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- EMDB-2800: A Novel Mechanism for Small Heat Shock Proteins to Function as Mo... -

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Basic information

Entry
Database: EMDB / ID: EMD-2800
TitleA Novel Mechanism for Small Heat Shock Proteins to Function as Molecular Chaperones
Map dataat low temperature, resting state
Sample
  • Sample: small heat shock protein 17 from C.elegans
  • Protein or peptide: Small heat shock protein 17 from C. elegans
Keywordssmall heat shock protein mechanism / oligomer
Function / homology
Function and homology information


VEGFA-VEGFR2 Pathway / unfolded protein binding / response to heat / protein refolding / nucleus / cytoplasm
Similarity search - Function
Alpha crystallin/Small heat shock protein, animal type / Alpha crystallin/Small heat shock protein, animal type / Hsp20/alpha crystallin family / Small heat shock protein (sHSP) domain profile. / Alpha crystallin/Hsp20 domain / Alpha crystallin/Hsp20 domain / HSP20-like chaperone / HSP20-like chaperone
Similarity search - Domain/homology
SHSP domain-containing protein
Similarity search - Component
Biological speciesCaenorhabditis elegans (invertebrata)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 14.5 Å
AuthorsKaiming Z / Anastasia NE / Zhao W / Hongli H / Xiaodong S / Chuang L / Xinping L / Xinmiao F / Zengyi C / Chang-cheng Y
CitationJournal: Sci Rep / Year: 2015
Title: A novel mechanism for small heat shock proteins to function as molecular chaperones.
Authors: Kaiming Zhang / Anastasia N Ezemaduka / Zhao Wang / Hongli Hu / Xiaodong Shi / Chuang Liu / Xinping Lu / Xinmiao Fu / Zengyi Chang / Chang-Cheng Yin /
Abstract: Small heat shock proteins (sHSPs) are molecular chaperones ubiquitously present in all forms of life, but their function mechanisms remain controversial. Here we show by cryo-electron microscopy and ...Small heat shock proteins (sHSPs) are molecular chaperones ubiquitously present in all forms of life, but their function mechanisms remain controversial. Here we show by cryo-electron microscopy and single particle 3D reconstruction that, at the low temperatures (4-25°C), CeHSP17 (a sHSP from Caenorhabditis elegans) exists as a 24-subunit spherical oligomer with tetrahedral symmetry. Our studies demonstrate that CeHSP17 forms large sheet-like super-molecular assemblies (SMAs) at the high temperatures (45-60°C), and such SMAs are apparently the form that exhibits chaperone-like activity. Our findings suggest a novel molecular mechanism for sHSPs to function as molecular chaperones.
History
DepositionOct 16, 2014-
Header (metadata) releaseOct 29, 2014-
Map releaseApr 1, 2015-
UpdateApr 1, 2015-
Current statusApr 1, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.97
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.97
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol

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Map

FileDownload / File: emd_2800.map.gz / Format: CCP4 / Size: 28.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationat low temperature, resting state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.36 Å/pix.
x 196 pix.
= 266.56 Å		1.36 Å/pix.
x 196 pix.
= 266.56 Å		1.36 Å/pix.
x 196 pix.
= 266.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.36 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.97 / Movie #1: 0.97
Minimum - Maximum-1.54168975 - 3.58621669
Average (Standard dev.)0.03756366 (±0.36322927)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-98-98-98
Dimensions196196196
Spacing196196196
CellA=B=C: 266.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z196196196
origin x/y/z0.0000.0000.000
length x/y/z266.560266.560266.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-98-98-98
NC/NR/NS196196196
D min/max/mean-1.5423.5860.038

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Supplemental data

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Sample components

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Entire : small heat shock protein 17 from C.elegans

EntireName: small heat shock protein 17 from C.elegans
Components
  • Sample: small heat shock protein 17 from C.elegans
  • Protein or peptide: Small heat shock protein 17 from C. elegans

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Supramolecule #1000: small heat shock protein 17 from C.elegans

SupramoleculeName: small heat shock protein 17 from C.elegans / type: sample / ID: 1000 / Details: monomer with his-tag is about 18.5kDa / Oligomeric state: 24 monomers / Number unique components: 1
Molecular weightExperimental: 460 KDa / Theoretical: 440 KDa
Method: Size exclusion chromatography, SDS-PAGE gel,Native gel

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Macromolecule #1: Small heat shock protein 17 from C. elegans

MacromoleculeName: Small heat shock protein 17 from C. elegans / type: protein_or_peptide / ID: 1 / Name.synonym: CeHSP17
Details: HSP17 encodes a heat shock protein that is a member of the HSP16/HSP20/alpha-crystallin family of heat shock proteins; by homology, HSP17 is predicted to function as a molecular chaperone ...Details: HSP17 encodes a heat shock protein that is a member of the HSP16/HSP20/alpha-crystallin family of heat shock proteins; by homology, HSP17 is predicted to function as a molecular chaperone that protects cells from heat-induced protein aggregation and denaturation. At present, the precise developmental and/or behavioral role of HSP17, as well as its expression pattern, are not yet known. [Source: WormBase]
Number of copies: 24 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Caenorhabditis elegans (invertebrata) / Strain: N2 Bristol
Molecular weightExperimental: 460 KDa / Theoretical: 440 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET21a
SequenceUniProtKB: SHSP domain-containing protein
InterPro: Alpha crystallin/Small heat shock protein, animal type, Alpha crystallin/Hsp20 domain, HSP20-like chaperone

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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

Concentration0.4 mg/mL
BufferpH: 8 / Details: 20 mM Tris-HCl, 150 mM NaCl
StainingType: NEGATIVE
Details: Grids were negatively stained with 2% (w/v) uranyl acetate for 10 s.
GridDetails: glow-discharged 200-mesh R1.2/1.3 Quantifoil grid
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV
Timed resolved state: Vitrified 30 msec after spraying with effector
Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureMin: 80 K / Max: 105 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateAug 13, 2013
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 106 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 80000
Sample stageSpecimen holder: 626 / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe image processing software package EMAN2, was used for the micrograph evaluation, particle picking, CTF correction, 2-D reference-free class averaging, initial model building and 3-D refinement.
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.5 Å / Resolution method: OTHER / Software - Name: EMAN2 / Details: gold stardard refinement / Number images used: 14217
Final two d classificationNumber classes: 8

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Atomic model buiding 1

Initial modelPDB ID:

1gme


Chain - Chain ID: B
SoftwareName: Chimera
Detailsusing one dimer of the homologue protein HSP16.9, applying the tetrahedral symmetry
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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