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- EMDB-1200: Automated cryoEM data acquisition and analysis of 284742 particle... -

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Entry
Database: EMDB / ID: EMD-1200
TitleAutomated cryoEM data acquisition and analysis of 284742 particles of GroEL.
Map dataThis is a three-dimensional volume of GroEL reconstructed to 7.8 Angstroms resolution
Sample
  • Sample: E.coli GroEL
  • Protein or peptide: GroEL
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsStagg SM / Pulokas J / Fellmann D / Cheng A / Quispe JD / Mallick SP / Avila RM / Carragher B / Potter CS
CitationJournal: J Struct Biol / Year: 2006
Title: Automated cryoEM data acquisition and analysis of 284742 particles of GroEL.
Authors: Scott M Stagg / Gabriel C Lander / James Pulokas / Denis Fellmann / Anchi Cheng / Joel D Quispe / Satya P Mallick / Radomir M Avila / Bridget Carragher / Clinton S Potter /
Abstract: One of the goals in developing our automated electron microscopy data acquisition system, Leginon, was to improve both the ease of use and the throughput of the process of acquiring low dose images ...One of the goals in developing our automated electron microscopy data acquisition system, Leginon, was to improve both the ease of use and the throughput of the process of acquiring low dose images of macromolecular specimens embedded in vitreous ice. In this article, we demonstrate the potential of the Leginon system for high-throughput data acquisition by describing an experiment in which we acquired images of more than 280,000 particles of GroEL in a single 25 h session at the microscope. We also demonstrate the potential for an automated pipeline for molecular microscopy by showing that these particles can be subjected to completely automated procedures to reconstruct a three-dimensional (3D) density map to a resolution better than 8 A. In generating the 3D maps, we used a variety of metadata associated with the data acquisition and processing steps to sort and select the particles. These metadata provide a number of insights into factors that affect the quality of the acquired images and the resulting reconstructions. In particular, we show that the resolution of the reconstructed 3D density maps improves with decreasing ice thickness. These data provide a basis for assessing the capabilities of high-throughput macromolecular microscopy.
History
DepositionFeb 28, 2006-
Header (metadata) releaseMar 1, 2006-
Map releaseJul 1, 2006-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 2.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1200.gif

Downloads & links

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Map

FileDownload / File: emd_1200.map.gz / Format: CCP4 / Size: 5.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a three-dimensional volume of GroEL reconstructed to 7.8 Angstroms resolution
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.26 Å/pix.
x 112 pix.
= 253.456 Å		2.26 Å/pix.
x 112 pix.
= 253.456 Å		2.26 Å/pix.
x 112 pix.
= 253.456 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.263 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 1.84 / Movie #1: 2.5
Minimum - Maximum-5.45532 - 7.65301
Average (Standard dev.)-0.000000000562777 (±0.735104)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-56-56-56
Dimensions112112112
Spacing112112112
CellA=B=C: 253.456 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.2632.2632.263
M x/y/z112112112
origin x/y/z0.0000.0000.000
length x/y/z253.456253.456253.456
α/β/γ90.00090.00090.000
start NX/NY/NZ-80-80-79
NX/NY/NZ160160160
MAP C/R/S123
start NC/NR/NS-56-56-56
NC/NR/NS112112112
D min/max/mean-5.4557.653-0.000

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Supplemental data

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Sample components

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Entire : E.coli GroEL

EntireName: E.coli GroEL
Components
  • Sample: E.coli GroEL
  • Protein or peptide: GroEL

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Supramolecule #1000: E.coli GroEL

SupramoleculeName: E.coli GroEL / type: sample / ID: 1000 / Oligomeric state: homotetradecamer / Number unique components: 1
Molecular weightExperimental: 800 KDa / Theoretical: 800 KDa / Method: Calculated molecular weight from sequence

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Macromolecule #1: GroEL

MacromoleculeName: GroEL / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Oligomeric state: homotetradecamer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / synonym: E.coli / Location in cell: cytosol
Molecular weightExperimental: 800 KDa / Theoretical: 800 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.2 mg/mL
BufferpH: 7.5 / Details: 100mM Hepes, 10mM Mg(OAc)2, 10mM KOAc, 2mM DTT
GridDetails: Protochips C-flat grid: holey carbon with 2um holes and 2um spacing 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: OTHER
Details: Vitrification instrument: FEI Vitrobot. Grid plasma cleaned for 20s with Fischione 1020 plasma cleaner using 75% Argon 25% Oxygen mix.
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.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 94 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 50,000X magnification
DateMay 19, 2005
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 552 / Average electron dose: 11.5 e/Å2 / Bits/pixel: 16
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.28 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Gatan 626 side entry cryo-stage / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe images were acquired using the Leginon automated data aquisition system. The particles were automatically selected using the Selexon package. The CTF was automatically estimated using the ACE package
CTF correctionDetails: Phase correction for each particle. Amplitude correction for the final volume
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 158390
Final two d classificationNumber classes: 333

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