0.00039991646)
SymmetrySpace group: 1
Details

EMDB XML:

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

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Supplemental data

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Mask #1

Fileemd_53420_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened postprocessed focussed cryo EM map from multibody...

Fileemd_53420_additional_1.map
AnnotationSharpened postprocessed focussed cryo EM map from multibody refinement of RalGAP2 complex (body2: alpha2 N-terminus)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 focussed cryo EM map from...

Fileemd_53420_half_map_1.map
AnnotationHalf map 1 focussed cryo EM map from multibody refinement of RalGAP2 complex (body2: alpha2 N-terminus)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 focussed cryo EM map from...

Fileemd_53420_half_map_2.map
AnnotationHalf map 2 focussed cryo EM map from multibody refinement of RalGAP2 complex (body2: alpha2 N-terminus)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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: RalGAP subunit alpha 2
    • Complex: RalGAP subunit beta
      • Protein or peptide: RalGAP 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: RalGAP subunit alpha 2

MacromoleculeName: RalGAP subunit alpha 2 / type: protein_or_peptide / ID: 1 / Details: N-terminal 3xFLAG tag MDYKDHDGDYKDHDIDYKDDDDKLAAA / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDYKDHDGDY KDHDIDYKDD DDKLAAAFSR RSHGDVKKST QKVLDPKKDV LTRLKHLRAL LDNVDANDLK QFFETNYSQI YFIFYENFIA LENSLKLKGN NKSQREELDS ILFLFEKILQ FLPERIFFRW HYQSIGSTLK KLLHTGNSIK IRCEGIRLFL LWLQALQTNC ...String:
MDYKDHDGDY KDHDIDYKDD DDKLAAAFSR RSHGDVKKST QKVLDPKKDV LTRLKHLRAL LDNVDANDLK QFFETNYSQI YFIFYENFIA LENSLKLKGN NKSQREELDS ILFLFEKILQ FLPERIFFRW HYQSIGSTLK KLLHTGNSIK IRCEGIRLFL LWLQALQTNC AEEQVLIFAC LVPGFPAVMS SRGPCTLETL INPSPSVADV KIYPEEITPL LPAISGEKIA EDQTCFFLQI LLKYMVIQAA SLEWKNKENQ DTGFKFLFTL FRKYYLPHLF PSFTKLTNIY KPVLDIPHLR PKPVYITTTR DNENIYSTKI PYMAARVVFI KWIVTFFLEK KYLTATQNTK NGVDVLPKII QTVGGGAVQE RAPELDGGGP TEQDKSHSNS STLSDRRLSN SSLCSIEEEH RMVYEMVQRI LLSTRGYVNF VNEVFHQAFL LPSCEIAVTR KVVQVYRKWI LQDKPVFMEE PDRKDVAQED AEKLGFSETD SKEASSESSG HKRSSSWGRT YSFTSAMSRG CVTEEENTNV KAGVQALLQV FLTNSANIFL LEPCAEVPVL LKEQVDACKA VLIIFRRMIM ELTMNKKTWE QMLQILLRIT EAVMQKPKDK QIKDLFAQSL AGLLFRTLMV AWIRANLCVY ISRELWDDFL GVLSSLTEWE ELINEWANIM DSLTAVLART VYGVEMTNLP LDKLSEQKEK KQRGKGCVLD PQKGTTVGRS FSLSWRSHPD VTEPMRFRSA TTSGAPGVEK ARNIVRQKAT EVEECQQSEN APAAGSGHLT VGQQQQVLRS SSTSDIPEPL CSDSSQGQKA ENTQNSSSSE PQPIQENKGH VKREHEGITI LVRRSSSPAE LDLKDDLQQT QGKCRERQKS ESTNSDTTLG CTNEAELSMG PWQTCEEDPE LNTPTDVVAD ADARHWLQLS PTDASNLTDS SECLTDDCSI IAGGSLTGWH PDSAAVLWRR VLGILGDVNN IQSPKIHARV FCYLYELWYK LAKIRDNLAI SLDNQSSPSP PVLIPPLRMF ASWLFKAATL PNEYKEGKLQ AYRLICAMMT RRQDVLPNSD FLVHFYLVMH LGLTSEDQDI LNTIIRHCPP RFFSLGFPGF SMLVGDFITA AARVLSTDIL TAPRSEAVTV LGSLVCFPNT YQEIPLLQSV PEVNEAITGT EDVKHYLINI LLKNATEEPN EYARCIAVCS LGVWICEELA QCTSHPQVKE AINVIGVTLK FPNKIVAQVA CDVLQLLVSY WEKLQMFETS LPRKMAEILV ATVAFLLPSA EYSSVETDKK FIVSLLLCLL DWCMALPVSV LLHPVSTAVL EEQHSARAPL LDYIYRVLHC CVCGSSTYTQ QSHYILTLAD LSSTDYDPFL PLANVKSSEP VQYHSSAELG NLLTVEEEKK RRSLELIPLT ARMVMAHLVN HLGHYPLSGG PAILHSLVSE NHDNAHVEGS ELSFEVFRSP NLQLFVFNDS TLISYLQTPT EGPVGGSPVG SLSDVRVIVR DISGKYSWDG KVLYGPLEGC LAPNGRNPSF LISSWHRDTF GPQKDSSQVE EGDDVLDKLL ENIGHTSPEC LLPSQLNLNE PSLTPCGMNY DQEKEIIEVI LRQNAQEDEY IQSHNFDSAM KVTSQGQPSP VEPRGPFYFC RLLLDDLGMN SWDRRKNFHL LKKNSKLLRE LKNLDSRQCR ETHKIAVFYI AEGQEDKCSI LSNERGSQAY EDFVAGLGWE VDLSTHCGFM GGLQRNGSTG QTAPYYATST VEVIFHVSTR MPSDSDDSLT KKLRHLGNDE VHIVWSEHSR DYRRGIIPTA FGDVSIIIYP MKNHMFFIAI TKKPEVPFFG PLFDGAIVSG KLLPSLVCAT CINASRAVKC LIPLYQSFYE ERALYLEAII QNHREVMTFE DFAAQVFSPS PSYSLSGTD

