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- EMDB-3407: Refinement of atomic models in high resolution EM reconstructions... -

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Basic information

Entry
Database: EMDB / ID: EMD-3407
TitleRefinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment
Map dataUnsharpened C7 reconstruction of GroEL
Sample
  • Sample: GroEL
  • Protein or peptide: GroEL
KeywordsCryo-EM / Refinement of atomic models / assessment
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 / 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 (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 3.26 Å
AuthorsJoseph AP / Malhotra S / Burnley T / Wood C / Clare DK / Winn M / Topf M
CitationJournal: Methods / Year: 2016
Title: Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment.
Authors: Agnel Praveen Joseph / Sony Malhotra / Tom Burnley / Chris Wood / Daniel K Clare / Martyn Winn / Maya Topf /
Abstract: As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high ...As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5-4.5Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders' overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite.
History
DepositionApr 14, 2016-
Header (metadata) releaseMay 18, 2016-
Map releaseJun 15, 2016-
UpdateJun 22, 2016-
Current statusJun 22, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3407.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened C7 reconstruction of GroEL
Voxel sizeX=Y=Z: 1.055 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.00854237 - 0.03263891
Average (Standard dev.)0.00035463 (±0.0021649)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin142142142
Dimensions300300300
Spacing300300300
CellA=B=C: 316.49997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0551.0551.055
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z316.500316.500316.500
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS142142142
NC/NR/NS300300300
D min/max/mean-0.0090.0330.000

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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: tetradecamer / Number unique components: 1
Molecular weightExperimental: 56 KDa / Theoretical: 56 KDa

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Macromolecule #1: GroEL

MacromoleculeName: GroEL / type: protein_or_peptide / ID: 1 / Name.synonym: 60 kDa Chaperonin / Oligomeric state: tetradecamer / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: cytoplasm
Molecular weightExperimental: 800 KDa / Theoretical: 800 KDa
SequenceUniProtKB: Chaperonin GroEL / GO: GroEL-GroES complex / InterPro: Chaperonin Cpn60/GroEL

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4 mg/mL
BufferpH: 7.4 / Details: 50 mM Tris-HCl, 50 mM KCl and 10 mM MgCl2
StainingType: NEGATIVE
Details: 3.5ul of protein was added to glow discharged C-flat r2/2 grid which were then blotted and plunge frozen
GridDetails: 400 mesh C-flat r2/2
VitrificationCryogen name: ETHANE / Chamber humidity: 40 % / Chamber temperature: 100 K / Instrument: HOMEMADE PLUNGER
Method: 3.5ul of protein was added to glow discharged C-flat r2/2 grid which were then blotted and plunge frozen

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 47400 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.8 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: Gatan / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 90 K / Max: 91 K / Average: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at the magnification used to collect the data
DetailsThe images were recorded using a dose rate of ~8 electrons/A2/s (~9 electrons/pixel/s) with 20 movie frames collected over a 4 second exposure
DateOct 9, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 370 / Average electron dose: 32 e/Å2
Details: Each image is summation of 20 frames recorded over 4 seconds
Bits/pixel: 32
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C7 (7 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.26 Å / Resolution method: OTHER / Software - Name: Relion, Ctffind3 / Number images used: 17400
DetailsGroEL was aligned and reconstructed in Relion
FSC plot (resolution estimation)

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