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- EMDB-5377: Direct electron detection yields cryo-EM reconstructions at resol... -

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Basic information

Entry
Database: EMDB / ID: EMD-5377
TitleDirect electron detection yields cryo-EM reconstructions at resolutions beyond .75 Nyquist frequency
Map dataIcosahedral reconstruction of bacteriophage epsilon 15
Sample
  • Sample: Bacteriophage Epsilon-15
  • Virus: Salmonella phage epsilon15 (virus)
KeywordsCryo-EM / Electron cryo-microscopy / Direct detection device / Active pixel sensor / CMOS detector / Nyquist frequency
Biological speciesSalmonella phage epsilon15 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.9 Å
AuthorsBammes BE / Rochat RH / Jakana J / Chen D / Chiu W
CitationJournal: J Struct Biol / Year: 2012
Title: Direct electron detection yields cryo-EM reconstructions at resolutions beyond 3/4 Nyquist frequency.
Authors: Benjamin E Bammes / Ryan H Rochat / Joanita Jakana / Dong-Hua Chen / Wah Chiu /
Abstract: One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-resolution signal from the image-recording media at the full-resolution limit of the transmission electron ...One limitation in electron cryo-microscopy (cryo-EM) is the inability to recover high-resolution signal from the image-recording media at the full-resolution limit of the transmission electron microscope. Direct electron detection using CMOS-based sensors for digitally recording images has the potential to alleviate this shortcoming. Here, we report a practical performance evaluation of a Direct Detection Device (DDD®) for biological cryo-EM at two different microscope voltages: 200 and 300 kV. Our DDD images of amorphous and graphitized carbon show strong per-pixel contrast with image resolution near the theoretical sampling limit of the data. Single-particle reconstructions of two frozen-hydrated bacteriophages, P22 and ε15, establish that the DDD is capable of recording usable signal for 3D reconstructions at about 4/5 of the Nyquist frequency, which is a vast improvement over the performance of conventional imaging media. We anticipate the unparalleled performance of this digital recording device will dramatically benefit cryo-EM for routine tomographic and single-particle structural determination of biological specimens.
History
DepositionJan 3, 2012-
Header (metadata) releaseJan 26, 2012-
Map releaseOct 24, 2012-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.17
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5377_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5377.map.gz / Format: CCP4 / Size: 122.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIcosahedral reconstruction of bacteriophage epsilon 15
Voxel sizeX=Y=Z: 3.04 Å
Density
Contour LevelBy AUTHOR: 0.17 / Movie #1: 0.17
Minimum - Maximum-0.62429988 - 0.85280883
Average (Standard dev.)0.00000793 (±0.09882288)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-163-163-163
Dimensions320320320
Spacing320320320
CellA=B=C: 972.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.043.043.04
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z972.800972.800972.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-163-163-163
NC/NR/NS320320320
D min/max/mean-0.6240.8530.000

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Supplemental data

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Sample components

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Entire : Bacteriophage Epsilon-15

EntireName: Bacteriophage Epsilon-15
Components
  • Sample: Bacteriophage Epsilon-15
  • Virus: Salmonella phage epsilon15 (virus)

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Supramolecule #1000: Bacteriophage Epsilon-15

SupramoleculeName: Bacteriophage Epsilon-15 / type: sample / ID: 1000 / Details: Sample was vitrified / Number unique components: 1

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Supramolecule #1: Salmonella phage epsilon15

SupramoleculeName: Salmonella phage epsilon15 / type: virus / ID: 1 / Name.synonym: Epsilon 15 / NCBI-ID: 215158 / Sci species name: Salmonella phage epsilon15 / Database: NCBI / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No / Syn species name: Epsilon 15
Host (natural)Organism: Salmonella (bacteria) / synonym: BACTERIA(EUBACTERIA)
Virus shellShell ID: 1 / Name: capsid / Diameter: 700 Å / T number (triangulation number): 7

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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: 7.5
GridDetails: 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 170 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 1.5 seconds before plunging

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Electron microscopy

MicroscopeJEOL 3200FSC
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 20800 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.1 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 20000
Specialist opticsEnergy filter - Name: Omega / Energy filter - Lower energy threshold: 15.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder: Eucentric / Specimen holder model: JEOL 3200FSC CRYOHOLDER
TemperatureMin: 100 K / Max: 100 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 400,000 times magnification
DateMar 6, 2010
Image recordingCategory: CCD / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Digitization - Sampling interval: 6 µm / Number real images: 75 / Average electron dose: 20 e/Å2
Details: Digital images were collected on a CMOS type direct electron detection device DE-12 from Direct Electron

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Image processing

CTF correctionDetails: Each Micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: MPSA
Details: Resolution was calculated at 7.9 Angstroms by the 0.5 FSC criterion and 6.8 Angstroms by the 0.143 FSC criterion both in comparison with the previously published 4.5 Angstrom resolution reconstruction.
Number images used: 1380
DetailsGrids were coated in thin carbon before applying sample

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