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- EMDB-13204: transcriptional activator PafBC bound to mycobacterial RNA polymerase -

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Basic information

Entry
Database: EMDB / ID: EMD-13204
Titletranscriptional activator PafBC bound to mycobacterial RNA polymerase
Map datafull map, sharpened
Sample
  • Complex: DNA-directed RNA polymerase with transcriptional activator PafBC
    • Complex: DNA-dependent RNA polymerase
    • Complex: RNA polymerase sigma factor SigA, RNA polymerase-binding protein RbpA and RNA polymerase-binding transcription factor CarD
    • Complex: PafB and PafC
Function / homologyPafC, HTH domain / PafC helix-turn-helix domain / Protein PafC / : / WYL domain / WYL domain / Protein pafC / Transcriptional regulator-like protein
Function and homology information
Biological speciesMycobacterium smegmatis (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsMueller AU / Kummer E / Schilling CM / Ban N / Weber-Ban E
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_185250 Switzerland
CitationJournal: Sci Adv / Year: 2021
Title: Transcriptional control of mycobacterial DNA damage response by sigma adaptation.
Authors: Andreas U Müller / Eva Kummer / Charlotte M Schilling / Nenad Ban / Eilika Weber-Ban /
Abstract: Transcriptional activator PafBC is the key regulator of the mycobacterial DNA damage response and controls around 150 genes, including genes involved in the canonical SOS response, through an unknown ...Transcriptional activator PafBC is the key regulator of the mycobacterial DNA damage response and controls around 150 genes, including genes involved in the canonical SOS response, through an unknown molecular mechanism. Using a combination of biochemistry and cryo–electron microscopy, we demonstrate that PafBC in the presence of single-stranded DNA activates transcription by reprogramming the canonical −10 and −35 promoter specificity of RNA polymerase associated with the housekeeping sigma subunit. We determine the structure of this transcription initiation complex, revealing a unique mode of promoter recognition, which we term “sigma adaptation.” PafBC inserts between DNA and sigma factor to mediate recognition of hybrid promoters lacking the −35 but featuring the canonical −10 and a PafBC-specific −26 element. Sigma adaptation may constitute a more general mechanism of transcriptional control in mycobacteria.
History
DepositionJul 15, 2021-
Header (metadata) releaseDec 22, 2021-
Map releaseDec 22, 2021-
UpdateDec 22, 2021-
Current statusDec 22, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.16
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.16
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13204.png

Downloads & links

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Map

FileDownload / File: emd_13204.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull map, sharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 400 pix.
= 336. Å		0.84 Å/pix.
x 400 pix.
= 336. Å		0.84 Å/pix.
x 400 pix.
= 336. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16 / Movie #1: 0.16
Minimum - Maximum-0.42262742 - 1.0527649
Average (Standard dev.)0.0011000502 (±0.026810348)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z336.000336.000336.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.4231.0530.001

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Supplemental data

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Mask #1

Fileemd_13204_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: full map, unsharpened

Fileemd_13204_additional_1.map
Annotationfull map, unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map

Fileemd_13204_half_map_1.map
Annotationhalf map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map

Fileemd_13204_half_map_2.map
Annotationhalf 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 : DNA-directed RNA polymerase with transcriptional activator PafBC

EntireName: DNA-directed RNA polymerase with transcriptional activator PafBC
Components
  • Complex: DNA-directed RNA polymerase with transcriptional activator PafBC
    • Complex: DNA-dependent RNA polymerase
    • Complex: RNA polymerase sigma factor SigA, RNA polymerase-binding protein RbpA and RNA polymerase-binding transcription factor CarD
    • Complex: PafB and PafC

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Supramolecule #1: DNA-directed RNA polymerase with transcriptional activator PafBC

SupramoleculeName: DNA-directed RNA polymerase with transcriptional activator PafBC
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#10

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Supramolecule #2: DNA-dependent RNA polymerase

SupramoleculeName: DNA-dependent RNA polymerase / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#5
Source (natural)Organism: Mycobacterium smegmatis (bacteria)

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Supramolecule #3: RNA polymerase sigma factor SigA, RNA polymerase-binding protein ...

SupramoleculeName: RNA polymerase sigma factor SigA, RNA polymerase-binding protein RbpA and RNA polymerase-binding transcription factor CarD
type: complex / ID: 3 / Parent: 1 / Macromolecule list: #6-#8
Source (natural)Organism: Mycobacterium smegmatis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #4: PafB and PafC

SupramoleculeName: PafB and PafC / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #9-#10
Source (natural)Organism: Mycobacterium smegmatis (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

BufferpH: 7.8
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 30575
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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