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- EMDB-3459: Hsp21 dodecamer, structural model based on cryo-EM and homology m... -

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Basic information

Entry
Database: EMDB / ID: EMD-3459
TitleHsp21 dodecamer, structural model based on cryo-EM and homology modelling
Map data
Sample
  • Complex: Hsp21 dodecamer
    • Protein or peptide: 25.3 kDa heat shock protein, chloroplastic
    • Protein or peptide: 25.3 kDa heat shock protein, chloroplastic
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.0 Å
AuthorsRutsdottir G / Harmark J / Koeck PJB / Hebert H / Soderberg CAG / 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 ...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
DepositionNov 7, 2016-
Header (metadata) releaseDec 28, 2016-
Map releaseMar 29, 2017-
UpdateMay 24, 2017-
Current statusMay 24, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5nms
  • Surface level: 1.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3459.png

Downloads & links

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Map

FileDownload / File: emd_3459.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.28 Å/pix.
x 192 pix.
= 245.76 Å		1.28 Å/pix.
x 192 pix.
= 245.76 Å		1.28 Å/pix.
x 192 pix.
= 245.76 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.28 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.1 / Movie #1: 1.1
Minimum - Maximum-1.1792046 - 5.7428164
Average (Standard dev.)0.039059773 (±0.24239069)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 245.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.281.281.28
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z245.760245.760245.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-1.1795.7430.039

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Supplemental data

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Sample components

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Entire : Hsp21 dodecamer

EntireName: Hsp21 dodecamer
Components
  • Complex: Hsp21 dodecamer
    • Protein or peptide: 25.3 kDa heat shock protein, chloroplastic
    • Protein or peptide: 25.3 kDa heat shock protein, chloroplastic

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Supramolecule #1: Hsp21 dodecamer

SupramoleculeName: Hsp21 dodecamer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pJC20
Molecular weightExperimental: 252 KDa

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Macromolecule #1: 25.3 kDa heat shock protein, chloroplastic

MacromoleculeName: 25.3 kDa heat shock protein, chloroplastic / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 16.393787 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
LLDPLSPMRT MRQMLDTMDR MFEDTMPVSG RNRGGSGVSE IRAPWDIKEE EHEIKMRFDM PGLSKEDVKI SVEDNVLVIK GEQKKEDSD DSWSGRSVSS YGTRLQLPDN CEKDKIKAEL KNGVLFITIP KTKVERKVID VQIQ

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Macromolecule #2: 25.3 kDa heat shock protein, chloroplastic

MacromoleculeName: 25.3 kDa heat shock protein, chloroplastic / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 11.777428 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
RAPWDIKEEE HEIKMRFDMP GLSKEDVKIS VEDNVLVIKG EQKKEDSDDS WSGRSVSSYG TRLQLPDNCE KDKIKAELKN GVLFITIPK TKVERKVIDV QIQ

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
GridModel: Quantifoil R2/4 / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL 2100F
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Average electron dose: 1.4 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 18407
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: COMMON LINE
FSC plot (resolution estimation)

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