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- EMDB-13026: Cryo-EM structure of the ATV RNAP Inhibitory Protein (RIP) bound ... -

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Basic information

Entry
Database: EMDB / ID: EMD-13026
TitleCryo-EM structure of the ATV RNAP Inhibitory Protein (RIP) bound to the DNA-binding channel of the host's RNA polymerase
Map data
SampleComplex of archaeal RNA-polymerase with the ATV inhibitory protein RIP
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 10
  • (DNA-directed RNA polymerase, subunit ...Polymerase) x 2
  • Conserved proteinConservation
  • RNAP inhibitory protein
  • (ligand) x 3
Function / homology
Function and homology information


suppression by virus of host transcription / RNA polymerase complex / virion component => GO:0044423 / DNA-templated transcription, initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / 4 iron, 4 sulfur cluster binding / transcription, DNA-templated / protein dimerization activity ...suppression by virus of host transcription / RNA polymerase complex / virion component => GO:0044423 / DNA-templated transcription, initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / 4 iron, 4 sulfur cluster binding / transcription, DNA-templated / protein dimerization activity / nucleotide binding / DNA binding / zinc ion binding / metal ion binding
Similarity search - Function
RNA polymerase Rpo13 subunit HTH domain / RNA polymerase Rpo13 subunit HTH domain / DNA-directed RNA polymerase, subunit G / DNA-directed RNA polymerase subunit Rpo8 / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo3 ...RNA polymerase Rpo13 subunit HTH domain / RNA polymerase Rpo13 subunit HTH domain / DNA-directed RNA polymerase, subunit G / DNA-directed RNA polymerase subunit Rpo8 / DNA-directed RNA polymerase subunit Rpo12 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo4 / DNA-directed RNA polymerase subunit Rpo3 / DNA-directed RNA polymerase subunit Rpo11 / DNA-directed RNA polymerase, subunit E/RPC8 / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / S1 domain profile. / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb7-like , N-terminal / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / HRDC-like superfamily / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / RNA polymerases N / 8 Kd subunits signature. / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / DNA-directed RNA polymerase subunit RPABC5/Rpb10 / RNA polymerases N / 8 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase subunit CX / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases L / 13 to 16 Kd subunits signature. / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / RPB5-like RNA polymerase subunit superfamily / RNA polymerase, subunit H/Rpb5 C-terminal / : / RNA polymerase Rpb5, C-terminal domain / : / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / RNA-binding domain, S1 / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / : / RNA polymerase Rpb6 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 3 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase, N-terminal / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, protrusion / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase beta subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / RNA polymerase Rpb2, domain 6 / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, OB-fold / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo8 / DNA-directed RNA polymerase subunit Rpo13 / DNA-directed RNA polymerase subunit Rpo12 / RNAP inhibitory protein / DNA-directed RNA polymerase subunit Rpo11 / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit Rpo3 / DNA-directed RNA polymerase subunit Rpo7 / DNA-directed RNA polymerase subunit Rpo6 ...DNA-directed RNA polymerase subunit Rpo10 / DNA-directed RNA polymerase subunit Rpo8 / DNA-directed RNA polymerase subunit Rpo13 / DNA-directed RNA polymerase subunit Rpo12 / RNAP inhibitory protein / DNA-directed RNA polymerase subunit Rpo11 / DNA-directed RNA polymerase subunit Rpo5 / DNA-directed RNA polymerase subunit Rpo3 / DNA-directed RNA polymerase subunit Rpo7 / DNA-directed RNA polymerase subunit Rpo6 / DNA-directed RNA polymerase subunit Rpo1C / DNA-directed RNA polymerase subunit Rpo2 / DNA-directed RNA polymerase subunit Rpo1N / DNA-directed RNA polymerase subunit Rpo4
Similarity search - Component
Biological speciesSulfolobus acidocaldarius DSM 639 (acidophilic) / Acidianus two-tailed virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsPilotto S / Fouqueau T / Lukoyanova N / Sheppard C / Lucas-Staat S / Diaz-Santin LM / Matelska D / Prangishvili D / Cheung ACM / Werner F
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust207446/Z/17/Z United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Structural basis of RNA polymerase inhibition by viral and host factors.
Authors: Simona Pilotto / Thomas Fouqueau / Natalya Lukoyanova / Carol Sheppard / Soizick Lucas-Staat / Luis Miguel Díaz-Santín / Dorota Matelska / David Prangishvili / Alan C M Cheung / Finn Werner /
Abstract: RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution ...RNA polymerase inhibition plays an important role in the regulation of transcription in response to environmental changes and in the virus-host relationship. Here we present the high-resolution structures of two such RNAP-inhibitor complexes that provide the structural bases underlying RNAP inhibition in archaea. The Acidianus two-tailed virus encodes the RIP factor that binds inside the DNA-binding channel of RNAP, inhibiting transcription by occlusion of binding sites for nucleic acid and the transcription initiation factor TFB. Infection with the Sulfolobus Turreted Icosahedral Virus induces the expression of the host factor TFS4, which binds in the RNAP funnel similarly to eukaryotic transcript cleavage factors. However, TFS4 allosterically induces a widening of the DNA-binding channel which disrupts trigger loop and bridge helix motifs. Importantly, the conformational changes induced by TFS4 are closely related to inactivated states of RNAP in other domains of life indicating a deep evolutionary conservation of allosteric RNAP inhibition.
History
DepositionJun 2, 2021-
Header (metadata) releaseAug 25, 2021-
Map releaseAug 25, 2021-
UpdateOct 13, 2021-
Current statusOct 13, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7oq4
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7oq4
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_13026.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 300 pix.
= 314.1 Å
1.05 Å/pix.
x 300 pix.
= 314.1 Å
1.05 Å/pix.
x 300 pix.
= 314.1 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.047 Å
Density
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.10906489 - 0.19450761
Average (Standard dev.)0.00018650922 (±0.0037748057)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 314.1 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z314.100314.100314.100
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ332332332
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1090.1950.000

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Supplemental data

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Half map: Half1 map obtained from 3D refinement in Relion...

