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- PDB-8u1l: Cryo-EM structure of the RAF1-HSP90-CDC37 complex in the closed state -

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Basic information

Entry
Database: PDB / ID: 8u1l
TitleCryo-EM structure of the RAF1-HSP90-CDC37 complex in the closed state
Components
  • Heat shock protein 83Heat shock response
  • Hsp90 co-chaperone Cdc37, N-terminally processed
  • RAF proto-oncogene serine/threonine-protein kinase
KeywordsSIGNALING PROTEIN/CHAPERONE / CRAF / RAF1 / HSP90 / CDC37 / SIGNALING PROTEIN-CHAPERONE complex
Function / homology
Function and homology information


regulation of type II interferon-mediated signaling pathway / HSP90-CDC37 chaperone complex / death-inducing signaling complex assembly / positive regulation of mitophagy in response to mitochondrial depolarization / intermediate filament cytoskeleton organization / type B pancreatic cell proliferation / protein kinase regulator activity / regulation of Rho protein signal transduction / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling ...regulation of type II interferon-mediated signaling pathway / HSP90-CDC37 chaperone complex / death-inducing signaling complex assembly / positive regulation of mitophagy in response to mitochondrial depolarization / intermediate filament cytoskeleton organization / type B pancreatic cell proliferation / protein kinase regulator activity / regulation of Rho protein signal transduction / SHOC2 M1731 mutant abolishes MRAS complex function / Gain-of-function MRAS complexes activate RAF signaling / Rap1 signalling / regulation of cell motility / protein folding chaperone complex / insulin secretion involved in cellular response to glucose stimulus / Negative feedback regulation of MAPK pathway / post-transcriptional regulation of gene expression / GP1b-IX-V activation signalling / IFNG signaling activates MAPKs / 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 / ERBB2-ERBB3 signaling pathway / regulation of cell differentiation / face development / regulation of cyclin-dependent protein serine/threonine kinase activity / pseudopodium / somatic stem cell population maintenance / neurotrophin TRK receptor signaling pathway / thyroid gland development / regulation of type I interferon-mediated signaling pathway / extrinsic apoptotic signaling pathway via death domain receptors / MAP kinase kinase kinase activity / protein targeting / negative regulation of protein-containing complex assembly / RHOBTB2 GTPase cycle / Schwann cell development / type II interferon-mediated signaling pathway / negative regulation of extrinsic apoptotic signaling pathway via death domain receptors / Signaling by ERBB2 / heat shock protein binding / response to muscle stretch / activation of adenylate cyclase activity / myelination / CD209 (DC-SIGN) signaling / Constitutive Signaling by Overexpressed ERBB2 / insulin-like growth factor receptor signaling pathway / thymus development / ATP-dependent protein folding chaperone / Signaling by ERBB2 TMD/JMD mutants / Hsp90 protein binding / RAF activation / Signaling by high-kinase activity BRAF mutants / Constitutive Signaling by EGFRvIII / wound healing / MAP2K and MAPK activation / negative regulation of cysteine-type endopeptidase activity involved in apoptotic process / Signaling by ERBB2 ECD mutants / Signaling by ERBB2 KD Mutants / Regulation of necroptotic cell death / Stimuli-sensing channels / Downregulation of ERBB2 signaling / kinase binding / Negative regulation of MAPK pathway / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / MAPK cascade / unfolded protein binding / Signaling by BRAF and RAF1 fusions / protein folding / Constitutive Signaling by Ligand-Responsive EGFR Cancer Variants / insulin receptor signaling pathway / positive regulation of peptidyl-serine phosphorylation / protein-folding chaperone binding / scaffold protein binding / regulation of apoptotic process / mitochondrial outer membrane / positive regulation of MAPK cascade / protein stabilization / non-specific serine/threonine protein kinase / protein kinase activity / negative regulation of cell population proliferation / protein phosphorylation / protein serine kinase activity / protein serine/threonine kinase activity / apoptotic process / negative regulation of apoptotic process / protein kinase binding / Golgi apparatus / enzyme binding / signal transduction / ATP hydrolysis activity
Similarity search - Function
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, 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 / Heat shock protein Hsp90, conserved site / Heat shock hsp90 proteins family signature. / C1-like domain superfamily / 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 / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / 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
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Heat shock protein 83 / RAF proto-oncogene serine/threonine-protein kinase / Hsp90 co-chaperone Cdc37
Similarity search - Component
Biological speciesHomo sapiens (human)
Trichoplusia ni (cabbage looper)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsFinci, L.I. / Simanshu, D.K.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)HHSN261200800001E United States
CitationJournal: Commun Biol / Year: 2024
Title: Structural dynamics of RAF1-HSP90-CDC37 and HSP90 complexes reveal asymmetric client interactions and key structural elements.
Authors: Lorenzo I Finci / Mayukh Chakrabarti / Gulcin Gulten / Joseph Finney / Carissa Grose / Tara Fox / Renbin Yang / Dwight V Nissley / Frank McCormick / Dominic Esposito / Trent E Balius / Dhirendra K Simanshu /
Abstract: RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular ...RAF kinases are integral to the RAS-MAPK signaling pathway, and proper RAF1 folding relies on its interaction with the chaperone HSP90 and the cochaperone CDC37. Understanding the intricate molecular interactions governing RAF1 folding is crucial for comprehending this process. Here, we present a cryo-EM structure of the closed-state RAF1-HSP90-CDC37 complex, where the C-lobe of the RAF1 kinase domain binds to one side of the HSP90 dimer, and an unfolded N-lobe segment of the RAF1 kinase domain threads through the center of the HSP90 dimer. CDC37 binds to the kinase C-lobe, mimicking the N-lobe with its HxNI motif. We also describe structures of HSP90 dimers without RAF1 and CDC37, displaying only N-terminal and middle domains, which we term the semi-open state. Employing 1 μs atomistic simulations, energetic decomposition, and comparative structural analysis, we elucidate the dynamics and interactions within these complexes. Our quantitative analysis reveals that CDC37 bridges the HSP90-RAF1 interaction, RAF1 binds HSP90 asymmetrically, and that HSP90 structural elements engage RAF1's unfolded region. Additionally, N- and C-terminal interactions stabilize HSP90 dimers, and molecular interactions in HSP90 dimers rearrange between the closed and semi-open states. Our findings provide valuable insight into the contributions of HSP90 and CDC37 in mediating client folding.
History
DepositionSep 1, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Heat shock protein 83
B: Heat shock protein 83
C: RAF proto-oncogene serine/threonine-protein kinase
D: Hsp90 co-chaperone Cdc37, N-terminally processed
hetero molecules


