|Entry||Database: PDB / ID: 2ynj|
|Title||GroEL at sub-nanometer resolution by Constrained Single Particle Tomography|
|Components||60 KDA CHAPERONIN|
|Function / homology||Chaperonin Cpn60 / Chaperonin Cpn60/TCP-1 family / Chaperonin Cpn60, conserved site / GroEL-like apical domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like equatorial domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonins cpn60 signature. / unfolded protein binding / protein refolding ...Chaperonin Cpn60 / Chaperonin Cpn60/TCP-1 family / Chaperonin Cpn60, conserved site / GroEL-like apical domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like equatorial domain superfamily / TCP-1/cpn60 chaperonin family / Chaperonins cpn60 signature. / unfolded protein binding / protein refolding / ATP binding / cytoplasm / 60 kDa chaperonin|
Function and homology information
|Specimen source||ESCHERICHIA COLI UTI89 (bacteria)|
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 8.4 Å resolution|
|Authors||Bartesaghi, A. / Lecumberry, F. / Sapiro, G. / Subramaniam, S.|
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Date||Deposition: Oct 15, 2012 / Release: Dec 12, 2012|
|Structure viewer||Molecule: |
Downloads & links
A: 60 KDA CHAPERONIN
B: 60 KDA CHAPERONIN
C: 60 KDA CHAPERONIN
D: 60 KDA CHAPERONIN
E: 60 KDA CHAPERONIN
F: 60 KDA CHAPERONIN
G: 60 KDA CHAPERONIN
H: 60 KDA CHAPERONIN
I: 60 KDA CHAPERONIN
J: 60 KDA CHAPERONIN
K: 60 KDA CHAPERONIN
L: 60 KDA CHAPERONIN
M: 60 KDA CHAPERONIN
N: 60 KDA CHAPERONIN
Mass: 55220.105 Da / Num. of mol.: 14 / Source: (gene. exp.) ESCHERICHIA COLI UTI89 (bacteria) / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: Q1R3B6
Mass: 523.247 Da / Num. of mol.: 14 / Formula: C10H16N5O12P3S
Comment: ATP-gamma-S (energy-carrying molecule analogue) *YM
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / Reconstruction method: single particle reconstruction|
|Component||Name: GROEL / Type: COMPLEX|
|Buffer solution||pH: 7.5|
|Specimen||Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: HOLEY CARBON|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Microscope model: FEI TITAN KRIOS / Date: Oct 7, 2011|
Details: THE TOTAL DOSE OF 25 (ELECTRONS PER SQUARE ANGSTROM) WAS FRACTIONATED EVENLY ACROSS 11 TILTED PROJECTIONS TAKEN BETWEEN 0 AND -20 DEGREES TILT (EVERY 2 DEGREES).
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 80 / Illumination mode: FLOOD BEAM|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 47000 / Calibrated magnification: 47000 / Nominal defocus max: 3000 / Nominal defocus min: 2000 / Cs: 2.7|
|Specimen holder||Temperature: 80 / Tilt angle max: 0 / Tilt angle min: -20|
|Image recording||Electron dose: 25 / Film or detector model: GENERIC CCD|
|Image scans||Number digital images: 1595|
|Radiation wavelength||Relative weight: 1|
|CTF correction||Details: 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|
|Symmetry||Point symmetry: D7|
|3D reconstruction||Method: CONSTRAINED SINGLE PARTICLE TOMOGRAPHY / Resolution: 8.4 / Number of particles: 10000 / Nominal pixel size: 1.74 / Actual pixel size: 1.74|
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2221. (DEPOSITION ID: 11174).
Symmetry type: POINT
|Atomic model building||Details: METHOD--RIGID BODY / Ref protocol: RIGID BODY FIT / Ref space: REAL / Target criteria: Cross-correlation coefficient|
|Atomic model building||PDB-ID: 3E76|
|Least-squares process||Highest resolution: 8.4|
|Refine hist #LAST||Highest resolution: 8.4|
|Number of atoms included #LAST||Protein: 53970 / Nucleic acid: 0 / Ligand: 504 / Solvent: 0 / Total: 54474|
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