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- EMDB-2791: Near-atomic resolution reconstruction of Nudaurelia capensis omeg... -

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Basic information

Entry
Database: EMDB / ID: EMD-2791
TitleNear-atomic resolution reconstruction of Nudaurelia capensis omega virus (NwV) using a mid-range electron microscope operated at 200 kV
Map dataReconstruction of Nudaurelia capensis omega virus (NwV). Binned in Fourier space from an original pixel size of 1.25
Sample
  • Sample: Near-atomic resolution reconstruction of NwV using a mid-range electron microscope operated at 200 kV
  • Virus: Nudaurelia capensis omega virus
KeywordsSingle-particle electron microscopy / Direct detectors / Near-atomic resolution
Biological speciesNudaurelia capensis omega virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsCampbell MG / Kearney BM / Cheng A / Potter CS / Johnson JE / Carragher B / Veesler D
CitationJournal: J Struct Biol / Year: 2014
Title: Near-atomic resolution reconstructions using a mid-range electron microscope operated at 200 kV.
Abstract: A new era has begun for single particle cryo-electron microscopy (cryoEM) which can now compete with X-ray crystallography for determination of protein structures. The development of direct detectors ...A new era has begun for single particle cryo-electron microscopy (cryoEM) which can now compete with X-ray crystallography for determination of protein structures. The development of direct detectors constitutes a revolution that has led to a wave of near-atomic resolution cryoEM reconstructions. However, regardless of the sample studied, virtually all high-resolution reconstructions reported to date have been achieved using high-end microscopes. We demonstrate that the new generation of direct detectors coupled to a widely used mid-range electron microscope also enables obtaining cryoEM maps of sufficient quality for de novo modeling of protein structures of different sizes and symmetries. We provide an outline of the strategy used to achieve a 3.7 Å resolution reconstruction of Nudaurelia capensis ω virus and a 4.2 Å resolution reconstruction of the Thermoplasma acidophilum T20S proteasome.
History
DepositionOct 7, 2014-
Header (metadata) releaseDec 24, 2014-
Map releaseDec 24, 2014-
UpdateMar 11, 2015-
Current statusMar 11, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.042
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.042
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.042
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2791.png

Downloads & links

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Map

FileDownload / File: emd_2791.map.gz / Format: CCP4 / Size: 500 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of Nudaurelia capensis omega virus (NwV). Binned in Fourier space from an original pixel size of 1.25
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.56 Å/pix.
x 512 pix.
= 798.72 Å		1.56 Å/pix.
x 512 pix.
= 798.72 Å		1.56 Å/pix.
x 512 pix.
= 798.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.56 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.042 / Movie #1: 0.042
Minimum - Maximum-0.10642233 - 0.18355425
Average (Standard dev.)0.0007166 (±0.007305)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-256-256-256
Dimensions512512512
Spacing512512512
CellA=B=C: 798.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.561.561.56
M x/y/z512512512
origin x/y/z0.0000.0000.000
length x/y/z798.720798.720798.720
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS-256-256-256
NC/NR/NS512512512
D min/max/mean-0.1060.1840.001

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Supplemental data

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Sample components

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Entire : Near-atomic resolution reconstruction of NwV using a mid-range el...

EntireName: Near-atomic resolution reconstruction of NwV using a mid-range electron microscope operated at 200 kV
Components
  • Sample: Near-atomic resolution reconstruction of NwV using a mid-range electron microscope operated at 200 kV
  • Virus: Nudaurelia capensis omega virus

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Supramolecule #1000: Near-atomic resolution reconstruction of NwV using a mid-range el...

SupramoleculeName: Near-atomic resolution reconstruction of NwV using a mid-range electron microscope operated at 200 kV
type: sample / ID: 1000 / Oligomeric state: Icosahedral / Number unique components: 1
Molecular weightTheoretical: 16.75 MDa

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Supramolecule #1: Nudaurelia capensis omega virus

SupramoleculeName: Nudaurelia capensis omega virus / type: virus / ID: 1 / Name.synonym: NwV / NCBI-ID: 12541 / Sci species name: Nudaurelia capensis omega virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No / Syn species name: NwV
Host (natural)Organism: Lepidoptera (butterflies and moths) / synonym: INVERTEBRATES
Host systemOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: SF21 / Recombinant plasmid: baculovirus
Molecular weightTheoretical: 16.75 MDa
Virus shellShell ID: 1 / Diameter: 410 Å / T number (triangulation number): 4

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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

Concentration60 mg/mL
BufferpH: 8 / Details: 250 mM NaCl, 50 mM Tris
GridDetails: 1.2/1.3 C-Flat grid, plasma cleaned
VitrificationCryogen name: ETHANE / Chamber temperature: 95 K / Instrument: GATAN CRYOPLUNGE 3 / Details: Vitrification carried out at room temperature / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 29,000 times magnification
DetailsCollected in counting mode
DateNov 16, 2013
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 625 / Average electron dose: 38 e/Å2
Details: Each movie was acquired over 5 s and was comprised of 25 frames
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 40000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 29000
Sample stageSpecimen holder: Liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsInitial data processing was performed using the Appion pipeline (including UCSF doseefgpu_driftcorr). Subsequent statistical refinement procedures and motion correction were performed using Relion.
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: I (icosahedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 14884

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