Theoretical massNumber of molelcules
Total (without water)286,3518
Polymers285,2884
Non-polymers1,0634
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Heat shock protein 83 / Heat shock response


Mass: 83322.133 Da / Num. of mol.: 2 / Mutation: 0 / Source method: isolated from a natural source / Source: (natural) Trichoplusia ni (cabbage looper) / References: UniProt: A0A7E5VSK5
#2: Protein RAF proto-oncogene serine/threonine-protein kinase


Mass: 74021.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RAF1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P04049
#3: Protein Hsp90 co-chaperone Cdc37, N-terminally processed


Mass: 44622.363 Da / Num. of mol.: 1 / Mutation: 0
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC37, CDC37A / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q16543
#4: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / 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
Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of RAF1-HSP90-CDC37COMPLEX#1-#30MULTIPLE SOURCES
2Heat shock protein 90Hsp90COMPLEX#11NATURAL
3RAF1 & HSP90 co-chaperone CDC37COMPLEX#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.286 MDaYES
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Trichoplusia ni (cabbage looper)7111
23Homo sapiens (human)9606
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium ChlorideNaClSodium chloride1
31 mMTCEPC9H15O6P1
45 w/vGlycerolC3H8O31
510 mMMagnesium ChlorideMgCl21
620 mMSodium MolybdateNa2MoO41
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Details: Grids were blotted for 4.5 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 FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
10RELION3.1.3initial Euler assignment
11RELION3.1.3final Euler assignment
12RELION3.1.3classification
13RELION3.1.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1877778
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97569 / Symmetry type: POINT

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