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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: 3407
TitleRefinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment
Map dataUnsharpened C7 reconstruction of GroEL
SampleGroEL:
KeywordsCryo-EM / Refinement of atomic models / assessment
Function / homologyChaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/TCP-1 family / Chaperonin Cpn60 / TCP-1/cpn60 chaperonin family / GroEL-like apical domain superfamily / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-GroES complex / Chaperonin Cpn60 ...Chaperonin Cpn60, conserved site / Chaperonins cpn60 signature. / Chaperonin Cpn60/TCP-1 family / Chaperonin Cpn60 / TCP-1/cpn60 chaperonin family / GroEL-like apical domain superfamily / GroEL-like equatorial domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-GroES complex / Chaperonin Cpn60 / chaperone cofactor-dependent protein refolding / protein binding involved in protein folding / response to radiation / virion assembly / protein folding / unfolded protein binding / response to heat / protein refolding / ATPase activity / cell cycle / cell division / magnesium ion binding / membrane / ATP binding / identical protein binding / cytosol / 60 kDa chaperonin
Function and homology information
SourceEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / 3.26 Å resolution
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.
DateDeposition: Apr 14, 2016 / Header (metadata) release: May 18, 2016 / Map release: Jun 15, 2016 / Last update: Jun 22, 2016

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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
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_3407.map.gz (map file in CCP4 format, 105470 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
300 pix
1.06 Å/pix.
= 316.5 Å
300 pix
1.06 Å/pix.
= 316.5 Å
300 pix
1.06 Å/pix.
= 316.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.055 Å
Density
Contour Level:0.01 (by author), 0.01 (movie #1):
Minimum - Maximum-0.00854237 - 0.03263891
Average (Standard dev.)0.00035463 (0.0021649)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions300300300
Origin142142142
Limit441441441
Spacing300300300
CellA=B=C: 316.49997 Å
α=β=γ: 90 deg.

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 / Number of components: 1 / Oligomeric State: tetradecamer
MassTheoretical: 56 kDa / Experimental: 56 kDa

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Component #1: protein, GroEL

ProteinName: GroEL / a.k.a: 60 kDa Chaperonin / Oligomeric Details: tetradecamer / Recombinant expression: No
MassTheoretical: 800 kDa / Experimental: 800 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (natural)Location in cell: cytoplasm
External referencesInterPro: Chaperonin Cpn60 / UniProt: 60 kDa chaperonin / Gene Ontology: GroEL-GroES complex

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: negative staining, cryo EM
Sample solutionSpecimen conc.: 4 mg/ml / Buffer solution: 50 mM Tris-HCl, 50 mM KCl and 10 mM MgCl2 / pH: 7.4
Support film400 mesh C-flat r2/2
Staining3.5ul of protein was added to glow discharged C-flat r2/2 grid which were then blotted and plunge frozen
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 100 K / Humidity: 40 %
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 imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Date: Oct 9, 2015
Details: The 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
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 32 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000 X (nominal), 47400 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at the magnification used to collect the data
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 2800 nm / Energy filter: Gatan / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 90 K ( 90 - 91 K)
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 370 / Sampling size: 5 microns / Bit depth: 32
Details: Each image is summation of 20 frames recorded over 4 seconds

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C7 (7 fold cyclic) / Number of projections: 17400 / Details: GroEL was aligned and reconstructed in Relion
3D reconstructionAlgorithm: Maximum likelihood / Software: Relion, Ctffind3 / CTF correction: Each particle / Resolution: 3.26 Å / Resolution method: FSC 0.143, gold-standard
FSC plot
(resolution estimation)

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