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- EMDB-1457: A test-bed for optimizing high-resolution single particle reconst... -

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Basic information

Entry
Database: EMDB / ID: EMD-1457
TitleA test-bed for optimizing high-resolution single particle reconstructions.
Map data
SampleGroEL
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 5.4 Å
AuthorsStagg SM / Lander GC / Quispe J / Voss NR / Cheng A / Bradlow H / Bradlow S / Carragher B / Potter CS
CitationJournal: J. Struct. Biol. / Year: 2008
Title: A test-bed for optimizing high-resolution single particle reconstructions.
Authors: Scott M Stagg / Gabriel C Lander / Joel Quispe / Neil R Voss / Anchi Cheng / Henry Bradlow / Steven Bradlow / Bridget Carragher / Clinton S Potter /
Abstract: It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of approximately 10A, but maps with resolutions of 5A or better are still ...It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of approximately 10A, but maps with resolutions of 5A or better are still celebrated events. The electron microscope has a resolving power to better than 2A, and thus should not be a limiting factor; instead the practical limitations in resolution most likely arise from a combination of specimen preparation methods, data collection parameters, and data analysis procedures. With the aid of a highly automated system for acquiring images, coupled to a relational database to keep track of all processing parameters, we have taken a systematic approach to optimizing parameters affecting the resolution of single particle reconstructions. Using GroEL as a test-bed, we performed a series of 3D reconstructions where we systematically varied the number of particles used in computing the map, the accelerating voltage of the microscope, and the electron dose used to acquire the images. We also investigated methods for excluding unacceptable or "bad" particles from contributing to the final 3D map. Using relatively standard instrumentation (Tecnai F20, 4K x 4K CCD, side entry cold stage) and a completely automated approach, these approaches resulted in a map with a nominal resolution of 5.4A (FSC(0.5)) in which secondary structure is clearly discernable and the handedness of some of the alpha-helices in the GroEL structure can be determined.
History
Current status-Processing site: PDBe / Status: Released
DepositionNov 9, 2007-
Header (metadata) releaseNov 9, 2007-
Map releaseJun 24, 2008-
UpdateMay 26, 2011-

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1457.map.gz / Format: CCP4 / Size: 14.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.63 Å/pix.
x 156 pix.
= 254.28 Å
1.63 Å/pix.
x 156 pix.
= 254.28 Å
1.63 Å/pix.
x 156 pix.
= 254.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.63 Å
Density
Contour LevelPrimary: 2.55 / Movie #1: 4
Minimum - Maximum-7.09001 - 10.484
Average (Standard dev.)0.0530646 (±1.33817)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-78-78-78
Dimensions156156156
Spacing156156156
CellA=B=C: 254.28 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.631.631.63
M x/y/z156156156
origin x/y/z0.0000.0000.000
length x/y/z254.280254.280254.280
α/β/γ90.00090.00090.000
start NX/NY/NZ-60-60-59
NX/NY/NZ120120120
MAP C/R/S123
start NC/NR/NS-78-78-78
NC/NR/NS156156156
D min/max/mean-7.09010.4840.053

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Supplemental data

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Sample components

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Entire GroEL

EntireName: GroEL / Oligomeric State: D7 14-mer / Number of components: 1
MassTheoretical: 800 kDa / Experimental: 800 kDa

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Component #1: protein, GroEL

ProteinName: GroEL / a.k.a: GroEL / Oligomeric Details: homotetradecamer / Recombinant expression: Yes / Number of Copies: 14
MassTheoretical: 800 kDa / Experimental: 800 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (natural)Location in cell: cytosol / Cell: E. coli

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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: 100mM Hepes, 10mM Mg(OAc)2, 10mM KOAc, 2mM DTT
pH: 7.5
Support filmProtochips C-flat grid: holey carbon with 2um holes and 2um spacing 400 mesh copper grid
Stainingnot stained
VitrificationInstrument: FEI VITROBOT / Cryogen name: ETHANE / Temperature: 93 K / Humidity: 100 %
Method: Temperature of chamber was 4 degrees C. 0 seconds drain time. Single blot. 0 mm offset. 4 ul sample applied to grid. Blot for 3.5 seconds before plunging.
Details: Vitrification instrument: Vitrobot. Grid plasma cleaned for 20s with Fischione 1020 plasma cleaner using 75% Argon 25% Oxygen mix.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Jul 12, 2006
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 13 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 100000 X (nominal), 100000 X (calibrated)
Astigmatism: objective lens astigmatism was corrected automatically using Leginon
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 3000 nm
Specimen HolderHolder: Side entry liquid nitrogen-cooled cryo specimen holder
Model: GATAN LIQUID NITROGEN / Temperature: 102
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of class averages: 1294 / Number of projections: 55351 / Applied symmetry: D7 (2x7 fold dihedral)
3D reconstructionAlgorithm: single particle reconstruction / Software: EMAN / CTF correction: Phase correction for each particle. / Resolution: 5.4 Å / Resolution method: FSC 0.5

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