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- PDB-5kne: CryoEM Reconstruction of Hsp104 Hexamer -

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Basic information

Entry
Database: PDB / ID: 5kne
TitleCryoEM Reconstruction of Hsp104 Hexamer
ComponentsHeat shock protein 104
KeywordsCHAPERONE / Hsp104 / AAA+ protein
Function / homology
Function and homology information


trehalose metabolic process / TRC complex / protein folding in endoplasmic reticulum / cellular heat acclimation / post-translational protein targeting to endoplasmic reticulum membrane / stress granule disassembly / : / protein unfolding / nuclear periphery / ADP binding ...trehalose metabolic process / TRC complex / protein folding in endoplasmic reticulum / cellular heat acclimation / post-translational protein targeting to endoplasmic reticulum membrane / stress granule disassembly / : / protein unfolding / nuclear periphery / ADP binding / unfolded protein binding / protein-folding chaperone binding / cellular response to heat / protein refolding / ATP hydrolysis activity / ATP binding / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp repeat (R) N-terminal domain / : / Clp, repeat (R) domain / Clp repeat (R) domain profile. ...ClpA/B, conserved site 2 / Chaperonins clpA/B signature 2. / ClpA/B, conserved site 1 / Chaperonins clpA/B signature 1. / ClpA/ClpB, AAA lid domain / AAA lid domain / Clp repeat (R) N-terminal domain / : / Clp, repeat (R) domain / Clp repeat (R) domain profile. / Clp, N-terminal domain superfamily / ClpA/B family / Clp ATPase, C-terminal / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / AAA domain (Cdc48 subfamily) / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Heat shock protein 104
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 5.64 Å
AuthorsYokom, A.L. / Gates, S.N. / Jackrel, M.E. / Mack, K.L. / Su, M. / Shorter, J. / Southworth, D.R.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM099836 United States
American Heart Association15PRE25090089 United States
National Science Foundation (NSF, United States)DGE-1321851 United States
Muscular Dystrophy AssociationMDA277268 United States
CitationJournal: Nat Struct Mol Biol / Year: 2016
Title: Spiral architecture of the Hsp104 disaggregase reveals the basis for polypeptide translocation.
Authors: Adam L Yokom / Stephanie N Gates / Meredith E Jackrel / Korrie L Mack / Min Su / James Shorter / Daniel R Southworth /
Abstract: Hsp104, a conserved AAA+ protein disaggregase, promotes survival during cellular stress. Hsp104 remodels amyloids, thereby supporting prion propagation, and disassembles toxic oligomers associated ...Hsp104, a conserved AAA+ protein disaggregase, promotes survival during cellular stress. Hsp104 remodels amyloids, thereby supporting prion propagation, and disassembles toxic oligomers associated with neurodegenerative diseases. However, a definitive structural mechanism for its disaggregase activity has remained elusive. We determined the cryo-EM structure of wild-type Saccharomyces cerevisiae Hsp104 in the ATP state, revealing a near-helical hexamer architecture that coordinates the mechanical power of the 12 AAA+ domains for disaggregation. An unprecedented heteromeric AAA+ interaction defines an asymmetric seam in an apparent catalytic arrangement that aligns the domains in a two-turn spiral. N-terminal domains form a broad channel entrance for substrate engagement and Hsp70 interaction. Middle-domain helices bridge adjacent protomers across the nucleotide pocket, thus explaining roles in ATP hydrolysis and protein disaggregation. Remarkably, substrate-binding pore loops line the channel in a spiral arrangement optimized for substrate transfer across the AAA+ domains, thereby establishing a continuous path for polypeptide translocation.
History
DepositionJun 28, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 27, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 3, 2016Group: Database references
Revision 1.2Aug 17, 2016Group: Database references
Revision 1.3Sep 21, 2016Group: Database references
Revision 1.4Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.5Nov 27, 2019Group: Author supporting evidence / Structure summary / Category: em_entity_assembly / pdbx_audit_support
Item: _em_entity_assembly.entity_id_list / _pdbx_audit_support.funding_organization
Revision 1.6Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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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)581,37317
Polymers575,8046
Non-polymers5,56811
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 95967.398 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: HSP104, YLL026W, L0948 / Production host: Escherichia coli (E. coli) / References: UniProt: P31539
#2: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*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 Complex of Hsp104 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pNOTAG
Buffer solutionpH: 7.5
SpecimenConc.: 0.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Clean protein sample incubated with AMP-PNP
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Details: Plunged into liquid ethane (FEI VITROBOT MARK IV)

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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 / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 1.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3661
Details: Movies contained 38 of 40 frames from an 8 sec exposure (200 milliseconds per frame).
Image scansMovie frames/image: 40 / Used frames/image: 2-40

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Processing

EM software
IDNameCategoryDetails
1Appionparticle selectionUsed template picker from 2D class averages of ~50,000 particles
11RELIONfinal Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 240005
Details: Particles were selected using automated picking within the Appion package.
3D reconstructionResolution: 5.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 172043 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingDetails: Backbone atoms fit into reconstruction density.

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