2025-08-13 REL PDBe PDBe PDBe 2025-04-15 2025-07-16 2025-07-16 2025-08-13 German Research Foundation (DFG) KU2531/2 Germany German Research Foundation (DFG) KU2531/6 Germany German Research Foundation (DFG) 496113311 Germany German Research Foundation (DFG) INST 211/667-1 Germany German Research Foundation (DFG) CRC 1430 A04 Germany German Research Foundation (DFG) CRC 1348 A15 Germany Focussed cryo EM map from multibody refinement of human RalGAP2 complex (body1: alpha2 beta heterodimer interface) Rasche R Klink BU Gatsogiannis C Kuemmel D Complex, GTPase activating protein, RAL, Asn thumb GAP, RalGAP, SIGNALING PROTEIN Rasche R Klink BU Apken LH Michalke E Chen M Oeckinghaus A Gatsogiannis C Kummel D Structure and mechanism of the RalGAP tumor suppressor complex. Nat Commun UK 16 7002 7002 2025 40738882 doi:10.1038/s41467-025-61743-9 2041-1723 Rasche R Apken LH Titze S Michalke E Singh RK Oeckinghaus A Kummel D The GTPase kappa B-Ras is an essential subunit of the RalGAP tumor suppressor complex. J.Biol.Chem. US 110460 110460 2025 40619001 doi:10.1016/j.jbc.2025.110460 1083-351X 0071 JBCHA3 EMDB EMD-53419 associated EM volume Homodimer of two RalGAPa2-RalGAPb heterodimers Homodimer of two RalGAPa2-RalGAPb heterodimers 0 1 2
co-expression of RalGAP subunit alpha2 and beta
Homo sapiens 0.17 RalGAP subunit alpha2 1 1 Homo sapiens RalGAP subunit beta 1 2 Homo sapiens RalGAP subunit alpha2 Homo sapiens
N-terminal 3xFLAG tag MDYKDHDGDYKDHDIDYKDDDDKLAAA
Homo sapiens LEVO MDYKDHDGDYKDHDIDYKDDDDKLAAAFSRRSHGDVKKSTQKVLDPKKDVLTRLKHLRALLDNVDANDLKQFFETNYSQIYFIFYENFIALENSLKLKGNNKSQREELDSILFLFEKILQFLPERIFFRWHYQSIGSTLKKLLHTGNSIKIRCEGIRLFLLWLQALQTNCAEEQVLIFACLVPGFPAVMSSRGPCTLETLINPSPSVADVKIYPEEITPLLPAISGEKIAEDQTCFFLQILLKYMVIQAASLEWKNKENQDTGFKFLFTLFRKYYLPHLFPSFTKLTNIYKPVLDIPHLRPKPVYITTTRDNENIYSTKIPYMAARVVFIKWIVTFFLEKKYLTATQNTKNGVDVLPKIIQTVGGGAVQERAPELDGGGPTEQDKSHSNSSTLSDRRLSNSSLCSIEEEHRMVYEMVQRILLSTRGYVNFVNEVFHQAFLLPSCEIAVTRKVVQVYRKWILQDKPVFMEEPDRKDVAQEDAEKLGFSETDSKEASSESSGHKRSSSWGRTYSFTSAMSRGCVTEEENTNVKAGVQALLQVFLTNSANIFLLEPCAEVPVLLKEQVDACKAVLIIFRRMIMELTMNKKTWEQMLQILLRITEAVMQKPKDKQIKDLFAQSLAGLLFRTLMVAWIRANLCVYISRELWDDFLGVLSSLTEWEELINEWANIMDSLTAVLARTVYGVEMTNLPLDKLSEQKEKKQRGKGCVLDPQKGTTVGRSFSLSWRSHPDVTEPMRFRSATTSGAPGVEKARNIVRQKATEVEECQQSENAPAAGSGHLTVGQQQQVLRSSSTSDIPEPLCSDSSQGQKAENTQNSSSSEPQPIQENKGHVKREHEGITILVRRSSSPAELDLKDDLQQTQGKCRERQKSESTNSDTTLGCTNEAELSMGPWQTCEEDPELNTPTDVVADADARHWLQLSPTDASNLTDSSECLTDDCSIIAGGSLTGWHPDSAAVLWRRVLGILGDVNNIQSPKIHARVFCYLYELWYKLAKIRDNLAISLDNQSSPSPPVLIPPLRMFASWLFKAATLPNEYKEGKLQAYRLICAMMTRRQDVLPNSDFLVHFYLVMHLGLTSEDQDILNTIIRHCPPRFFSLGFPGFSMLVGDFITAAARVLSTDILTAPRSEAVTVLGSLVCFPNTYQEIPLLQSVPEVNEAITGTEDVKHYLINILLKNATEEPNEYARCIAVCSLGVWICEELAQCTSHPQVKEAINVIGVTLKFPNKIVAQVACDVLQLLVSYWEKLQMFETSLPRKMAEILVATVAFLLPSAEYSSVETDKKFIVSLLLCLLDWCMALPVSVLLHPVSTAVLEEQHSARAPLLDYIYRVLHCCVCGSSTYTQQSHYILTLADLSSTDYDPFLPLANVKSSEPVQYHSSAELGNLLTVEEEKKRRSLELIPLTARMVMAHLVNHLGHYPLSGGPAILHSLVSENHDNAHVEGSELSFEVFRSPNLQLFVFNDSTLISYLQTPTEGPVGGSPVGSLSDVRVIVRDISGKYSWDGKVLYGPLEGCLAPNGRNPSFLISSWHRDTFGPQKDSSQVEEGDDVLDKLLENIGHTSPECLLPSQLNLNEPSLTPCGMNYDQEKEIIEVILRQNAQEDEYIQSHNFDSAMKVTSQGQPSPVEPRGPFYFCRLLLDDLGMNSWDRRKNFHLLKKNSKLLRELKNLDSRQCRETHKIAVFYIAEGQEDKCSILSNERGSQAYEDFVAGLGWEVDLSTHCGFMGGLQRNGSTGQTAPYYATSTVEVIFHVSTRMPSDSDDSLTKKLRHLGNDEVHIVWSEHSRDYRRGIIPTAFGDVSIIIYPMKNHMFFIAITKKPEVPFFGPLFDGAIVSGKLLPSLVCATCINASRAVKCLIPLYQSFYEERALYLEAIIQNHREVMTFEDFAAQVFSPSPSYSLSGTD Q2PPJ7 RalGAP subunit beta Homo sapiens
N-terminal 3xHA tag MYPYDVPDYAGSYPYDVPDYAGSYPYDVPDYAGS
Homo sapiens LEVO MYPYDVPDYAGSYPYDVPDYAGSYPYDVPDYAGSMYSEWRSLHLVIQNDQGHTSVLHSYPESVGREVANAVVRPLGQVLGTPSVAGSENLLKTDKEVKWTMEVICYGLTLPLDGETVKYCVDVYTDWIMALVLPKDSIPLPVIKEPNQYVQTILKHLQNLFVPRQEQGSSQIRLCLQVLRAIQKLARESSLMARETWEVLLLFLLQINDILLAPPTVQGGIAENLAEKLIGVLFEVWLLACTRCFPTPPYWKTAKEMVANWRHHPAVVEQWSKVICALTSRLLRFTYGPSFPAFKVPDEDASLIPPEMDNECVAQTWFRFLHMLSNPVDLSNPAIISSTPKFQEQFLNVSGMPQELNQYPCLKHLPQIFFRAMRGISCLVDAFLGISRPRSDSAPPTPVNRLSMPQSAAVSTTPPHNRRHRAVTVNKATMKTSTVSTAHASKVQHQTSSTSPLSSPNQTSSEPRPLPAPRRPKVNSILNLFGSWLFDAAFVHCKLHNGINRDSSMTAITTQASMEFRRKGSQMSTDTMVSNPMFDASEFPDNYEAGRAEACGTLCRIFCSKKTGEEILPAYLSRFYMLLIQGLQINDYVCHPVLASVILNSPPLFCCDLKGIDVVVPYFISALETILPDRELSKFKSYVNPTELRRSSINILLSLLPLPHHFGTVKSEVVLEGKFSNDDSSSYDKPITFLSLKLRLVNILIGALQTETDPNNTQMILGAMLNIVQDSALLEAIGCQMEMGGGENNLKSHSRTNSGISSASGGSTEPTTPDSERPAQALLRDYALNTDSAAGLLIRSIHLVTQRLNSQWRQDMSISLAALELLSGLAKVKVMVDSGDRKRAISSVCTYIVYQCSRPAPLHSRDLHSMIVAAFQCLCVWLTEHPDMLDEKDCLKEVLEIVELGISGSKSKNNEQEVKYKGDKEPNPASMRVKDAAEATLTCIMQLLGAFPSPSGPASPCSLVNETTLIKYSRLPTINKHSFRYFVLDNSVILAMLEQPLGNEQNDFFPSVTVLVRGMSGRLAWAQQLCLLPRGAKANQKLFVPEPRPVPKNDVGFKYSVKHRPFPEEVDKIPFVKADLSIPDLHEIVTEELEERHEKLRSGMAQQIAYEIHLEQQSEEELQKRSFPDPVTDCKPPPPAQEFQTARLFLSHFGFLSLEALKEPANSRLPPHLIALDSTIPGFFDDIGYLDLLPCRPFDTVFIFYMKPGQKTNQEILKNVESSRTVQPHFLEFLLSLGWSVDVGRHPGWTGHVSTSWSINCCDDGEGSQQEVISSEDIGASIFNGQKKVLYYADALTEIAFVVPSPVESLTDSLESNISDQDSDSNMDLMPGILKQPSLTLELFPNHTDNLNSSQRLSPSSRMRKLPQGRPVPPLGPETRVSVVWVERYDDIENFPLSELMTEISTGVETTANSSTSLRSTTLEKEVPVIFIHPLNTGLFRIKIQGATGKFNMVIPLVDGMIVSRRALGFLVRQTVINICRRKRLESDSYSPPHVRRKQKITDIVNKYRNKQLEPEFYTSLFQEVGLKNCSS Q86X10 singleParticle particle 0.7 7.5 20.0 C8H18N2O4S HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 150.0 NaCl sodium chloride 2.0 MgCl2 magnesium chloride 1.0 C9H15O6P TCEP (tris(2-carboxyethyl)phosphine) Quantifoil R2/1 COPPER 300 GLOW DISCHARGE AIR ETHANE 100 286 FEI VITROBOT MARK IV
