|Entry||Database: EMDB / ID: 5754|
|Title||Maximizing the potential of electron cryomicroscopy data collected using direct detectors|
|Map data||Reconstruction of mature STIV virion|
|Sample||Mature Sulfolobus Turreted Icosahedral Virus:|
|Keywords||Electron microscopy / Direct detectors / Near-atomic resolution / Sulfolobus turreted icosahedral virus|
|Source||Sulfolobus turreted icosahedral virus|
|Method||single particle reconstruction / cryo EM / 5.8 Å resolution|
|Authors||Veesler D / Campbell MG / Cheng A / Fu CY / Murez Z / Johnson JE / Potter CS / Carragher B|
|Citation||Journal: 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.
|Date||Deposition: Sep 18, 2013 / Header (metadata) release: Mar 19, 2014 / Map release: Mar 19, 2014 / Last update: Mar 19, 2014|
|Structure viewer||EM map: |
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|File||emd_5754.map.gz (map file in CCP4 format, 4194305 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.21 Å|
CCP4 map header:
-Entire Mature Sulfolobus Turreted Icosahedral Virus
|Entire||Name: Mature Sulfolobus Turreted Icosahedral Virus / Number of components: 1 / Oligomeric State: icosahedral|
|Mass||Theoretical: 75 MDa|
-Component #1: virus, Sulfolobus turreted icosahedral virus
|Virus||Name: Sulfolobus turreted icosahedral virus / Class: VIRION / Empty: No / Enveloped: Yes / Isolate: STRAIN|
|Mass||Theoretical: 75 MDa|
|Species||Species: Sulfolobus turreted icosahedral virus / Strain: YNPRC179|
|Source (natural)||Host Species: Sulfolobus solfataricus (archaea) / Host category: ARCHAEA / Host species strain: 2-2-12|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Buffer solution: 23 mM KH2PO4, 19 mM (NH4)2SO4, 1 mM MgSO4, 2 mM CaCl2|
|Support film||plasma cleaned C-flat holey carbon grids (CF-1.2/1.3, Protochips)|
|Vitrification||Instrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temperature: 94 K / Method: Blot for 3 seconds before plunging. / Details: Vitrification was carried out at room temperature.|
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20 / Date: Nov 30, 2012|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 22 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 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 Holder||Holder: Nitrogen cooled / Model: GATAN LIQUID NITROGEN / Temperature: 90 K|
|Camera||Detector: GATAN K2 (4k x 4k)|
|Image acquisition||Number of digital images: 754|
Details: Every movie is composed of sixteen frames recorded by the direct electron detector.
|Processing||Method: 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 reconstruction||Algorithm: 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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