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- PDB-7z38: Structure of the RAF1-HSP90-CDC37 complex (RHC-I) -

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Basic information

Entry
Database: PDB / ID: 7z38
TitleStructure of the RAF1-HSP90-CDC37 complex (RHC-I)
Components
  • Heat shock protein HSP 90-beta
  • Hsp90 co-chaperone Cdc37
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsTRANSFERASE / RAF1-HSP90-CDC37 / complex / proto-oncogene / serine/threonine kinase / cancer / chaperone
Function / homology
Function and homology information


regulation of type II interferon-mediated signaling pathway / HSP90-CDC37 chaperone complex / death-inducing signaling complex assembly / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / aryl hydrocarbon receptor complex / intermediate filament cytoskeleton organization / histone methyltransferase binding / dynein axonemal particle / receptor ligand inhibitor activity ...regulation of type II interferon-mediated signaling pathway / HSP90-CDC37 chaperone complex / death-inducing signaling complex assembly / negative regulation of proteasomal protein catabolic process / Aryl hydrocarbon receptor signalling / aryl hydrocarbon receptor complex / intermediate filament cytoskeleton organization / histone methyltransferase binding / dynein axonemal particle / receptor ligand inhibitor activity / regulation of Rho protein signal transduction / positive regulation of type 2 mitophagy / type B pancreatic cell proliferation / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / regulation of cyclin-dependent protein serine/threonine kinase activity / ATP-dependent protein binding / Rap1 signalling / positive regulation of protein localization to cell surface / protein kinase regulator activity / insulin secretion involved in cellular response to glucose stimulus / protein folding chaperone complex / Negative feedback regulation of MAPK pathway / IFNG signaling activates MAPKs / post-transcriptional regulation of gene expression / GP1b-IX-V activation signalling / Respiratory syncytial virus genome replication / telomerase holoenzyme complex assembly / ERBB2-ERBB3 signaling pathway / positive regulation of transforming growth factor beta receptor signaling pathway / Uptake and function of diphtheria toxin / neurotrophin TRK receptor signaling pathway / Drug-mediated inhibition of ERBB2 signaling / Resistance of ERBB2 KD mutants to trastuzumab / Resistance of ERBB2 KD mutants to sapitinib / Resistance of ERBB2 KD mutants to tesevatinib / Resistance of ERBB2 KD mutants to neratinib / Resistance of ERBB2 KD mutants to osimertinib / Resistance of ERBB2 KD mutants to afatinib / Resistance of ERBB2 KD mutants to AEE788 / Resistance of ERBB2 KD mutants to lapatinib / Drug resistance in ERBB2 TMD/JMD mutants / regulation of type I interferon-mediated signaling pathway / pseudopodium / dendritic growth cone / TPR domain binding / face development / Assembly and release of respiratory syncytial virus (RSV) virions / regulation of cell differentiation / thyroid gland development / The NLRP3 inflammasome / protein phosphatase activator activity / Sema3A PAK dependent Axon repulsion / extrinsic apoptotic signaling pathway via death domain receptors / somatic stem cell population maintenance / regulation of protein ubiquitination / positive regulation of peptidyl-serine phosphorylation / HSF1-dependent transactivation / MAP kinase kinase kinase activity / response to unfolded protein / HSF1 activation / type II interferon-mediated signaling pathway / telomere maintenance via telomerase / Attenuation phase / chaperone-mediated protein complex assembly / axonal growth cone / protein targeting / negative regulation of protein-containing complex assembly / Schwann cell development / RHOBTB2 GTPase cycle / Purinergic signaling in leishmaniasis infection / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / : / supramolecular fiber organization / DNA polymerase binding / heat shock protein binding / response to muscle stretch / Signaling by ERBB2 / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / myelination / protein folding chaperone / peptide binding / CD209 (DC-SIGN) signaling / cellular response to interleukin-4 / insulin-like growth factor receptor signaling pathway / ESR-mediated signaling / HSP90 chaperone cycle for steroid hormone receptors (SHR) in the presence of ligand / Constitutive Signaling by Overexpressed ERBB2 / thymus development / adenylate cyclase activator activity / placenta development / nitric-oxide synthase regulator activity / positive regulation of cell differentiation / Hsp90 protein binding / ATP-dependent protein folding chaperone / wound healing / Signaling by ERBB2 TMD/JMD mutants / RAF activation / Signaling by high-kinase activity BRAF mutants / Constitutive Signaling by EGFRvIII
Similarity search - Function
Cdc37, Hsp90 binding domain / Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 N terminal kinase binding / Cdc37 ...Cdc37, Hsp90 binding domain / Cdc37, C-terminal / Cdc37, Hsp90 binding / Cdc37, Hsp90-binding domain superfamily / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 C terminal domain / Cdc37 Hsp90 binding domain / Cdc37 N terminal kinase binding / Cdc37 / Cdc37, N-terminal domain / Cdc37 N terminal kinase binding / Raf-like Ras-binding domain / Raf-like Ras-binding / Ras-binding domain (RBD) profile. / Raf-like Ras-binding domain / Diacylglycerol/phorbol-ester binding / : / Phorbol esters/diacylglycerol binding domain (C1 domain) / Zinc finger phorbol-ester/DAG-type signature. / Zinc finger phorbol-ester/DAG-type profile. / Protein kinase C conserved region 1 (C1) domains (Cysteine-rich domains) / Protein kinase C-like, phorbol ester/diacylglycerol-binding domain / C1-like domain superfamily / Heat shock protein Hsp90, conserved site / Heat shock hsp90 proteins family signature. / HSP90, C-terminal domain / Heat shock protein Hsp90, N-terminal / Heat shock protein Hsp90 family / Hsp90 protein / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Methane Monooxygenase Hydroxylase; Chain G, domain 1 / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase superfamily / Ubiquitin-like domain superfamily / Ribosomal protein S5 domain 2-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / RAF proto-oncogene serine/threonine-protein kinase / Heat shock protein HSP 90-beta / Hsp90 co-chaperone Cdc37
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.16 Å
AuthorsMesa, P. / Garcia-Alonso, S. / Barbacid, M. / Montoya, G.
Funding support Denmark, 4items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
Novo Nordisk FoundationNNF0024386 Denmark
Novo Nordisk FoundationNNF17SA0030214 Denmark
Novo Nordisk FoundationNNF18OC0055061 Denmark
CitationJournal: Mol Cell / Year: 2022
Title: Structure of the RAF1-HSP90-CDC37 complex reveals the basis of RAF1 regulation.
Authors: Sara García-Alonso / Pablo Mesa / Laura de la Puente Ovejero / Gonzalo Aizpurua / Carmen G Lechuga / Eduardo Zarzuela / Clara M Santiveri / Manuel Sanclemente / Javier Muñoz / Mónica ...Authors: Sara García-Alonso / Pablo Mesa / Laura de la Puente Ovejero / Gonzalo Aizpurua / Carmen G Lechuga / Eduardo Zarzuela / Clara M Santiveri / Manuel Sanclemente / Javier Muñoz / Mónica Musteanu / Ramón Campos-Olivas / Jorge Martínez-Torrecuadrada / Mariano Barbacid / Guillermo Montoya /
Abstract: RAF 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- ...RAF 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.
History
DepositionMar 1, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 14, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 28, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 9, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Heat shock protein HSP 90-beta
B: Heat shock protein HSP 90-beta
C: RAF proto-oncogene serine/threonine-protein kinase
D: Hsp90 co-chaperone Cdc37
hetero molecules