UniProtKB: Ral GTPase-activating protein subunit alpha-2

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Macromolecule #2: RalGAP subunit beta

MacromoleculeName: RalGAP subunit beta / type: protein_or_peptide / ID: 2
Details: N-terminal 3xHA tag MYPYDVPDYAGSYPYDVPDYAGSYPYDVPDYAGS
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MYPYDVPDYA GSYPYDVPDY AGSYPYDVPD YAGSMYSEWR SLHLVIQNDQ GHTSVLHSYP ESVGREVANA VVRPLGQVLG TPSVAGSENL LKTDKEVKWT MEVICYGLTL PLDGETVKYC VDVYTDWIMA LVLPKDSIPL PVIKEPNQYV QTILKHLQNL FVPRQEQGSS ...String:
MYPYDVPDYA GSYPYDVPDY AGSYPYDVPD YAGSMYSEWR SLHLVIQNDQ GHTSVLHSYP ESVGREVANA VVRPLGQVLG TPSVAGSENL LKTDKEVKWT MEVICYGLTL PLDGETVKYC VDVYTDWIMA LVLPKDSIPL PVIKEPNQYV QTILKHLQNL FVPRQEQGSS QIRLCLQVLR AIQKLARESS LMARETWEVL LLFLLQINDI LLAPPTVQGG IAENLAEKLI GVLFEVWLLA CTRCFPTPPY WKTAKEMVAN WRHHPAVVEQ WSKVICALTS RLLRFTYGPS FPAFKVPDED ASLIPPEMDN ECVAQTWFRF LHMLSNPVDL SNPAIISSTP KFQEQFLNVS GMPQELNQYP CLKHLPQIFF RAMRGISCLV DAFLGISRPR SDSAPPTPVN RLSMPQSAAV STTPPHNRRH RAVTVNKATM KTSTVSTAHA SKVQHQTSST SPLSSPNQTS SEPRPLPAPR RPKVNSILNL FGSWLFDAAF VHCKLHNGIN RDSSMTAITT QASMEFRRKG SQMSTDTMVS NPMFDASEFP DNYEAGRAEA CGTLCRIFCS KKTGEEILPA YLSRFYMLLI QGLQINDYVC HPVLASVILN SPPLFCCDLK GIDVVVPYFI SALETILPDR ELSKFKSYVN PTELRRSSIN ILLSLLPLPH HFGTVKSEVV LEGKFSNDDS SSYDKPITFL SLKLRLVNIL IGALQTETDP NNTQMILGAM LNIVQDSALL EAIGCQMEMG GGENNLKSHS RTNSGISSAS GGSTEPTTPD SERPAQALLR DYALNTDSAA GLLIRSIHLV TQRLNSQWRQ DMSISLAALE LLSGLAKVKV MVDSGDRKRA ISSVCTYIVY QCSRPAPLHS RDLHSMIVAA FQCLCVWLTE HPDMLDEKDC LKEVLEIVEL GISGSKSKNN EQEVKYKGDK EPNPASMRVK DAAEATLTCI MQLLGAFPSP SGPASPCSLV NETTLIKYSR LPTINKHSFR YFVLDNSVIL AMLEQPLGNE QNDFFPSVTV LVRGMSGRLA WAQQLCLLPR GAKANQKLFV PEPRPVPKND VGFKYSVKHR PFPEEVDKIP FVKADLSIPD LHEIVTEELE ERHEKLRSGM AQQIAYEIHL EQQSEEELQK RSFPDPVTDC KPPPPAQEFQ TARLFLSHFG FLSLEALKEP ANSRLPPHLI ALDSTIPGFF DDIGYLDLLP CRPFDTVFIF YMKPGQKTNQ EILKNVESSR TVQPHFLEFL LSLGWSVDVG RHPGWTGHVS TSWSINCCDD GEGSQQEVIS SEDIGASIFN GQKKVLYYAD ALTEIAFVVP SPVESLTDSL ESNISDQDSD SNMDLMPGIL KQPSLTLELF PNHTDNLNSS QRLSPSSRMR KLPQGRPVPP LGPETRVSVV WVERYDDIEN FPLSELMTEI STGVETTANS STSLRSTTLE KEVPVIFIHP LNTGLFRIKI QGATGKFNMV IPLVDGMIVS RRALGFLVRQ TVINICRRKR LESDSYSPPH VRRKQKITDI VNKYRNKQLE PEFYTSLFQE VGLKNCSS

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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 286 K / Instrument: FEI VITROBOT MARK IV / Details: double blotting.
DetailsCo-expression of RalGAP subunit 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: 3.93 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

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