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- EMDB-2290: First 3D model of wild type MjHSP16.5 at 60 degree Celsius by CryoEM. -

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Basic information

Entry
Database: EMDB / ID: EMD-2290
TitleFirst 3D model of wild type MjHSP16.5 at 60 degree Celsius by CryoEM.
Map dataFirst model for reconstruction of wild type MjHSP16.5 at 60 degree Celsius
Sample
  • Sample: wild type small heat shock protein (sHSP) HSP16.5 from Methanocaldococcus jannaschii (MjHSP16.5)
  • Protein or peptide: MjHSP16.5
KeywordsSmall heat shock protein / HSP16.5
Function / homology
Function and homology information


response to stress / protein complex oligomerization / response to salt stress / protein folding chaperone / response to hydrogen peroxide / unfolded protein binding / protein folding / response to heat / protein stabilization / protein-containing complex ...response to stress / protein complex oligomerization / response to salt stress / protein folding chaperone / response to hydrogen peroxide / unfolded protein binding / protein folding / response to heat / protein stabilization / protein-containing complex / identical protein binding / cytoplasm
Similarity search - Function
: / 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
Small heat shock protein HSP16.5
Similarity search - Component
Biological speciesMethanocaldococcus jannaschii (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 14.0 Å
AuthorsRoy A Q / Yan Z / Andrew L / Ian W / Ehmke P / Fei S
CitationJournal: Philos Trans R Soc Lond B Biol Sci / Year: 2013
Title: Changes in the quaternary structure and function of MjHSP16.5 attributable to deletion of the IXI motif and introduction of the substitution, R107G, in the α-crystallin domain.
Authors: Roy A Quinlan / Yan Zhang / Andrew Lansbury / Ian Williamson / Ehmke Pohl / Fei Sun /
Abstract: The archael small heat-shock protein (sHSP), MjHSP16.5, forms a 24-subunit oligomer with octahedral symmetry. Here, we demonstrate that the IXI motif present in the C-terminal domain is necessary for ...The archael small heat-shock protein (sHSP), MjHSP16.5, forms a 24-subunit oligomer with octahedral symmetry. Here, we demonstrate that the IXI motif present in the C-terminal domain is necessary for the oligomerization of MjHSP16.5. Removal increased the in vitro chaperone activity with citrate synthase as the client protein. Less predictable were the effects of the R107G substitution in MjHSP16.5 because of the differences in the oligomerization of metazoan and non-metazoan sHSPs. We present the crystal structure for MjHSP16.5 R107G and compare this with an improved (2.5 Å) crystal structure for wild-type (WT) MjHSP16.5. Although no significant structural differences were found in the crystal, using cryo-electron microscopy, we identified two 24mer species with octahedral symmetry for the WT MjHSP16.5 both at room temperature and at 60°C, all showing two major species with the same diameter of 12.4 nm. Similarly, at room temperature, there are also two kinds of 12.4 nm oligomers for R107G MjHSP16.5, but in the 60°C sample, a larger 24mer species with a diameter of 13.6 nm was observed with significant changes in the fourfold symmetry axis and dimer-dimer interface. This highly conserved arginine, therefore, contributes to the quaternary organization of non-metazoan sHSP oligomers. Potentially, the R107G substitution has functional consequences as R107G MjHSP16.5 was far superior to the WT protein in protecting βL-crystallin against heat-induced aggregation.
History
DepositionJan 21, 2013-
Header (metadata) releaseFeb 27, 2013-
Map releaseMar 13, 2013-
UpdateJul 24, 2013-
Current statusJul 24, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.73
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.73
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

2290_EMDB-22...

Downloads & links

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Map

FileDownload / File: emd_2290.map.gz / Format: CCP4 / Size: 62.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFirst model for reconstruction of wild type MjHSP16.5 at 60 degree Celsius
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 256 pix.
= 238.848 Å		0.93 Å/pix.
x 256 pix.
= 238.848 Å		0.93 Å/pix.
x 256 pix.
= 238.848 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.933 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.73 / Movie #1: 1.73
Minimum - Maximum-6.68931007 - 8.134586329999999
Average (Standard dev.)0.00061734 (±1.02556777)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions256256256
Spacing256256256
CellA=B=C: 238.848 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.9330.9330.933
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z238.848238.848238.848
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS256256256
D min/max/mean-6.6898.1350.001

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Supplemental data

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Sample components

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Entire : wild type small heat shock protein (sHSP) HSP16.5 from Methanocal...

EntireName: wild type small heat shock protein (sHSP) HSP16.5 from Methanocaldococcus jannaschii (MjHSP16.5)
Components
  • Sample: wild type small heat shock protein (sHSP) HSP16.5 from Methanocaldococcus jannaschii (MjHSP16.5)
  • Protein or peptide: MjHSP16.5

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Supramolecule #1000: wild type small heat shock protein (sHSP) HSP16.5 from Methanocal...

SupramoleculeName: wild type small heat shock protein (sHSP) HSP16.5 from Methanocaldococcus jannaschii (MjHSP16.5)
type: sample / ID: 1000 / Oligomeric state: 24mer / Number unique components: 1
Molecular weightExperimental: 396 KDa / Theoretical: 396 KDa

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Macromolecule #1: MjHSP16.5

MacromoleculeName: MjHSP16.5 / type: protein_or_peptide / ID: 1 / Name.synonym: small heat shock protein (sHSP) from HSP16.5 / Number of copies: 24 / Oligomeric state: 24mer / Recombinant expression: Yes
Source (natural)Organism: Methanocaldococcus jannaschii (archaea) / Location in cell: cytoplasmic
Molecular weightExperimental: 16.5 KDa / Theoretical: 16.5 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET23b
SequenceUniProtKB: Small heat shock protein HSP16.5 / GO: cytoplasm, response to stress
InterPro: Alpha crystallin/Hsp20 domain, HSP20-like chaperone

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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

Concentration0.8 mg/mL
BufferpH: 7.4 / Details: 10mM HEPES, 100mM NaCl.
GridDetails: GIG holey grids (LifeTrust, China) were treated with a glow discharge machine (Master Plasmer)
VitrificationCryogen name: ETHANE / Chamber temperature: 9 K / Instrument: FEI VITROBOT MARK IV
Method: The samples were first heated to 60 degree Celsius in the boiler, then 3.5 microlitre samples were added to the grid and blotted for 1.5 s with blot force 2 at 100% humidity;

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureAverage: 85 K
DateOct 11, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 2119 / Average electron dose: 20 e/Å2
Details: Electron micrograph exposures were made with the automatic collection package Leginon.
Bits/pixel: 32
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 96000
Sample stageSpecimen holder: liquid nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected using an automatic selection program Gautomatch developed in Fei Sun lab (to be published). Octahedron symmetry were imposed during 3D reconstructing.
CTF correctionDetails: Each image
Final reconstructionApplied symmetry - Point group: O (octahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 14.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN1
Details: The final reconstructed density map was further sharpened by application of an amplitude correction algorithm in the program BFACTOR.
Number images used: 4842
Final two d classificationNumber classes: 193

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