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- EMDB-24763: Cryo-EM map of the phage AR9 non-virion RNA polymerase holoenzyme... -

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Basic information

Entry
Database: EMDB / ID: EMD-24763
TitleCryo-EM map of the phage AR9 non-virion RNA polymerase holoenzyme in complex with DNA containing the AR9 P077 promoter
Map dataCryo-EM map of bacteriophage AR9 non-virion RNA polymerase in complex with two oligonucleotides containing two P077 promoters.
Sample
  • Complex: AR9 nvRNAP promoter complex
Function / homology
Function and homology information


RNA polymerase II activity / DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / 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: 3.8 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_24763.png

Downloads & links

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Map

FileDownload / File: emd_24763.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of bacteriophage AR9 non-virion RNA polymerase in complex with two oligonucleotides containing two P077 promoters.
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-6.6733656 - 17.809258
Average (Standard dev.)5.0541194e-12 (±0.99999994)
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_24763_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_24763_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 : AR9 nvRNAP promoter complex

EntireName: AR9 nvRNAP promoter complex
Components
  • Complex: AR9 nvRNAP promoter complex

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Supramolecule #1: AR9 nvRNAP promoter complex

SupramoleculeName: AR9 nvRNAP promoter complex / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1
Details: The complex consists of the AR9 nvRNAP holoenzyme and two copies of the same oligonucleotide containing the P077 promoter. Only three bases in one of the oligonucleotides are sufficiently ...Details: The complex consists of the AR9 nvRNAP holoenzyme and two copies of the same oligonucleotide containing the P077 promoter. Only three bases in one of the oligonucleotides are sufficiently ordered for atomic model building.
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

Concentration10 mg/mL
BufferpH: 6.8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 43.68 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: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 106867
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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