double blotting.
Co-expression of Monodisperse sample from size exclusion chromatography
 TFS KRIOS FLOOD BEAM BRIGHT FIELD FIELD EMISSION GUN 300 50.0 2.7 0.5 4.2 215000.0 FEI TITAN KRIOS AUTOGRID HOLDER NITROGEN TFS Selectris X 10 FEI FALCON IV (4k x 4k) 4096 4096 4 64606 3.2 60.0
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.
 1 5519589 RELION 5 PHASE FLIPPING AND AMPLITUDE CORRECTION A model of the RGalpha2/RGbeta complex was calculated using AlphaFold2 1 C1 FOURIER SPACE 3.79 FSC 0.143 CUT-OFF RELION 5 420975 MAXIMUM LIKELIHOOD RELION 5 MAXIMUM LIKELIHOOD RELION 5 4 221930.0 RELION 5 emd_53419.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 360 360 360 0 0 0 360 360 360 417.59998 417.59998 417.59998 90.0 90.0 90.0 X Y Z -0.009386701 0.024716135 -0.000011364501 0.0004733285 1.16 1.16 1.16 0.00693 AUTHOR Focussed cryo EM map from multibody refinement of RalGAP2 complex (body1: alpha2 beta heterodimer interface) AlphaFold in silico model AB INITIO MODEL
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.
cross correlation REAL 129.719999999999999 emd_53419_msk_1.map emd_53419_additional_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 360 360 360 0 0 0 360 360 360 417.59998 417.59998 417.59998 90.0 90.0 90.0 X Y Z -0.03401246 0.06353883 -0.000011362318 0.0010396449 1.16 1.16 1.16 AUTHOR Sharpened postprocessed focussed cryo EM map from multibody refinement of RalGAP2 complex (body1: alpha2 beta heterodimer interface) emd_53419_half_map_2.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 360 360 360 0 0 0 360 360 360 417.59998 417.59998 417.59998 90.0 90.0 90.0 X Y Z -0.012320158 0.026978556 -0.000011421999 0.0008478314 1.16 1.16 1.16 AUTHOR Half map 1 focussed cryo EM map from multibody refinement of RalGAP2 complex (body1: alpha2 beta heterodimer interface) emd_53419_half_map_1.map.gz 1 IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) 360 360 360 0 0 0 360 360 360 417.59998 417.59998 417.59998 90.0 90.0 90.0 X Y Z -0.011523971 0.027719984 -0.000011302637 0.0008492205 1.16 1.16 1.16 AUTHOR Half map 2 focussed cryo EM map from multibody refinement of RalGAP2 complex (body1: alpha2 beta heterodimer interface)