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- PDB-7pe3: Pseudo-atomic model of the tetrahedral 24mer of Hsp17 from Caenor... -

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Basic information

Entry
Database: PDB / ID: 7pe3
TitlePseudo-atomic model of the tetrahedral 24mer of Hsp17 from Caenorhabditis elegans
ComponentsSHSP domain-containing protein
KeywordsCHAPERONE / small heat shock protein / Caenorhabditis elegans / proteostasis
Function / homologyAlpha crystallin/Small heat shock protein, animal type / Hsp20/alpha crystallin family / Small heat shock protein (sHSP) domain profile. / Alpha crystallin/Hsp20 domain / HSP20-like chaperone / unfolded protein binding / response to heat / protein refolding / SHSP domain-containing protein
Function and homology information
Biological speciesCaenorhabditis elegans (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.49 Å
AuthorsRossa, B. / Weinkauf, S. / Buchner, J.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB1035 Germany
CitationJournal: J Biol Chem / Year: 2023
Title: The permanently chaperone-active small heat shock protein Hsp17 from Caenorhabditis elegans exhibits topological separation of its N-terminal regions.
Authors: Annika Strauch / Benjamin Rossa / Fabian Köhler / Simon Haeussler / Moritz Mühlhofer / Florian Rührnößl / Caroline Körösy / Yevheniia Bushman / Barbara Conradt / Martin Haslbeck / ...Authors: Annika Strauch / Benjamin Rossa / Fabian Köhler / Simon Haeussler / Moritz Mühlhofer / Florian Rührnößl / Caroline Körösy / Yevheniia Bushman / Barbara Conradt / Martin Haslbeck / Sevil Weinkauf / Johannes Buchner /
Abstract: Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, ...Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under nonstress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps.
History
DepositionAug 9, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 14, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 4, 2023Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: SHSP domain-containing protein
B: SHSP domain-containing protein


Theoretical massNumber of molelcules
Total (without water)34,8852
Polymers34,8852
Non-polymers00
Water00
1
A: SHSP domain-containing protein
B: SHSP domain-containing protein
x 12


Theoretical massNumber of molelcules
Total (without water)418,62024
Polymers418,62024
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation11

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Components

#1: Protein SHSP domain-containing protein


Mass: 17442.510 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Caenorhabditis elegans (invertebrata) / Gene: hsp-17, CELE_F52E1.7, F52E1.7 / Plasmid: pET21a / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q7JP52

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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: Tetrahedral oligomer (24mer) of Hsp17 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.422 MDa / Experimental value: NO
Source (natural)Organism: Caenorhabditis elegans (invertebrata)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Plasmid: pET21a
Buffer solutionpH: 7.4 / Details: PBS
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 70 % / Chamber temperature: 298 K
Details: waiting time 0, blotting force 5, blotting time 2.5

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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: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2568
Details: Images were recorded in CDS mode as tif-stacks with 30 frames.
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2cryoSPARC2particle selectiontemplate picking
5Gctf1.06CTF correction
8UCSF Chimera1.14model fittingfit_in_map was used for rigid body fitting
9NAMD2.14model fittingMDFF was used for flexible fitting
11PHENIX1.19.2model refinementphenix.real_space_refinement was used iteratively
12Coot0.9.5model refinementcoot was used to rebuild Ramachandran- and sidechain outliers
13cryoSPARC2initial Euler assignment
14cryoSPARC2final Euler assignment
16cryoSPARC23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 663141
Details: Gautomatch automatic picking yielded an initial dataset that was used to generate templates for cryoSPARC2 template picking.
SymmetryPoint symmetry: T (tetrahedral)
3D reconstructionResolution: 6.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 187116 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0032295
ELECTRON MICROSCOPYf_angle_d0.6773106
ELECTRON MICROSCOPYf_dihedral_angle_d6.172299
ELECTRON MICROSCOPYf_chiral_restr0.064330
ELECTRON MICROSCOPYf_plane_restr0.004418

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