Theoretical massNumber of molelcules
Total (without water)290,7518
Polymers289,6884
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration, light scattering
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area22190 Å2
ΔGint-130 kcal/mol
Surface area78110 Å2

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Components

#1: Protein Heat shock protein HSP 90-beta / HSP 90 / Heat shock 84 kDa / HSP 84 / HSP84


Mass: 84371.281 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-terminal HA-tag + HSP90-beta / Source: (gene. exp.) Homo sapiens (human) / Gene: HSP90AB1, HSP90B, HSPC2, HSPCB / Plasmid: pcDNA3 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: P08238
#2: Protein RAF proto-oncogene serine/threonine-protein kinase / Proto-oncogene c-RAF / cRaf / Raf-1


Mass: 74409.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: RAF1 + linker Gly-Ser-Ala + C-terminal StrepTagII / Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1, RAF / Plasmid: pcDNA3 / Cell line (production host): Expi293F / Production host: Homo sapiens (human)
References: UniProt: P04049, non-specific serine/threonine protein kinase
#3: Protein Hsp90 co-chaperone Cdc37 / Hsp90 chaperone protein kinase-targeting subunit / p50Cdc37


Mass: 46535.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: CDC37 + C-terminal Myc-DDK tag / Source: (gene. exp.) Homo sapiens (human) / Gene: CDC37, CDC37A / Plasmid: pCMV6 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q16543
#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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

ComponentName: RAF1-HSP90-CDC37 complex, RHC-I / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.28913380 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F cells
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris bufferTris-HCl1
2150 mMsodium chlorideNaCl1
310 mMmagnesium chlorideMgCl21
410 mMpotassium chlorideKCl1
520 mMsodium molybdateNa2MoO41
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: UltrAuFoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8138
Details: 8138 images (4430 non-tilted and 3708 30 degrees tilted)
Image scansWidth: 4096 / Height: 4096

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20rc3_4406refinement
PHENIX1.20rc3_4406refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.1particle selectionblob picker protocol
2EPUimage acquisition
4cryoSPARC3.3.1CTF correctionpatch CTF protocol
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10ISOLDEmodel refinement
11RELION3.1.3initial Euler assignment
12RELION3.1.3final Euler assignment
14RELION3.1.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3760000
Details: 3760k in total: 1353k RAF1:HSP90:CDC37 complex, 1127k 14-3-3 dimer, RAF1:14-3-3 complex 1245k
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 266000 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 62.68 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004614507
ELECTRON MICROSCOPYf_angle_d0.571619501
ELECTRON MICROSCOPYf_chiral_restr0.04162136
ELECTRON MICROSCOPYf_plane_restr0.00412492
ELECTRON MICROSCOPYf_dihedral_angle_d5.47741901

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