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

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Basic information

Entry
Database: PDB / ID: 2ynj
TitleGroEL at sub-nanometer resolution by Constrained Single Particle Tomography
Components60 KDA CHAPERONIN
KeywordsCHAPERONE
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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / THALLIUM (I) ION / Chaperonin GroEL
Similarity search - Component
Biological speciesESCHERICHIA COLI UTI89 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / 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, 2012Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 12, 2012Provider: repository / Type: Initial release
Revision 1.1Dec 19, 2012Group: Database references
Revision 1.2Aug 23, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id ..._em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-2221
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)792,19398
Polymers773,08114
Non-polymers19,11184
Water0
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein
60 KDA CHAPERONIN / GROEL PROTEIN / PROTEIN CPN60


Mass: 55220.105 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI UTI89 (bacteria) / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: Q1R3B6
#2: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#3: Chemical...
ChemComp-TL / THALLIUM (I) ION / Thallium


Mass: 204.383 Da / Num. of mol.: 56 / Source method: obtained synthetically / Formula: Tl
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 14 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: GROEL / Type: COMPLEX
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: 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 gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 80 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 47000 X / Calibrated magnification: 47000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm
Specimen holderTemperature: 80 K / Tilt angle max: 0 ° / Tilt angle min: -20 °
Image recordingElectron dose: 25 e/Å2 / Film or detector model: GENERIC CCD
Image scansNum. digital images: 1595
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2FREALIGN3D reconstruction
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
SymmetryPoint symmetry: D7 (2x7 fold dihedral)
3D reconstructionMethod: CONSTRAINED SINGLE PARTICLE TOMOGRAPHY / Resolution: 8.4 Å / Num. of particles: 10000 / Nominal pixel size: 1.74 Å / Actual pixel size: 1.74 Å
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2221. (DEPOSITION ID: 11174).
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient / Details: METHOD--RIGID BODY
Atomic model buildingPDB-ID: 3.0E+76
RefinementHighest resolution: 8.4 Å
Refinement stepCycle: LAST / Highest resolution: 8.4 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms53970 0 504 0 54474

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