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- EMDB-14037: Cryo-electron tomogram of EIAV CASPNC VLPs, acquired on Krios 3Gi... -

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Basic information

Entry
Database: EMDB / ID: EMD-14037
TitleCryo-electron tomogram of EIAV CASPNC VLPs, acquired on Krios 3Gi, Bioquantum K3
Map dataExample tomogram of EIAV Gag VLPs
Sample
  • Virus: Equine infectious anemia virus
    • Protein or peptide: EIAV CASPNC
KeywordsRetrovirus / Equine infectious anemia virus / capsid protein / VIRAL PROTEIN
Biological speciesEquine infectious anemia virus
Methodelectron tomography / cryo EM
AuthorsObr M / Hagen WJH / Dick RA / Yu L / Kotecha A / Schur FKM
Funding support Austria, United States, 2 items
OrganizationGrant numberCountry
Austrian Science FundP31445 Austria
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI147890 United States
CitationJournal: J Struct Biol / Year: 2022
Title: Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs.
Authors: Martin Obr / Wim J H Hagen / Robert A Dick / Lingbo Yu / Abhay Kotecha / Florian K M Schur /
Abstract: The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for ...The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4-6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes.
History
DepositionDec 17, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Movie viewer
Supplemental images

emd_14037.png

Downloads & links

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Map

FileDownload / File: emd_14037.map.gz / Format: CCP4 / Size: 166.6 MB / Type: IMAGE STORED AS SIGNED INTEGER (2 BYTES)
AnnotationExample tomogram of EIAV Gag VLPs
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.58 Å/pix.
x 237 pix.
= 1558.512 Å		6.58 Å/pix.
x 720 pix.
= 4734.72 Å		6.58 Å/pix.
x 512 pix.
= 3366.912 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 6.576 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-205.0 - 290.0
Average (Standard dev.)77.921899999999994 (±17.389506999999998)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin104-104104
Dimensions720512237
Spacing512720237
CellA: 3366.912 Å / B: 4734.72 Å / C: 1558.5121 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Integer*27
Å/pix. X/Y/Z6.5766.5766.576
M x/y/z512720237
origin x/y/z0.0000.0000.000
length x/y/z3366.9124734.7201558.512
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS-104104104
NC/NR/NS512720237
D min/max/mean-205.000290.00077.922

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Supplemental data

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Sample components

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Entire : Equine infectious anemia virus

EntireName: Equine infectious anemia virus
Components
  • Virus: Equine infectious anemia virus
    • Protein or peptide: EIAV CASPNC

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Supramolecule #1: Equine infectious anemia virus

SupramoleculeName: Equine infectious anemia virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 11665 / Sci species name: Equine infectious anemia virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Equus caballus (horse)
Virus shellShell ID: 1 / Name: Immature capsid

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Macromolecule #1: EIAV CASPNC

MacromoleculeName: EIAV CASPNC / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: SPIMIDGAGN RNFRPLTPRG YTTWVNTIQT NGLLNEASQN LFGILSVDCT SEEMNAFLDV V PGQAGQKQ ILLDAIDKIA DDWDNRHPLP NAPLVAPPQG PIPMTARFIR GLGVPRERQM EP AFDQFRQ TYRQWIIEAM SEGIKVMIGK PKAQNIRQGA KEPYPEFVDR ...String:
SPIMIDGAGN RNFRPLTPRG YTTWVNTIQT NGLLNEASQN LFGILSVDCT SEEMNAFLDV V PGQAGQKQ ILLDAIDKIA DDWDNRHPLP NAPLVAPPQG PIPMTARFIR GLGVPRERQM EP AFDQFRQ TYRQWIIEAM SEGIKVMIGK PKAQNIRQGA KEPYPEFVDR LLSQIKSEGH PQE ISKFLT DTLTIQNANE ECRNAMRHLR PEDTLEEKMY ACRDIGTTKQ KMML LAKAL QTGLAGPFK GGALKGGPLK AAQTCYNCGK PGHLSSQCRA PKVCFKCKQP GHFSKQCRSV P KNGKQGAQ GRPQKQTF

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 6.2
Component:
ConcentrationFormulaName
20.0 mMMES2-(N-morpholino)ethanesulfonic acid
100.0 mMNaClsodium chloride
2.0 mMTCEPtris(2-carboxyethyl)phosphine
10.0 microMIP6inositol hexakisphosphate
GridModel: C-flat-2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: LEICA EM GP
Details: 3.5 seconds blotting time, using back-side blotting.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Aurion / Diameter: 10 nm

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Average exposure time: 0.096 sec. / Average electron dose: 3.7 e/Å2 / Details: dose-fractionation: 7 frames / tilted image
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.25 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Software - Name: eTomo (ver. 4.9.10)
Details: Tomogram reconstructed in Etomo using weighted back-projection with SIRT-like lowpass filter.
Number images used: 41

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