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- EMDB-18163: consensus map of the ASFV RNA polymerase in open conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-18163
Titleconsensus map of the ASFV RNA polymerase in open conformation
Map data3D refinement map (not sharpened)
Sample
  • Complex: apo-form of the 8-subunit RNA polymerase from African Swine Fever Virus
KeywordsRNA polymerase / ASFV / transcription / eukaryotic virus
Biological speciesAfrican swine fever virus BA71V
Methodsingle particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsPilotto S / Sykora M / Cackett G / Werner F
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/X017028/1 United Kingdom
Wellcome TrustWT207446/Z/17/Z United Kingdom
Wellcome TrustWT108877/B/15/Z United Kingdom
CitationJournal: Nat Commun / Year: 2024
Title: Structure of the recombinant RNA polymerase from African Swine Fever Virus.
Authors: Simona Pilotto / Michal Sýkora / Gwenny Cackett / Christopher Dulson / Finn Werner /
Abstract: African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of ...African Swine Fever Virus is a Nucleo-Cytoplasmic Large DNA Virus that causes an incurable haemorrhagic fever in pigs with a high impact on global food security. ASFV replicates in the cytoplasm of the infected cell and encodes its own transcription machinery that is independent of cellular factors, however, not much is known about how this system works at a molecular level. Here, we present methods to produce recombinant ASFV RNA polymerase, functional assays to screen for inhibitors, and high-resolution cryo-electron microscopy structures of the ASFV RNAP in different conformational states. The ASFV RNAP bears a striking resemblance to RNAPII with bona fide homologues of nine of its twelve subunits. Key differences include the fusion of the ASFV assembly platform subunits RPB3 and RPB11, and an unusual C-terminal domain of the stalk subunit vRPB7 that is related to the eukaryotic mRNA cap 2´-O-methyltransferase 1. Despite the high degree of structural conservation with cellular RNA polymerases, the ASFV RNAP is resistant to the inhibitors rifampicin and alpha-amanitin. The cryo-EM structures and fully recombinant RNAP system together provide an important tool for the design, development, and screening of antiviral drugs in a low biosafety containment environment.
History
DepositionAug 8, 2023-
Header (metadata) releaseFeb 14, 2024-
Map releaseFeb 14, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18163.png

Downloads & links

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Map

FileDownload / File: emd_18163.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D refinement map (not sharpened)
Voxel sizeX=Y=Z: 0.828 Å
Density
Contour LevelBy AUTHOR: 0.005
Minimum - Maximum-0.011981491 - 0.033042848
Average (Standard dev.)-0.000019435762 (±0.0011425354)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 248.40001 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half 2 map

Fileemd_18163_half_map_1.map
Annotationhalf 2 map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half 1 map

Fileemd_18163_half_map_2.map
Annotationhalf 1 map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : apo-form of the 8-subunit RNA polymerase from African Swine Fever...

EntireName: apo-form of the 8-subunit RNA polymerase from African Swine Fever Virus
Components
  • Complex: apo-form of the 8-subunit RNA polymerase from African Swine Fever Virus

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Supramolecule #1: apo-form of the 8-subunit RNA polymerase from African Swine Fever...

SupramoleculeName: apo-form of the 8-subunit RNA polymerase from African Swine Fever Virus
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 450 KDa

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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

Concentration0.05 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3tris
250.0 mMNaClSodium chloridesodium chloride
1.0 mMMgCl2magnesium chloride
0.1 mMZnSO4zinc sulfate
1.0 mMC4H10O2S2DTT
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisparse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 14638 / Average exposure time: 2.6 sec. / Average electron dose: 48.152 e/Å2
Details: Images were collected in movie-mode for a total of 50 frames per image. The data collection was carried out in super-resolution mode and binned 2 on-the-fly.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4842857
Startup modelType of model: NONE / Details: Initial model in cryoSPARC v3 and relion v4
Initial angle assignmentType: NOT APPLICABLE
Software:
Namedetails
cryoSPARC (ver. 3)2/3D classifications and 3DVA
RELION (ver. 4)3D refinement and post-processing

Details: Both relion and cryoSPARC were used
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 4) / Software - details: 3D refinement
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4) / Software - details: 3D refinement / Number images used: 352192
DetailsImages were motion corrected in Relion v4
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8q3b


Chain - Source name: Other / Chain - Initial model type: experimental model
Details: The complete biological assembly for the PDB entry 8Q3B was used for the docking in the new map
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 84.5995 / Target criteria: cross-correlation coefficient

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