Fileemd_13026_half_map_1.map
AnnotationHalf1 map obtained from 3D refinement in Relion 3.0. The A/pix has been corrected later during post-processing.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half2 map obtained from 3D refinement in Relion...

Fileemd_13026_half_map_2.map
AnnotationHalf2 map obtained from 3D refinement in Relion 3.0. The A/pix has been corrected later during post-processing.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Complex of archaeal RNA-polymerase with the ATV inhibitory protein RIP

EntireName: Complex of archaeal RNA-polymerase with the ATV inhibitory protein RIP
Details: The RNA polymerase was incubated with a 10-fold excess of RIP for 5 min at 338 K followed by cross-linking with BS3
Number of Components: 18

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Component #1: protein, Complex of archaeal RNA-polymerase with the ATV inhibito...

ProteinName: Complex of archaeal RNA-polymerase with the ATV inhibitory protein RIP
Details: The RNA polymerase was incubated with a 10-fold excess of RIP for 5 min at 338 K followed by cross-linking with BS3
Recombinant expression: No
MassExperimental: 421 kDa

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Component #2: protein, DNA-directed RNA polymerase subunit A'

ProteinName: DNA-directed RNA polymerase subunit A'Polymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 99.929055 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #3: protein, DNA-directed RNA polymerase subunit B

ProteinName: DNA-directed RNA polymerase subunit BPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 126.647102 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #4: protein, DNA-directed RNA polymerase subunit A''

ProteinName: DNA-directed RNA polymerase subunit A''Polymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 44.50743 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #5: protein, DNA-directed RNA polymerase subunit D

ProteinName: DNA-directed RNA polymerase subunit DPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 29.858752 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #6: protein, DNA-directed RNA polymerase subunit E

ProteinName: DNA-directed RNA polymerase subunit EPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 20.49675 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #7: protein, DNA-directed RNA polymerase, subunit F

ProteinName: DNA-directed RNA polymerase, subunit FPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 13.070987 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #8: protein, DNA-directed RNA polymerase, subunit G

ProteinName: DNA-directed RNA polymerase, subunit GPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 15.292758 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)
Source (engineered)Expression System: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #9: protein, DNA-directed RNA polymerase subunit H

ProteinName: DNA-directed RNA polymerase subunit HPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.477059 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #10: protein, DNA-directed RNA polymerase subunit K

ProteinName: DNA-directed RNA polymerase subunit KPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.25199 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #11: protein, DNA-directed RNA polymerase subunit L

ProteinName: DNA-directed RNA polymerase subunit LPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.061791 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #12: protein, DNA-directed RNA polymerase subunit N

ProteinName: DNA-directed RNA polymerase subunit NPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 7.673108 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #13: protein, DNA-directed RNA polymerase subunit P

ProteinName: DNA-directed RNA polymerase subunit PPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 5.662912 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #14: protein, Conserved protein

ProteinName: Conserved proteinConservation / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 12.331662 kDa
SourceSpecies: Sulfolobus acidocaldarius DSM 639 (acidophilic)

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Component #15: protein, RNAP inhibitory protein

ProteinName: RNAP inhibitory protein
Details: Residual sequence from the thrombin cleavage site at the C-terminus of RIP (LVPR)
Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 17.321936 kDa
SourceSpecies: Acidianus two-tailed virus
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)

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Component #16: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #17: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #18: ligand, FE3-S4 CLUSTER

LigandName: FE3-S4 CLUSTER / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.295795 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen State: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.06 mg/mL
Buffer solution: DTT was added fresh before the incubation of the two species
pH: 7
Support filmafter glow discharged, the grid was coated with graphene oxide
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen Name: ETHANE / Temperature: 277 K / Humidity: 94 %
Details: blotting force: -10 blotting time: 2 sec wait time: 0 sec.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Details: sample holder was tilted of -30 degrees for the entire data collection
Electron gunElectron Source: FIELD EMISSION GUN / Accelerating Voltage: 300 kV / Electron Dose: 45.5 e/Å2 / Illumination Mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging Mode: BRIGHT FIELD / Defocus: -1000.0 - -2500.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of Digital Images: 2130

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Image processing

ProcessingMethod: single particle reconstruction / Applied Symmetry: C1 (asymmetric) / Number of Projections: 151237
Details: The movie-stacks were aligned and summed using MotionCor2 within Relion 3.0
3D reconstructionSoftware: RELION / Resolution: 3.27 Å / Resolution Method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target Criteria: correlation coefficient / Refinement space: REAL
Details: The C-terminal tail of RIP was manually built in Coot after the first refinement run.
Input PDB model: 7OH0, 3FAJ
Chain ID: A

Overall BValue: 81.4405
Output model

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