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- PDB-5nms: Hsp21 dodecamer, structural model based on cryo-EM and homology m... -

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Basic information

Entry
Database: PDB / ID: 5nms
TitleHsp21 dodecamer, structural model based on cryo-EM and homology modelling
Components(25.3 kDa heat shock protein, chloroplastic) x 2
KeywordsCHAPERONE / stress response / heat shock protein / all-beta greek key
Function / homology
Function and homology information


chloroplast nucleoid / chloroplast organization / protein folding chaperone complex / response to light stimulus / : / response to heat / regulation of DNA-templated transcription
Similarity search - Function
Heat shock protein 21-like / Hsp20/alpha crystallin family / Small heat shock protein (sHSP) domain profile. / Alpha crystallin/Hsp20 domain / HSP20-like chaperone
Similarity search - Domain/homology
Heat shock protein 21, chloroplastic
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 10 Å
AuthorsRutsdottir, G. / Harmark, J. / Koeck, P.J.B. / Hebert, H. / Soderberg, C.A.G. / Emanuelsson, C.
Funding support Sweden, Denmark, 4items
OrganizationGrant numberCountry
Carl Tryggers Foundation14-127 Sweden
Crafoord Foundation20140873 Sweden
Danish Council for Independent Research0602-02380B Denmark
Center for Innovative Medicine, KI Sweden
CitationJournal: J Biol Chem / Year: 2017
Title: Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity.
Authors: Gudrun Rutsdottir / Johan Härmark / Yoran Weide / Hans Hebert / Morten I Rasmussen / Sven Wernersson / Michal Respondek / Mikael Akke / Peter Højrup / Philip J B Koeck / Christopher A G ...Authors: Gudrun Rutsdottir / Johan Härmark / Yoran Weide / Hans Hebert / Morten I Rasmussen / Sven Wernersson / Michal Respondek / Mikael Akke / Peter Højrup / Philip J B Koeck / Christopher A G Söderberg / Cecilia Emanuelsson /
Abstract: Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have ...Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IVI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IVI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.
History
DepositionApr 7, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2017Provider: repository / Type: Initial release
SupersessionMay 24, 2017ID: 5MB8
Revision 1.1May 24, 2017Group: Database references
Revision 1.2Aug 30, 2017Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.name
Revision 1.3Sep 13, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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  • Deposited structure unit
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: 25.3 kDa heat shock protein, chloroplastic
B: 25.3 kDa heat shock protein, chloroplastic
C: 25.3 kDa heat shock protein, chloroplastic
E: 25.3 kDa heat shock protein, chloroplastic
D: 25.3 kDa heat shock protein, chloroplastic
F: 25.3 kDa heat shock protein, chloroplastic
G: 25.3 kDa heat shock protein, chloroplastic
H: 25.3 kDa heat shock protein, chloroplastic
I: 25.3 kDa heat shock protein, chloroplastic
K: 25.3 kDa heat shock protein, chloroplastic
J: 25.3 kDa heat shock protein, chloroplastic
L: 25.3 kDa heat shock protein, chloroplastic


Theoretical massNumber of molelcules
Total (without water)169,02712
Polymers169,02712
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area22350 Å2
ΔGint-71 kcal/mol
Surface area93570 Å2
MethodPISA

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Components

#1: Protein
25.3 kDa heat shock protein, chloroplastic / AtHsp25.3


Mass: 16393.787 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: HSP25.3, At4g27670, T29A15.160 / Production host: Escherichia coli (E. coli) / References: UniProt: P31170
#2: Protein
25.3 kDa heat shock protein, chloroplastic / AtHsp25.3


Mass: 11777.428 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: HSP25.3, At4g27670, T29A15.160 / Production host: Escherichia coli (E. coli) / References: UniProt: P31170

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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: Hsp21 dodecamer, a chloroplast small heat shock protein chaperone
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL 2100F
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 1.4 e/Å2 / Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k)

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
2EMAN2image acquisition
4EMAN2CTF correction
7UCSF Chimera11model fitting
9EMAN2initial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 33456
SymmetryPoint symmetry: D3 (2x3 fold dihedral)
3D reconstructionResolution: 10 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 18407 / Symmetry type: POINT

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