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- EMDB-5754: Maximizing the potential of electron cryomicroscopy data collecte... -

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Basic information

Entry
Database: EMDB / ID: EMD-5754
TitleMaximizing the potential of electron cryomicroscopy data collected using direct detectors
Map dataReconstruction of mature STIV virion
Sample
  • Sample: Mature Sulfolobus Turreted Icosahedral Virus
  • Virus: Sulfolobus turreted icosahedral virus
KeywordsElectron microscopy / Direct detectors / Near-atomic resolution / Sulfolobus turreted icosahedral virus
Biological speciesSulfolobus turreted icosahedral virus
Methodsingle particle reconstruction / cryo EM / Resolution: 5.8 Å
AuthorsVeesler D / Campbell MG / Cheng A / Fu CY / Murez Z / Johnson JE / Potter CS / Carragher B
CitationJournal: J Struct Biol / Year: 2013
Title: Maximizing the potential of electron cryomicroscopy data collected using direct detectors.
Authors: David Veesler / Melody G Campbell / Anchi Cheng / Chi-Yu Fu / Zachary Murez / John E Johnson / Clinton S Potter / Bridget Carragher /
Abstract: Single-particle electron cryomicroscopy is undergoing a technical revolution due to the recent developments of direct detectors. These new recording devices detect electrons directly (i.e. without ...Single-particle electron cryomicroscopy is undergoing a technical revolution due to the recent developments of direct detectors. These new recording devices detect electrons directly (i.e. without conversion into light) and feature significantly improved detective quantum efficiencies and readout rates as compared to photographic films or CCDs. We evaluated here the potential of one such detector (Gatan K2 Summit) to enable the achievement of near-atomic resolution reconstructions of biological specimens when coupled to a widely used, mid-range transmission electron microscope (FEI TF20 Twin). Compensating for beam-induced motion and stage drift provided a 4.4Å resolution map of Sulfolobus turreted icosahedral virus (STIV), which we used as a test particle in this study. Several motion correction and dose fractionation procedures were explored and we describe their influence on the resolution of the final reconstruction. We also compared the quality of this data to that collected with a FEI Titan Krios microscope equipped with a Falcon I direct detector, which provides a benchmark for data collected using a high-end electron microscope.
History
DepositionSep 18, 2013-
Header (metadata) releaseMar 19, 2014-
Map releaseMar 19, 2014-
UpdateMar 19, 2014-
Current statusMar 19, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5754.gif

Downloads & links

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Map

FileDownload / File: emd_5754.map.gz / Format: CCP4 / Size: 3.9 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of mature STIV virion
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.21 Å/pix.
x 1024 pix.
= 1239.04 Å		1.21 Å/pix.
x 1024 pix.
= 1239.04 Å		1.21 Å/pix.
x 1024 pix.
= 1239.04 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.21 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.28681743 - 0.33031565
Average (Standard dev.)-0.00033474 (±0.01979297)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-512-512-512
Dimensions102410241024
Spacing102410241024
CellA=B=C: 1239.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.211.211.21
M x/y/z102410241024
origin x/y/z0.0000.0000.000
length x/y/z1239.0401239.0401239.040
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-512-512-512
NC/NR/NS102410241024
D min/max/mean-0.2870.330-0.000

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Supplemental data

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Sample components

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Entire : Mature Sulfolobus Turreted Icosahedral Virus

EntireName: Mature Sulfolobus Turreted Icosahedral Virus
Components
  • Sample: Mature Sulfolobus Turreted Icosahedral Virus
  • Virus: Sulfolobus turreted icosahedral virus

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Supramolecule #1000: Mature Sulfolobus Turreted Icosahedral Virus

SupramoleculeName: Mature Sulfolobus Turreted Icosahedral Virus / type: sample / ID: 1000 / Oligomeric state: icosahedral / Number unique components: 1
Molecular weightTheoretical: 75 MDa

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Supramolecule #1: Sulfolobus turreted icosahedral virus

SupramoleculeName: Sulfolobus turreted icosahedral virus / type: virus / ID: 1 / NCBI-ID: 269145 / Sci species name: Sulfolobus turreted icosahedral virus / Sci species strain: YNPRC179 / Database: NCBI / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Sulfolobus solfataricus (archaea) / Strain: 2-2-12 / synonym: ARCHAEA
Molecular weightTheoretical: 75 MDa
Virus shellShell ID: 1 / Diameter: 730 Å

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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

BufferpH: 3.5
Details: 23 mM KH2PO4, 19 mM (NH4)2SO4, 1 mM MgSO4, 2 mM CaCl2
GridDetails: plasma cleaned C-flat holey carbon grids (CF-1.2/1.3, Protochips)
VitrificationCryogen name: ETHANE / Chamber temperature: 94 K / Instrument: GATAN CRYOPLUNGE 3 / Details: Vitrification was carried out at room temperature. / Method: Blot for 3 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 41322 times magnification
DateNov 30, 2012
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 754 / Average electron dose: 22 e/Å2
Details: Every movie is composed of sixteen frames recorded by the direct electron detector.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 41322 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.7 µm / Nominal defocus min: 0.45 µm
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe final reconstruction was sharpened with a negative temperature factor of 650 A^2.
CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 5.8 Å / Resolution method: OTHER / Software - Name: Frealign
Details: The final reconstruction was computed using the first ten frames of each movie and sharpened with a negative temperature factor of 650 A^2.
Number images used: 4446

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