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- EMDB-5143: Ab initio reconstruction of GroEL via the asymmetric random-model... -

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Basic information

Entry
Database: EMDB / ID: EMD-5143
TitleAb initio reconstruction of GroEL via the asymmetric random-model method
Map data
SampleGroEL
Keywordsrandom-model method / ab initio reconstruction / GroEL
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 18 Å
AuthorsSanz E / Stewart AB / Belnap DM
Citation
Journal: J Struct Biol / Year: 2010
Title: The random-model method enables ab initio 3D reconstruction of asymmetric particles and determination of particle symmetry.
Authors: Eduardo Sanz-García / Aaron B Stewart / David M Belnap /
Abstract: Model-based, 3D reconstruction techniques depend on reliable starting models. We present an extension of the random-model method (RMM) that allows the ab initio generation of suitable starting models ...Model-based, 3D reconstruction techniques depend on reliable starting models. We present an extension of the random-model method (RMM) that allows the ab initio generation of suitable starting models directly from un-averaged, experimental images of asymmetric or symmetric particles. Therefore, the asymmetric RMM can also be used to determine point-group symmetry. The procedure is facilitated by the use of (a) variable angular step-sizes during iterative origin and orientation searches, (b) high numbers of particle images, and (c) highly defocused images. The method is inhibited by mixed-handedness orientation assignments and by particles with inconspicuous features. For symmetric particles, symmetric RMMs can overcome these deficiencies.
#1: Journal: J.STRUCT.BIOL. / Year: 2008
Title: A test-bed for optimizing high-resolution single particle reconstructions
Authors: Stagg SM / Lander GC / Quispe J / Voss NR / Cheng A / Bradlow H / Bradlow S / Carragher B / Potter CS
History
DepositionNov 21, 2009-
Header (metadata) releaseApr 26, 2010-
Map releaseApr 26, 2010-
UpdateJun 21, 2010-
Current statusJun 21, 2010Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • 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_5143.map.gz / Format: CCP4 / Size: 13.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.64 Å/pix.
x 155 pix.
= 254.2 Å
1.64 Å/pix.
x 155 pix.
= 254.2 Å
1.64 Å/pix.
x 155 pix.
= 254.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.64 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.0942693 - 0.0825672
Average (Standard dev.)-0.00000000384914 (±0.0126561)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-77-77-77
Dimensions155155155
Spacing155155155
CellA=B=C: 254.2 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.641.641.64
M x/y/z155155155
origin x/y/z0.0000.0000.000
length x/y/z254.200254.200254.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-34-26-72
NX/NY/NZ6953145
MAP C/R/S123
start NC/NR/NS-77-77-77
NC/NR/NS155155155
D min/max/mean-0.0940.083-0.000

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Supplemental data

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Sample components

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Entire GroEL

EntireName: GroEL
Details: S.M. Stagg, G.C. Lander, J. Quispe, N.R. Voss, A. Cheng, H. Bradlow, S. Bradlow, B. Carragher, C.S. Potter, A test-bed for optimizing high-resolution single particle reconstructions, J. ...Details: S.M. Stagg, G.C. Lander, J. Quispe, N.R. Voss, A. Cheng, H. Bradlow, S. Bradlow, B. Carragher, C.S. Potter, A test-bed for optimizing high-resolution single particle reconstructions, J. Struct. Biol. 163 (2008) 29-39.Publicly available data set.
Number of components: 14

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Component #1: cellular-component, GroEL

Cellular-componentName: GroEL / a.k.a: GroEL / Oligomeric Details: homo-tetradecamer
Details: S.M. Stagg, G.C. Lander, J. Quispe, N.R. Voss, A. Cheng, H. Bradlow, S. Bradlow, B. Carragher, C.S. Potter, A test-bed for optimizing high-resolution single particle reconstructions, J. ...Details: S.M. Stagg, G.C. Lander, J. Quispe, N.R. Voss, A. Cheng, H. Bradlow, S. Bradlow, B. Carragher, C.S. Potter, A test-bed for optimizing high-resolution single particle reconstructions, J. Struct. Biol. 163 (2008) 29-39.Publicly available data set.
Recombinant expression: No / Number of Copies: 14
SourceSpecies: Escherichia coli (E. coli)
Source (natural)Cell: Escherichia coli

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 3.2 mg/mL
Buffer solution: 100 mM Hepes, pH 7.5, 10 mM Mg(OAc)2, 10 mM KOAc, and 2 mM DTT
pH: 7.5
Support film400 mesh copper grids Cflat
VitrificationInstrument: FEI VITROBOT / Cryogen name: ETHANE / Details: Vitrification instrument: Vitrobot

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Details: S.M. Stagg, G.C. Lander, J. Quispe, N.R. Voss, A. Cheng, H. Bradlow, S. Bradlow, B. Carragher, C.S. Potter, A test-bed for optimizing high-resolution single particle reconstructions, J. ...Details: S.M. Stagg, G.C. Lander, J. Quispe, N.R. Voss, A. Cheng, H. Bradlow, S. Bradlow, B. Carragher, C.S. Potter, A test-bed for optimizing high-resolution single particle reconstructions, J. Struct. Biol. 163 (2008) 29-39.Publicly available data set.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
LensMagnification: 100000 X (nominal) / Imaging mode: BRIGHT FIELD / Defocus: 2000 - 9000 nm
Specimen HolderHolder: Eucentric / Model: GATAN LIQUID NITROGEN
CameraDetector: GENERIC CCD

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Image acquisition

Image acquisitionDetails: S.M. Stagg, G.C. Lander, J. Quispe, N.R. Voss, A. Cheng, H. Bradlow, S. Bradlow, B. Carragher, C.S. Potter, A test-bed for optimizing high-resolution single particle reconstructions, J. ...Details: S.M. Stagg, G.C. Lander, J. Quispe, N.R. Voss, A. Cheng, H. Bradlow, S. Bradlow, B. Carragher, C.S. Potter, A test-bed for optimizing high-resolution single particle reconstructions, J. Struct. Biol. 163 (2008) 29-39.Publicly available data set.

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 6613
Details: S.M. Stagg, G.C. Lander, J. Quispe, N.R. Voss, A. Cheng, H. Bradlow, S. Bradlow, B. Carragher, C.S. Potter, A test-bed for optimizing high-resolution single particle reconstructions, J. ...Details: S.M. Stagg, G.C. Lander, J. Quispe, N.R. Voss, A. Cheng, H. Bradlow, S. Bradlow, B. Carragher, C.S. Potter, A test-bed for optimizing high-resolution single particle reconstructions, J. Struct. Biol. 163 (2008) 29-39.Publicly available data set.
Applied symmetry: C1 (asymmetric)
3D reconstructionAlgorithm: Projection matching / Software: PFT3DR, Bsoft / CTF correction: Phase-flipped
Details: Random-model method. Angular step-size was initially set to 20 deg. in the first iteration and gradually decreased by 0.19 deg. in each successive iteration, until a lower limit of 1 deg. was reached.
Resolution: 18 Å / Resolution method: FSC 0.5

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