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- EMDB-14032: Structure of the EIAV CA-SP2 hexamer (C2), acquired on Krios G4, ... -

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Basic information

Entry
Database: EMDB / ID: EMD-14032
TitleStructure of the EIAV CA-SP2 hexamer (C2), acquired on Krios G4, Selectris/Falcon4, processed with Relion/M
Map dataEIAV CA hexamer, C2 symmetrized, sharpened and denoised map
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
Methodsubtomogram averaging / cryo EM / Resolution: 3.2 Å
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) releaseApr 6, 2022-
Map releaseApr 6, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14032.png

Downloads & links

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Map

FileDownload / File: emd_14032.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEIAV CA hexamer, C2 symmetrized, sharpened and denoised map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.18 Å/pix.
x 340 pix.
= 400.86 Å		1.18 Å/pix.
x 340 pix.
= 400.86 Å		1.18 Å/pix.
x 340 pix.
= 400.86 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.179 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0027
Minimum - Maximum-0.009793695 - 0.015021782
Average (Standard dev.)0.00003151212 (±0.0006658925)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 400.86002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14032_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EIAV CA hexamer, C2 symmetrized, half map 1

Fileemd_14032_half_map_1.map
AnnotationEIAV CA hexamer, C2 symmetrized, half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EIAV CA hexamer, C2 symmetrized, half map 2

Fileemd_14032_half_map_2.map
AnnotationEIAV CA hexamer, C2 symmetrized, half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
Source (natural)Organism: Equine infectious anemia virus
Recombinant expressionOrganism: Escherichia coli (E. coli)
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
Processingsubtomogram averaging
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.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Average exposure time: 1.02 sec. / Average electron dose: 3.5 e/Å2 / Details: Images acquired in the EER format.
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
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 reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Warp (ver. nb20201104) / Number subtomograms used: 53665
ExtractionNumber tomograms: 69 / Number images used: 53665 / Software - Name: Warp (ver. nb20201104)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: Warp (ver. nb20201104)
FSC plot (resolution estimation)

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