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- EMDB-12771: Single particle cryo-EM reconstruction of a 40-mer assembly of re... -

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Basic information

Entry
Database: EMDB / ID: EMD-12771
TitleSingle particle cryo-EM reconstruction of a 40-mer assembly of recombinant yeast Hsp26 S207E mutant.
Map dataCryo-EM Single particle Reconstruction of a 40mer oligomer of yeast Hsp26 S207E mutant
Sample
  • Complex: 40mer complex of S207E mutant of yeast Hsp26
Function / homology
Function and homology information


cytoplasmic stress granule / unfolded protein binding / protein folding / cellular response to heat / mRNA binding / mitochondrion / identical protein binding / nucleus / cytoplasm
Similarity search - Function
: / 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 26
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.76 Å
AuthorsMuehlhofer M / Peters C / Kriehuber T / Kreuzeder M / Kazman P / Rodina N / Reif B / Haslbeck M / Weinkauf S / Buchner J
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB 1035 Germany
CitationJournal: Nat Commun / Year: 2021
Title: Phosphorylation activates the yeast small heat shock protein Hsp26 by weakening domain contacts in the oligomer ensemble.
Authors: Moritz Mühlhofer / Carsten Peters / Thomas Kriehuber / Marina Kreuzeder / Pamina Kazman / Natalia Rodina / Bernd Reif / Martin Haslbeck / Sevil Weinkauf / Johannes Buchner /
Abstract: Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. Its chaperone activity is activated by oligomer dissociation at heat shock temperatures. Hsp26 contains 9 phosphorylation sites in ...Hsp26 is a small heat shock protein (sHsp) from S. cerevisiae. Its chaperone activity is activated by oligomer dissociation at heat shock temperatures. Hsp26 contains 9 phosphorylation sites in different structural elements. Our analysis of phospho-mimetic mutations shows that phosphorylation activates Hsp26 at permissive temperatures. The cryo-EM structure of the Hsp26 40mer revealed contacts between the conserved core domain of Hsp26 and the so-called thermosensor domain in the N-terminal part of the protein, which are targeted by phosphorylation. Furthermore, several phosphorylation sites in the C-terminal extension, which link subunits within the oligomer, are sensitive to the introduction of negative charges. In all cases, the intrinsic inhibition of chaperone activity is relieved and the N-terminal domain becomes accessible for substrate protein binding. The weakening of domain interactions within and between subunits by phosphorylation to activate the chaperone activity in response to proteotoxic stresses independent of heat stress could be a general regulation principle of sHsps.
History
DepositionApr 19, 2021-
Header (metadata) releaseNov 24, 2021-
Map releaseNov 24, 2021-
UpdateDec 1, 2021-
Current statusDec 1, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.274
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.274
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12771.png

Downloads & links

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Map

FileDownload / File: emd_12771.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM Single particle Reconstruction of a 40mer oligomer of yeast Hsp26 S207E mutant
Voxel sizeX=Y=Z: 1.33 Å
Density
Contour LevelBy AUTHOR: 0.274 / Movie #1: 0.274
Minimum - Maximum-0.23992974 - 0.7651763
Average (Standard dev.)0.006303423 (±0.05936104)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 383.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.331.331.33
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z383.040383.040383.040
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ384384384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.2400.7650.006

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Supplemental data

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Sample components

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Entire : 40mer complex of S207E mutant of yeast Hsp26

EntireName: 40mer complex of S207E mutant of yeast Hsp26
Components
  • Complex: 40mer complex of S207E mutant of yeast Hsp26

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Supramolecule #1: 40mer complex of S207E mutant of yeast Hsp26

SupramoleculeName: 40mer complex of S207E mutant of yeast Hsp26 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: HB101 / Recombinant plasmid: pQE60::HSP26
Molecular weightTheoretical: 955.2 KDa

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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: 7.4
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 294 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 5010 / Average exposure time: 8.0 sec. / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 688419
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: ab initio reconstruction with cryosparc
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 8.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 165455
FSC plot (resolution estimation)

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