|Entry||Database: PDB / ID: 5nms|
|Title||Hsp21 dodecamer, structural model based on cryo-EM and homology modelling|
|Components||(25.3 kDa heat shock protein, chloroplastic) x 2|
|Keywords||CHAPERONE / stress response / heat shock protein / chaperone / all-beta greek key|
|Function / homology||Alpha crystallin/Hsp20 domain / HSP20-like chaperone / Small heat shock protein HSP20 / Hsp20/alpha crystallin family / Small heat shock protein (sHSP) domain profile. / chloroplast nucleoid / chloroplast organization / response to high light intensity / chaperone complex / response to light stimulus ...Alpha crystallin/Hsp20 domain / HSP20-like chaperone / Small heat shock protein HSP20 / Hsp20/alpha crystallin family / Small heat shock protein (sHSP) domain profile. / chloroplast nucleoid / chloroplast organization / response to high light intensity / chaperone complex / 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 source||Arabidopsis thaliana (thale cress)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 10 Å resolution|
|Authors||Rutsdottir, G. / Harmark, J. / Koeck, P.J.B. / Hebert, H. / Soderberg, C.A.G. / Emanuelsson, C.|
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Date||Deposition: Apr 7, 2017 / Release: May 3, 2017|
|Structure viewer||Molecule: |
Downloads & links
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
Mass: 16393.787 Da / Num. of mol.: 6 / Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: HSP25.3, At4g27670, T29A15.160 / Production host: Escherichia coli (E. coli) / References: UniProt: P31170
Mass: 11777.428 Da / Num. of mol.: 6 / Source: (gene. exp.) Arabidopsis thaliana (thale cress) / Gene: HSP25.3, At4g27670, T29A15.160 / Production host: Escherichia coli (E. coli) / References: UniProt: P31170
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: Hsp21 dodecamer, a chloroplast small heat shock protein chaperone|
Type: COMPLEX / Entity ID: 1,
|Source (natural)||Organism: Arabidopsis thaliana (thale cress)||Source (recombinant)||Organism: Escherichia coli (E. coli)||Buffer solution||pH: 8||Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES||Vitrification||Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE|
-Electron microscopy imaging
|Microscopy||Microscope model: JEOL 2100F|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 1.4 e/Å2 / Film or detector model: DIRECT ELECTRON DE-20 (5k x 3k)|
|CTF correction||Type: PHASE FLIPPING ONLY|
|Particle selection||Number of particles selected: 33456|
|Symmetry||Point symmetry: D3|
|3D reconstruction||Resolution: 1 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 18407 / Symmetry type: POINT|
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