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- PDB-6n8t: Hsp104DWB closed conformation -

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Basic information

Entry
Database: PDB / ID: 6n8t
TitleHsp104DWB closed conformation
ComponentsHeat shock protein 104Heat shock response
KeywordsCHAPERONE / Hsp104 / ClpB / protein disaggregase / molecular chaperone / AAA+ / ATPase / cryo-EM
Function / homology
Function and homology information


inheritance of oxidatively modified proteins involved in replicative cell aging / trehalose metabolism in response to heat stress / TRC complex / cellular heat acclimation / protein folding in endoplasmic reticulum / stress granule disassembly / protein metabolic process / chaperone cofactor-dependent protein refolding / protein unfolding / nuclear periphery ...inheritance of oxidatively modified proteins involved in replicative cell aging / trehalose metabolism in response to heat stress / TRC complex / cellular heat acclimation / protein folding in endoplasmic reticulum / stress granule disassembly / protein metabolic process / chaperone cofactor-dependent protein refolding / protein unfolding / nuclear periphery / ADP binding / ATPase activity, coupled / unfolded protein binding / chaperone binding / ATP binding / identical protein binding / nucleus / cytoplasm
AAA lid domain / ClpA/B family / C-terminal, D2-small domain, of ClpB protein / AAA domain (Cdc48 subfamily) / Clp amino terminal domain, pathogenicity island component / ATPase family associated with various cellular activities (AAA) / ClpA/ClpB, AAA lid domain / Clp, N-terminal domain superfamily / ClpA/B, conserved site 2 / P-loop containing nucleoside triphosphate hydrolase ...AAA lid domain / ClpA/B family / C-terminal, D2-small domain, of ClpB protein / AAA domain (Cdc48 subfamily) / Clp amino terminal domain, pathogenicity island component / ATPase family associated with various cellular activities (AAA) / ClpA/ClpB, AAA lid domain / Clp, N-terminal domain superfamily / ClpA/B, conserved site 2 / P-loop containing nucleoside triphosphate hydrolase / Clp ATPase, C-terminal / ClpA/B, conserved site 1 / Clp, N-terminal / ATPase, AAA-type, core / AAA+ ATPase domain
Heat shock protein 104
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.7 Å
AuthorsLee, S. / Rho, S.H. / Lee, J. / Sung, N. / Liu, J. / Tsai, F.T.F.
CitationJournal: Cell Rep / Year: 2019
Title: Cryo-EM Structures of the Hsp104 Protein Disaggregase Captured in the ATP Conformation.
Authors: Sukyeong Lee / Soung Hun Roh / Jungsoon Lee / Nuri Sung / Jun Liu / Francis T F Tsai /
Abstract: Hsp104 is a ring-forming, ATP-driven molecular machine that recovers functional protein from both stress-denatured and amyloid-forming aggregates. Although Hsp104 shares a common architecture with ...Hsp104 is a ring-forming, ATP-driven molecular machine that recovers functional protein from both stress-denatured and amyloid-forming aggregates. Although Hsp104 shares a common architecture with Clp/Hsp100 protein unfoldases, different and seemingly conflicting 3D structures have been reported. Examining the structure of Hsp104 poses considerable challenges because Hsp104 readily hydrolyzes ATP, whereas ATP analogs can be slowly turned over and are often contaminated with other nucleotide species. Here, we present the single-particle electron cryo-microscopy (cryo-EM) structures of a catalytically inactive Hsp104 variant (Hsp104) in the ATP-bound state determined between 7.7 Å and 9.3 Å resolution. Surprisingly, we observe that the Hsp104 hexamer adopts distinct ring conformations (closed, extended, and open) despite being in the same nucleotide state. The latter underscores the structural plasticity of Hsp104 in solution, with different conformations stabilized by nucleotide binding. Our findings suggest that, in addition to ATP hydrolysis-driven conformational changes, Hsp104 uses stochastic motions to translocate unfolded polypeptides.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 30, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 2, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 16, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Nov 20, 2019Group: Database references / Category: pdbx_database_related / Item: _pdbx_database_related.db_name

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Assembly

Deposited unit
A: Heat shock protein 104
B: Heat shock protein 104
C: Heat shock protein 104
D: Heat shock protein 104
E: Heat shock protein 104
F: Heat shock protein 104
hetero molecules


Theoretical massNumber of molelcules
Total (without water)600,36718
Polymers594,2816
Non-polymers6,08612
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area52710 Å2
ΔGint-82 kcal/mol
Surface area197250 Å2

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Components

#1: Protein
Heat shock protein 104 / Heat shock response / Protein aggregation-remodeling factor HSP104


Mass: 99046.859 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: HSP104, YLL026W, L0948 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21-CodonPlus (DE3)-RIL cells / References: UniProt: P31539
#2: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM

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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: Hexamer of Hsp104 with ATP / Type: COMPLEX / Details: Hsp 104 double Walker B mutant / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.6 MDa / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: Buffer solution is freshly prepared.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMMOPSC7H15NO4S1
210 mMMagnesium ChlorideMgCl21
35 mMATPAdenosine triphosphateC10H16N5O13P31
SpecimenConc.: 0.3 mg/ml / Details: This sample was mono disperse. / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 K / Details: blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 7.6 sec. / Electron dose: 38 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1394

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 7.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56859 / Symmetry type: POINT

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