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- EMDB-4185: Human GroEL obtained by grid-blotting from blue native PAGE -

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Basic information

Entry
Database: EMDB / ID: EMD-4185
TitleHuman GroEL obtained by grid-blotting from blue native PAGE
Map dataGrid blotted from blue native PAGE human GroEL
Sample
  • Complex: GroEL only
Function / homology
Function and homology information


GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat ...GroEL-GroES complex / chaperonin ATPase / virion assembly / chaperone cofactor-dependent protein refolding / isomerase activity / ATP-dependent protein folding chaperone / response to radiation / unfolded protein binding / protein folding / response to heat / protein refolding / magnesium ion binding / ATP hydrolysis activity / ATP binding / membrane / identical protein binding / cytosol
Similarity search - Function
Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/GroEL / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like apical domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonin Cpn60/GroEL/TCP-1 family
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsKirykowicz AM / Woodward JD
CitationJournal: To Be Published
Title: Human GroEL obtained by grid-blotting from blue native PAGE
Authors: Kirykowicz AM / Woodward JD
History
DepositionNov 30, 2017-
Header (metadata) releaseDec 13, 2017-
Map releaseDec 5, 2018-
UpdateDec 5, 2018-
Current statusDec 5, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.45
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.45
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4185.png

Downloads & links

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Map

FileDownload / File: emd_4185.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGrid blotted from blue native PAGE human GroEL
Voxel sizeX=Y=Z: 4.22 Å
Density
Contour LevelBy AUTHOR: 2.45 / Movie #1: 2.45
Minimum - Maximum-3.3466055 - 6.095054
Average (Standard dev.)0.000000006981233 (±1.)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 270.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.224.224.22
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z270.080270.080270.080
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-3.3476.0950.000

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Supplemental data

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Sample components

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Entire : GroEL only

EntireName: GroEL only
Components
  • Complex: GroEL only

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Supramolecule #1: GroEL only

SupramoleculeName: GroEL only / type: complex / ID: 1 / Parent: 0 / Details: Obtained from Sigma-Aldrich
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 840 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7
Details: Diluted in 50 mM Tris-HCl, 200 mM NaCl (pH 8.0). 1-5 ug of GroEL run on blue native page: Anode buffer: 25 mM Imidazole Cathode buffer A: 50 mM Tricine, 7.5 mM Imidazole, 0.02% Coomassie ...Details: Diluted in 50 mM Tris-HCl, 200 mM NaCl (pH 8.0). 1-5 ug of GroEL run on blue native page: Anode buffer: 25 mM Imidazole Cathode buffer A: 50 mM Tricine, 7.5 mM Imidazole, 0.02% Coomassie G250 (pH 7.0) Cathode buffer B: 50 mM Tricine, 7.5 mM Imidazole, 0.002% Coomassie G250 (pH 7.0)
StainingType: NEGATIVE / Material: Uranyl Acetate
Details: 1-5 ug of groEL was run on a blue native PAGE gel. Sample was transferred to grid by grid blotting procedure and washed/stained with 2% UA.
GridMaterial: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
DetailsBought from Sigma-Aldrich (Germany)

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.2 mm / Nominal magnification: 50000
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average exposure time: 0.5 sec. / Average electron dose: 20.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 463 / Details: Manual picking
Startup modelType of model: OTHER
Details: Starting model generated from EMAN Common Lines with C7 symmetry imposed
Initial angle assignmentType: COMMON LINE / Details: EMAN
Final angle assignmentType: PROJECTION MATCHING
Details: EMAN procedure: 5 iteration of 10 degrees (19 classes generated), 5 iterations of 8 degrees (23 classes generated), 10 iterations of 5 degrees (61 classes generated), and 6 iterations of 3 ...Details: EMAN procedure: 5 iteration of 10 degrees (19 classes generated), 5 iterations of 8 degrees (23 classes generated), 10 iterations of 5 degrees (61 classes generated), and 6 iterations of 3 degrees (155 classes generated)
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Details: EMAN procedure / Number images used: 463

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