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- PDB-8u1m: Cryo-EM structure of the HSP90 dimer (NTD-MD) in the semi-open state -

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Basic information

Entry
Database: PDB / ID: 8u1m
TitleCryo-EM structure of the HSP90 dimer (NTD-MD) in the semi-open state
ComponentsHeat shock protein 83
KeywordsCHAPERONE / HSP / HSP90
Function / homology
Function and homology information


ATP-dependent protein folding chaperone / unfolded protein binding / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
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 / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily / Ribosomal protein S5 domain 2-type fold
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / Heat shock protein 83
Similarity search - Component
Biological speciesTrichoplusia ni (cabbage looper)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)167,7076
Polymers166,6442
Non-polymers1,0634
Water00
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


Mass: 83322.133 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Trichoplusia ni (cabbage looper) / References: UniProt: A0A7E5VSK5
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#3: 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

ComponentName: Heat shock protein 90 / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.166 MDa / Experimental value: NO
Source (natural)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMSodium ChlorideNaCl1
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 FIELD / 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: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 325732 / Symmetry type: POINT

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