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- EMDB-2366: Electron cryo-microscopy of phosphorylation-mimicking mutants of ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2366
TitleElectron cryo-microscopy of phosphorylation-mimicking mutants of alphaB-crystallin: the hexamer
Map dataC3 reconstruction of hexameric alphaB phosphorylation mutants
Sample
  • Sample: Hexameric phosphorylation-mimicking mutant of alphaB-crystallin
  • Protein or peptide: Hexameric phosphorylation-mimicking mutant of human alphaB-crystallin
Keywordscryo electron microscopy / small heat shock protein / phosphorylation
Function / homology
Function and homology information


microtubule polymerization or depolymerization / negative regulation of intracellular transport / apoptotic process involved in morphogenesis / regulation of programmed cell death / cardiac myofibril / structural constituent of eye lens / tubulin complex assembly / negative regulation of amyloid fibril formation / lens development in camera-type eye / M band ...microtubule polymerization or depolymerization / negative regulation of intracellular transport / apoptotic process involved in morphogenesis / regulation of programmed cell death / cardiac myofibril / structural constituent of eye lens / tubulin complex assembly / negative regulation of amyloid fibril formation / lens development in camera-type eye / M band / muscle organ development / actin filament bundle / negative regulation of reactive oxygen species metabolic process / HSF1-dependent transactivation / stress-activated MAPK cascade / negative regulation of protein-containing complex assembly / muscle contraction / synaptic membrane / glutathione metabolic process / response to hydrogen peroxide / cellular response to gamma radiation / negative regulation of cell growth / Z disc / : / response to estradiol / amyloid-beta binding / response to heat / protein refolding / protein folding / microtubule binding / dendritic spine / perikaryon / response to hypoxia / lysosome / protein stabilization / negative regulation of gene expression / axon / negative regulation of DNA-templated transcription / negative regulation of apoptotic process / protein-containing complex binding / structural molecule activity / cell surface / protein homodimerization activity / protein-containing complex / mitochondrion / extracellular exosome / nucleoplasm / metal ion binding / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
Alpha-crystallin B chain, ACD domain / : / Alpha-crystallin, N-terminal / Alpha crystallin A chain, N terminal / Alpha crystallin/Small heat shock protein, animal type / Hsp20/alpha crystallin family / Small heat shock protein (sHSP) domain profile. / Alpha crystallin/Hsp20 domain / HSP20-like chaperone
Similarity search - Domain/homology
Alpha-crystallin B chain
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 17.0 Å
AuthorsPeschek J / Braun N / Rohrberg J / Back K / Kriehuber T / Kastenmueller A / Weinkauf S / Buchner J
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Regulated structural transitions unleash the chaperone activity of αB-crystallin.
Authors: Jirka Peschek / Nathalie Braun / Julia Rohrberg / Katrin Christiane Back / Thomas Kriehuber / Andreas Kastenmüller / Sevil Weinkauf / Johannes Buchner /
Abstract: The small heat shock protein αB-crystallin is an oligomeric molecular chaperone that binds aggregation-prone proteins. As a component of the proteostasis system, it is associated with cataract, ...The small heat shock protein αB-crystallin is an oligomeric molecular chaperone that binds aggregation-prone proteins. As a component of the proteostasis system, it is associated with cataract, neurodegenerative diseases, and myopathies. The structural determinants for the regulation of its chaperone function are still largely elusive. Combining different experimental approaches, we show that phosphorylation-induced destabilization of intersubunit interactions mediated by the N-terminal domain (NTD) results in the remodeling of the oligomer ensemble with an increase in smaller, activated species, predominantly 12-mers and 6-mers. Their 3D structures determined by cryo-electron microscopy and biochemical analyses reveal that the NTD in these species gains flexibility and solvent accessibility. These modulated properties are accompanied by an increase in chaperone activity in vivo and in vitro and a more efficient cooperation with the heat shock protein 70 system in client folding. Thus, the modulation of the structural flexibility of the NTD, as described here for phosphorylation, appears to regulate the chaperone activity of αB-crystallin rendering the NTD a conformational sensor for nonnative proteins.
History
DepositionApr 21, 2013-
Header (metadata) releaseMay 29, 2013-
Map releaseSep 25, 2013-
UpdateOct 9, 2013-
Current statusOct 9, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.087
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.087
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2366.png

Downloads & links

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Map

FileDownload / File: emd_2366.map.gz / Format: CCP4 / Size: 4.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC3 reconstruction of hexameric alphaB phosphorylation mutants
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.8 Å/pix.
x 108 pix.
= 194.4 Å		1.8 Å/pix.
x 108 pix.
= 194.4 Å		1.8 Å/pix.
x 108 pix.
= 194.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.087 / Movie #1: 0.087
Minimum - Maximum0.0 - 0.24658167
Average (Standard dev.)0.00632407 (±0.02604847)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions108108108
Spacing108108108
CellA=B=C: 194.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.81.81.8
M x/y/z108108108
origin x/y/z0.0000.0000.000
length x/y/z194.400194.400194.400
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS108108108
D min/max/mean0.0000.2470.006

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Supplemental data

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Sample components

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Entire : Hexameric phosphorylation-mimicking mutant of alphaB-crystallin

EntireName: Hexameric phosphorylation-mimicking mutant of alphaB-crystallin
Components
  • Sample: Hexameric phosphorylation-mimicking mutant of alphaB-crystallin
  • Protein or peptide: Hexameric phosphorylation-mimicking mutant of human alphaB-crystallin

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Supramolecule #1000: Hexameric phosphorylation-mimicking mutant of alphaB-crystallin

SupramoleculeName: Hexameric phosphorylation-mimicking mutant of alphaB-crystallin
type: sample / ID: 1000 / Details: The sample was polydisperse / Oligomeric state: Hexameric / Number unique components: 1
Molecular weightTheoretical: 121 KDa

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Macromolecule #1: Hexameric phosphorylation-mimicking mutant of human alphaB-crystallin

MacromoleculeName: Hexameric phosphorylation-mimicking mutant of human alphaB-crystallin
type: protein_or_peptide / ID: 1
Name.synonym: Alpha(B)-crystallin, Heat shock protein beta-5
Details: Hexameric phosphorylation-mimicking mutant of human alphaB-crystallin
Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 121 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Alpha-crystallin B chain

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.4 / Details: PBS
StainingType: NEGATIVE / Details: Cryo
VitrificationCryogen name: ETHANE / Instrument: OTHER

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Electron microscopy

MicroscopeOTHER
DateSep 2, 2010
Image recordingDigitization - Scanner: OTHER
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 49415 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

CTF correctionDetails: Each negative
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic / Number images used: 5617

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