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

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Basic information

Entry
Database: EMDB / ID: 5754
TitleMaximizing the potential of electron cryomicroscopy data collected using direct detectors
Map dataReconstruction of mature STIV virion
SampleMature Sulfolobus Turreted Icosahedral Virus:
virus
KeywordsElectron microscopy / Direct detectors / Near-atomic resolution / Sulfolobus turreted icosahedral virus
SourceSulfolobus turreted icosahedral virus
Methodsingle particle reconstruction / cryo EM / 5.8 Å resolution
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.
DateDeposition: Sep 18, 2013 / Header (metadata) release: Mar 19, 2014 / Map release: Mar 19, 2014 / Last update: Mar 19, 2014

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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

Downloads & links

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Map

Fileemd_5754.map.gz (map file in CCP4 format, 4194305 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1024 pix
1.21 Å/pix.
= 1239.04 Å
1024 pix
1.21 Å/pix.
= 1239.04 Å
1024 pix
1.21 Å/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
Contour Level:0.06 (by emdb), 0.06 (movie #1):
Minimum - Maximum-0.28681743 - 0.33031565
Average (Standard dev.)-0.00033474 (0.01979297)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions102410241024
Origin-512-512-512
Limit511511511
Spacing102410241024
CellA=B=C: 1239.04 Å
α=β=γ: 90 deg.

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 / Number of components: 1 / Oligomeric State: icosahedral
MassTheoretical: 75 MDa

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Component #1: virus, Sulfolobus turreted icosahedral virus

VirusName: Sulfolobus turreted icosahedral virus / Class: VIRION / Empty: No / Enveloped: Yes / Isolate: STRAIN
MassTheoretical: 75 MDa
SpeciesSpecies: Sulfolobus turreted icosahedral virus / Strain: YNPRC179
Source (natural)Host Species: Sulfolobus solfataricus (archaea) / Host category: ARCHAEA / Host species strain: 2-2-12

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionBuffer solution: 23 mM KH2PO4, 19 mM (NH4)2SO4, 1 mM MgSO4, 2 mM CaCl2
pH: 3.5
Support filmplasma cleaned C-flat holey carbon grids (CF-1.2/1.3, Protochips)
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temperature: 94 K / Method: Blot for 3 seconds before plunging. / Details: Vitrification was carried out at room temperature.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20 / Date: Nov 30, 2012
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 22 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 41322 X (calibrated)
Astigmatism: Objective lens astigmatism was corrected at 41322 times magnification
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 450 - 3700 nm
Specimen HolderHolder: Nitrogen cooled / Model: GATAN LIQUID NITROGEN / Temperature: 90 K
CameraDetector: GATAN K2 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 754
Details: Every movie is composed of sixteen frames recorded by the direct electron detector.

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 4446
Details: The final reconstruction was sharpened with a negative temperature factor of 650 A^2.
3D reconstructionAlgorithm: Projection matching / Software: Frealign / CTF correction: Each particle
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.
Resolution: 5.8 Å
Resolution method: FSC at 0.143 cut-off. The reported resolution is for the entire reconstruction. The resolution of the coat subunit region (B345) is estimated to 4.35 A using the same criterion.

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