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- EMDB-2221: GroEL at sub-nanometer resolution by Constrained Single Particle ... -

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Basic information

Entry
Database: EMDB / ID: EMD-2221
TitleGroEL at sub-nanometer resolution by Constrained Single Particle Tomography
Map dataReconstruction of GroEL
Sample
  • Sample: GroEL
  • Protein or peptide: GroEL
Function / homology
Function and homology information


chaperonin ATPase / isomerase activity / ATP-dependent protein folding chaperone / unfolded protein binding / protein refolding / ATP binding / cytoplasm
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 speciesEscherichia coli UTI89 (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 8.4 Å
AuthorsBartesaghi A / Lecumberry F / Sapiro G / Subramaniam S
CitationJournal: Structure / Year: 2012
Title: Protein secondary structure determination by constrained single-particle cryo-electron tomography.
Authors: Alberto Bartesaghi / Federico Lecumberry / Guillermo Sapiro / Sriram Subramaniam /
Abstract: Cryo-electron microscopy (cryo-EM) is a powerful technique for 3D structure determination of protein complexes by averaging information from individual molecular images. The resolutions that can be ...Cryo-electron microscopy (cryo-EM) is a powerful technique for 3D structure determination of protein complexes by averaging information from individual molecular images. The resolutions that can be achieved with single-particle cryo-EM are frequently limited by inaccuracies in assigning molecular orientations based solely on 2D projection images. Tomographic data collection schemes, however, provide powerful constraints that can be used to more accurately determine molecular orientations necessary for 3D reconstruction. Here, we propose "constrained single-particle tomography" as a general strategy for 3D structure determination in cryo-EM. A key component of our approach is the effective use of images recorded in tilt series to extract high-resolution information and correct for the contrast transfer function. By incorporating geometric constraints into the refinement to improve orientational accuracy of images, we reduce model bias and overrefinement artifacts and demonstrate that protein structures can be determined at resolutions of ∼8 Å starting from low-dose tomographic tilt series.
History
DepositionOct 15, 2012-
Header (metadata) releaseOct 24, 2012-
Map releaseDec 12, 2012-
UpdateDec 19, 2012-
Current statusDec 19, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.64
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.64
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-2ynj
  • Surface level: 1.64
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2221.png

Downloads & links

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Map

FileDownload / File: emd_2221.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of GroEL
Voxel sizeX=Y=Z: 1.74 Å
Density
Contour LevelBy AUTHOR: 1.64 / Movie #1: 1.64
Minimum - Maximum-1.52058649 - 3.54873729
Average (Standard dev.)0.0 (±0.28805894)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-89-89-89
Dimensions180180180
Spacing180180180
CellA=B=C: 313.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.741.741.74
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z313.200313.200313.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS-89-89-89
NC/NR/NS180180180
D min/max/mean-1.5213.549-0.000

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Supplemental data

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Sample components

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Entire : GroEL

EntireName: GroEL
Components
  • Sample: GroEL
  • Protein or peptide: GroEL

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Supramolecule #1000: GroEL

SupramoleculeName: GroEL / type: sample / ID: 1000 / Oligomeric state: 14-mer / Number unique components: 1
Molecular weightTheoretical: 800 KDa

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Macromolecule #1: GroEL

MacromoleculeName: GroEL / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: 14-mer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli UTI89 (bacteria)
Molecular weightTheoretical: 800 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration3 mg/mL
BufferpH: 7.5
Details: 100 mM Hepes, pH 7.5, 10 mM Mg(OAc)2, 10 mM KOAc, 2 mM DTT
GridDetails: 400 mesh C-flat, 2um hole size (CF/2/2 grids), plasma cleaned for 6s with a Gatan Solarus plasma cleaner.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 4 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 80 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 47000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 47000
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt series - Axis1 - Min angle: -20 ° / Tilt series - Axis1 - Max angle: 0 °
TemperatureMin: 80 K / Max: 93 K / Average: 80 K
Alignment procedureLegacy - Astigmatism: Astigmatism corrected at 76000x
DetailsThe total dose of 25 (electrons/square Angstrom) was fractionated evenly across 11 tilted projections taken between 0 and -20 degrees tilt (every 2 degrees).
DateOct 7, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Number real images: 1595 / Average electron dose: 25 e/Å2
Details: The 1595 micrographs corresponded to 145 tilt-series each containing 11 projections with tilt-angles between 0 and -20 degrees (every 2-degrees).
Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Defocus values were assigned to each particle projection based on the defocus at the untilted plane of each tilt-series and a correction according to the relative height of each particle. to this plane
Final angle assignmentDetails: FREALIGN
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.4 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: FREALIGN
Details: Final map was amplitude corrected with BSOFT's command 'bampweight' using a density map generated from the PDB ID 3e76 coordinates as a reference.
DetailsParticles were manually selected in 3D from reconstructed tomograms and their corresponding raw 2D projections extracted for further processing using Constrained Single Particle Tomography. Average number of projections used in the 3D reconstructions: 10000.

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Atomic model buiding 1

Initial modelPDB ID:

3e76


Chain - Chain ID: H
SoftwareName: Chimera
DetailsProtocol: Rigid body. Coordinates of chain H from 3e76 were fit using D7-symmetric fitting operation in Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation
Output model

PDB-2ynj:
GroEL at sub-nanometer resolution by Constrained Single Particle Tomography

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