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- EMDB-24765: Cryo-EM map of the phage AR9 non-virion RNA polymerase holoenzyme -

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Basic information

Entry
Database: EMDB / ID: EMD-24765
TitleCryo-EM map of the phage AR9 non-virion RNA polymerase holoenzyme
Map dataCryo-EM map of the phage AR9 non-virion RNA polymerase holoenzyme
Sample
  • Complex: Bacteriophage AR9 non-virion RNA polymerase holoenzyme
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / DNA-templated transcription / DNA binding / metal ion binding
Similarity search - Function
DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6
Similarity search - Domain/homology
DNA-directed RNA polymerase / DNA-directed RNA polymerase beta subunit / DNA-directed RNA polymerase / DNA-directed RNA polymerase subunit / DNA-directed RNA polymerase
Similarity search - Component
Biological speciesBacillus phage AR9 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsFraser A / Leiman PG / Sokolova ML
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis of template strand deoxyuridine promoter recognition by a viral RNA polymerase.
Authors: Alec Fraser / Maria L Sokolova / Arina V Drobysheva / Julia V Gordeeva / Sergei Borukhov / John Jumper / Konstantin V Severinov / Petr G Leiman /
Abstract: Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the -10 promoter element, is located in the non-template ...Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the -10 promoter element, is located in the non-template strand of the double-stranded DNA molecule ~10 nucleotides upstream of the transcription start site. Here, we explain the mechanism by which the phage AR9 non-virion RNAP (nvRNAP), a bacterial RNAP homolog, recognizes the -10 element of its deoxyuridine-containing promoter in the template strand. The AR9 sigma-like subunit, the nvRNAP enzyme core, and the template strand together form two nucleotide base-accepting pockets whose shapes dictate the requirement for the conserved deoxyuridines. A single amino acid substitution in the AR9 sigma-like subunit allows one of these pockets to accept a thymine thus expanding the promoter consensus. Our work demonstrates the extent to which viruses can evolve host-derived multisubunit enzymes to make transcription of their own genes independent of the host.
History
DepositionAug 27, 2021-
Header (metadata) releaseAug 31, 2022-
Map releaseAug 31, 2022-
UpdateMar 15, 2023-
Current statusMar 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24765.png

Downloads & links

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Map

FileDownload / File: emd_24765.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the phage AR9 non-virion RNA polymerase holoenzyme
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 200 pix.
= 216. Å		1.08 Å/pix.
x 200 pix.
= 216. Å		1.08 Å/pix.
x 200 pix.
= 216. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.0
Minimum - Maximum-17.973656 - 22.294903
Average (Standard dev.)4.95839e-09 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 216.00002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_24765_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_24765_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Bacteriophage AR9 non-virion RNA polymerase holoenzyme

EntireName: Bacteriophage AR9 non-virion RNA polymerase holoenzyme
Components
  • Complex: Bacteriophage AR9 non-virion RNA polymerase holoenzyme

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Supramolecule #1: Bacteriophage AR9 non-virion RNA polymerase holoenzyme

SupramoleculeName: Bacteriophage AR9 non-virion RNA polymerase holoenzyme
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bacillus phage AR9 (virus)

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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

Concentration20 mg/mL
BufferpH: 6.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 43.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio reconstruction.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 104471
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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