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

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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 / chaperone / all-beta greek key
Function/homologychaperone complex / chloroplast nucleoid / chloroplast organization / response to high light intensity / Small heat shock protein HSP20 / Alpha crystallin/Hsp20 domain / Small heat shock protein (sHSP) domain profile. / Hsp20/alpha crystallin family / HSP20-like chaperone / response to light stimulus ...chaperone complex / chloroplast nucleoid / chloroplast organization / response to high light intensity / Small heat shock protein HSP20 / Alpha crystallin/Hsp20 domain / Small heat shock protein (sHSP) domain profile. / Hsp20/alpha crystallin family / HSP20-like chaperone / response to light stimulus / protein self-association / response to hydrogen peroxide / response to heat / regulation of transcription, DNA-templated / transcription, DNA-templated / Heat shock protein 21, chloroplastic
Function and homology information
Specimen sourceArabidopsis thaliana / plant / thale cress / /
MethodElectron microscopy (10 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsRutsdottir, G. / Harmark, J. / Koeck, P.J.B. / Hebert, H. / Soderberg, C.A.G. / Emanuelsson, C.
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 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.
Copyright: 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Apr 7, 2017 / Release: May 3, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0May 3, 2017Structure modelrepositoryInitial release
1.1May 24, 2017Structure modelDatabase references
1.2Aug 30, 2017Structure modelData collectionem_image_scans / em_software_em_software.name
1.3Sep 13, 2017Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization

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Structure visualization

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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
Polyers169,02712
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)22350
ΔGint (kcal/M)-71
Surface area (Å2)93570
MethodPISA

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Components

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


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


Mass: 11777.428 Da / Num. of mol.: 6
Source: (gene. exp.) Arabidopsis thaliana / plant / thale cress / /
Gene: HSP25.3, At4g27670, T29A15.160 / Production host: Escherichia coli / References: UniProt:P31170

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Hsp21 dodecamer, a chloroplast small heat shock protein chaperone
Type: COMPLEX / Entity ID: 1, 2 / Source: RECOMBINANT
Source (natural)Organism: Arabidopsis thaliana
Source (recombinant)Organism: Escherichia 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

MicroscopyMicroscope model: JEOL 2100F
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
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
12RELION3RECONSTRUCTION
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNumber of particles selected: 33456
SymmetryPoint symmetry: D3
3D reconstructionResolution: 1 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 18407 / Symmetry type: POINT

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