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

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Entry
Database: PDB / ID: 2ynj
TitleGroEL at sub-nanometer resolution by Constrained Single Particle Tomography
Components60 KDA CHAPERONIN
KeywordsCHAPERONE
Function / homologyChaperonin 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 sourceESCHERICHIA COLI UTI89 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 8.4 Å resolution
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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 15, 2012 / Release: Dec 12, 2012
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 12, 2012Structure modelrepositoryInitial release
1.1Dec 19, 2012Structure modelDatabase references
1.2Aug 23, 2017Structure modelData collection / Refinement descriptionem_3d_fitting / em_software_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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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
Polyers773,08114
Non-polymers19,11184
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

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


Mass: 55220.105 Da / Num. of mol.: 14 / 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 / Formula: C10H16N5O12P3S
Comment: ATP-gamma-S (energy-carrying molecule analogue) *YM
#3: Chemical...
ChemComp-TL / THALLIUM (I) ION


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


Mass: 24.305 Da / Num. of mol.: 14 / Formula: Mg / Magnesium

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 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
MicroscopyMicroscope 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 gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 80 / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 47000 / Calibrated magnification: 47000 / Nominal defocus max: 3000 / Nominal defocus min: 2000 / Cs: 2.7
Specimen holderTemperature: 80 / Tilt angle max: 0 / Tilt angle min: -20
Image recordingElectron dose: 25 / Film or detector model: GENERIC CCD
Image scansNumber 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
3D reconstructionMethod: 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 buildingDetails: METHOD--RIGID BODY / Ref protocol: RIGID BODY FIT / Ref space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingPDB-ID: 3E76
Least-squares processHighest resolution: 8.4
Refine hist #LASTHighest resolution: 8.4
Number of atoms included #LASTProtein: 53970 / Nucleic acid: 0 / Ligand: 504 / Solvent: 0 / Total: 54474

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