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- EMDB-5375: Direct electron detection yields cryo-EM reconstructions at resol... -

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Basic information

Entry
Database: EMDB / ID: EMD-5375
TitleDirect electron detection yields cryo-EM reconstructions at resolutions beyond .75 Nyquist frequency
Map dataIcosahedral reconstruction of bacteriophage P22
Sample
  • Sample: Bacteriophage P22 procapsid
  • Virus: Enterobacteria phage P22 (virus)
KeywordsCryo-EM / Electron cryo-microscopy / Direct detection device / Active pixel sensor / CMOS detector / Nyquist frequency
Biological speciesEnterobacteria phage P22 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.5 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
DepositionDec 20, 2011-
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: 0.8
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5375_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5375.map.gz / Format: CCP4 / Size: 210.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIcosahedral reconstruction of bacteriophage P22
Voxel sizeX=Y=Z: 3.48 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum-0.99926412 - 1.86862397
Average (Standard dev.)0.0 (±0.18091138)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-128-128-128
Dimensions384384384
Spacing384384384
CellA=B=C: 1336.3201 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.483.483.48
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z1336.3201336.3201336.320
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-128-128-128
NC/NR/NS384384384
D min/max/mean-0.9991.869-0.000

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Supplemental data

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Sample components

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Entire : Bacteriophage P22 procapsid

EntireName: Bacteriophage P22 procapsid
Components
  • Sample: Bacteriophage P22 procapsid
  • Virus: Enterobacteria phage P22 (virus)

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Supramolecule #1000: Bacteriophage P22 procapsid

SupramoleculeName: Bacteriophage P22 procapsid / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: Enterobacteria phage P22

SupramoleculeName: Enterobacteria phage P22 / type: virus / ID: 1 / Name.synonym: P22 / NCBI-ID: 10754 / Sci species name: Enterobacteria phage P22 / Database: NCBI / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: Yes / Syn species name: P22
Host (natural)Organism: Salmonella (bacteria) / synonym: BACTERIA(EUBACTERIA)
Virus shellShell ID: 1 / Name: Procapsid / Diameter: 525 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

Concentration1 mg/mL
BufferpH: 7.6 / Details: 50 mM Tris pH 7.6, 25 mM NaCl, 2mM EDTA
GridDetails: 400 mesh copper grid
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 170 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeJEOL 2010F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 17200 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 15000
Sample stageSpecimen holder: Side Entry / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureMin: 100 K / Max: 100 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
DateJun 10, 2010
Image recordingCategory: CCD / Film or detector model: DIRECT ELECTRON DE-12 (4k x 3k) / Digitization - Sampling interval: 6 µm / Number real images: 200 / Average electron dose: 17 e/Å2
Details: Images were collected on a CMOS type detector DE-12 from Direct Electron

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Image processing

CTF correctionDetails: Each Micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: MPSA
Details: Resolution was calculated to be 8.5 Angstroms at 0.5 FSC and 7.3 Angstroms at 0.143 FSC as compared against the 3.8 Angstrom reconstruction of P22.
Number images used: 7500

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