García-Alonso, SaraMesa, PabloOvejero, Laura de la PuenteAizpurua, GonzaloLechuga, Carmen GZarzuela, EduardoSantiveri, Clara MSanclemente, ManuelMuñoz, JavierMusteanu, MónicaCampos-Olivas, RamónMartínez-Torrecuadrada, JorgeBarbacid, MarianoMontoya, Guillermo2025-07-012025-07-012022-09-15Mol Cell . 2022 Sep 15;82(18):3438-3452https://hdl.handle.net/20.500.12105/26791We thank the Danish Cryo-EM National Facility in CFIM at the University of Co-penhagen for support during cryo-EM data collection. We also thank the Pro-tein Expression Unit at CPR for assistance in protein expression and purification. S.G.-A. is a recipient of a postdoctoral fellowship from the Spanish Association against Cancer Scientific Foundation (FCAECC) . This work was supported by grants from the European Research Council (ERC-2015-AdG/695566, THERACAN) , the Spanish Ministry of Science, Innovation and Universities (RTC-2017-6576-1) , the Autonomous Community of Madrid (B2017/BMD-3884 iLUNG-CM) , the CRIS Cancer Foundation, and the AECC (GC16173694BARB) to M.B. and by a grant from the Spanish Ministry of Science, Innovation and Universities (RTI2018-094664-B-I00) to M.B. and M.M.; M.B. is a recipient of an Endowed Chair from the AXA Research Fund. The Experimental Oncology group is a member of CIBERONC. G.M. is part of the Novo Nordisk Foundation Center for Protein Research (CPR) , which is sup-ported financially by the Novo Nordisk Foundation (grant NNF14CC0001) . This work was also supported by grant NNF0024386, grant NNF17SA0030214, and Distinguished Investigator grant NNF18OC0055061 to G.M., who is a member of the Integrative Structural Biology Cluster (ISBUC) at the University of CopenhagenRAF kinases are RAS-activated enzymes that initiate signaling through the MAPK cascade to control cellular proliferation, differentiation, and survival. Here, we describe the structure of the full-length RAF1 protein in complex with HSP90 and CDC37 obtained by cryoelectron microscopy. The reconstruction reveals a RAF1 kinase with an unfolded N-lobe separated from its C-lobe. The hydrophobic core of the N-lobe is trapped in the HSP90 dimer, while CDC37 wraps around the chaperone and interacts with the N- and C-lobes of the kinase. The structure indicates how CDC37 can discriminate between the different members of the RAF family. Our structural analysis also reveals that the folded RAF1 assembles with 14-3-3 dimers, suggesting that after folding RAF1 follows a similar activation as B-RAF. Finally, disruption of the interaction between CDC37 and the DFG segment of RAF1 unveils potential vulnerabilities in attempting the pharmacological degradation of RAF1 for therapeutic purposes.engVoRhttp://creativecommons.org/licenses/by-nc-nd/4.0/MAPK signalingRAF kinasesRAF1cancercryo-EMAttribution-NonCommercial-NoDerivatives 4.0 International3605523582183438-3452Molecular Cellopen access