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- PDB-6d00: Calcarisporiella thermophila Hsp104 -

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Basic information

Entry
Database: PDB / ID: 6d00
TitleCalcarisporiella thermophila Hsp104
ComponentsCalcarisporiella thermophila Hsp104
KeywordsCHAPERONE / Spiral hexamer / antagonize toxin / ADP-binding state
Function / homology
Function and homology information


cellular heat acclimation / : / protein unfolding / unfolded protein binding / protein-folding chaperone binding / protein refolding / ATP hydrolysis activity / ATP binding / cytosol
Similarity search - Function
Double Clp-N motif / Clp, N-terminal domain / 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 amino terminal domain, pathogenicity island component / : ...Double Clp-N motif / Clp, N-terminal domain / 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 amino terminal domain, pathogenicity island component / : / Clp, repeat (R) domain / Clp repeat (R) domain profile. / Clp, N-terminal domain superfamily / ClpA/B family / Clp ATPase, C-terminal / AAA domain (Cdc48 subfamily) / C-terminal, D2-small domain, of ClpB protein / C-terminal, D2-small domain, of ClpB protein / Helicase, Ruva Protein; domain 3 - #60 / Helicase, Ruva Protein; domain 3 / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Heat shock protein Hsp104
Similarity search - Component
Biological speciesCalcarisporiella thermophila (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsZhang, K. / Pintilie, G.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)T32GM071399 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)F31NS101807 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R01GM099836 United States
Department of Energy (DOE, United States)GM094585 United States
Department of Energy (DOE, United States)GM115586 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)P41GM103832 United States
CitationJournal: Structure / Year: 2019
Title: Structure of Calcarisporiella thermophila Hsp104 Disaggregase that Antagonizes Diverse Proteotoxic Misfolding Events.
Authors: Karolina Michalska / Kaiming Zhang / Zachary M March / Catherine Hatzos-Skintges / Grigore Pintilie / Lance Bigelow / Laura M Castellano / Leann J Miles / Meredith E Jackrel / Edward Chuang ...Authors: Karolina Michalska / Kaiming Zhang / Zachary M March / Catherine Hatzos-Skintges / Grigore Pintilie / Lance Bigelow / Laura M Castellano / Leann J Miles / Meredith E Jackrel / Edward Chuang / Robert Jedrzejczak / James Shorter / Wah Chiu / Andrzej Joachimiak /
Abstract: Hsp104 is an AAA+ protein disaggregase with powerful amyloid-remodeling activity. All nonmetazoan eukaryotes express Hsp104 while eubacteria express an Hsp104 ortholog, ClpB. However, most studies ...Hsp104 is an AAA+ protein disaggregase with powerful amyloid-remodeling activity. All nonmetazoan eukaryotes express Hsp104 while eubacteria express an Hsp104 ortholog, ClpB. However, most studies have focused on Hsp104 from Saccharomyces cerevisiae and ClpB orthologs from two eubacterial species. Thus, the natural spectrum of Hsp104/ClpB molecular architectures and protein-remodeling activities remains largely unexplored. Here, we report two structures of Hsp104 from the thermophilic fungus Calcarisporiella thermophila (CtHsp104), a 2.70Å crystal structure and 4.0Å cryo-electron microscopy structure. Both structures reveal left-handed, helical assemblies with all domains clearly resolved. We thus provide the highest resolution and most complete view of Hsp104 hexamers to date. We also establish that CtHsp104 antagonizes several toxic protein-misfolding events in vivo where S. cerevisiae Hsp104 is ineffective, including rescue of TDP-43, polyglutamine, and α-synuclein toxicity. We suggest that natural Hsp104 variation is an invaluable, untapped resource for illuminating therapeutic disaggregases for fatal neurodegenerative diseases.
History
DepositionApr 9, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 3, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Jul 14, 2021Group: Database references / Category: citation / Item: _citation.journal_id_ISSN
Revision 1.3Mar 13, 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
1: Calcarisporiella thermophila Hsp104
2: Calcarisporiella thermophila Hsp104
3: Calcarisporiella thermophila Hsp104
4: Calcarisporiella thermophila Hsp104
5: Calcarisporiella thermophila Hsp104
6: Calcarisporiella thermophila Hsp104
hetero molecules


Theoretical massNumber of molelcules
Total (without water)598,59318
Polymers593,4676
Non-polymers5,12612
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: homology
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area46090 Å2
ΔGint-68 kcal/mol
Surface area228650 Å2

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Components

#1: Protein
Calcarisporiella thermophila Hsp104


Mass: 98911.109 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Calcarisporiella thermophila (fungus)
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A452CSQ7*PLUS
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Comment: ADP, 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: Calcarisporiella thermophila Hsp104 with ADP / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.6 MDa / Experimental value: YES
Source (natural)Organism: Calcarisporiella thermophila (fungus)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
Details: 20 mM HEPES-KOH, 100 mM KCl, 2 mM MgCl2, 2 mM ADP and 4 mM DTT
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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 magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 31.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3786
EM imaging opticsEnergyfilter name: GIF Quantum LS
Image scansMovie frames/image: 30 / Used frames/image: 3-30

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Processing

Software
NameVersionClassification
v1.9.1refinement
NAMDv2.12)refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
11RELION2.1classification
12RELION2.13D reconstruction
CTF correctionDetails: Gctf / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 372498
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 224915 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT

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