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- EMDB-50590: Transcriptional activator PafBC bound to mycobacterial RNA polymerase -

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Basic information

Entry
Database: EMDB / ID: EMD-50590
TitleTranscriptional activator PafBC bound to mycobacterial RNA polymerase
Map dataSharpened map.
Sample
  • Complex: Transcriptional activator PafBC bound to mycobacterial RNA polymerase
    • Complex: PafBC
      • Protein or peptide: Transcriptional regulator-like protein
      • Protein or peptide: PafC
    • Complex: DNA-directed RNA polymerase
    • Complex: RNAP polymerase sigma factor A and RNA polymerase-binding protein RbpA
KeywordsPafBC / sigma adaptation / WYL domain / transcriptional activator / TRANSCRIPTION
Function / homologyPafC, HTH domain / PafC helix-turn-helix domain / Protein PafC / : / WYL domain profile. / WYL domain / WYL domain / Protein pafC / Transcriptional regulator-like protein
Function and homology information
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsZdanowicz R / Schilling CM / Rabl J / Mueller AU / Boehringer D / Glockshuber R / Weber-Ban E
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation310030_215606 Switzerland
CitationJournal: Sci Adv / Year: 2025
Title: Single-stranded DNA binding to the transcription factor PafBC triggers the mycobacterial DNA damage response.
Authors: Charlotte M Schilling / Rafal Zdanowicz / Julius Rabl / Andreas U Müller / Daniel Boehringer / Rudi Glockshuber / Eilika Weber-Ban /
Abstract: The DNA damage response in mycobacteria is controlled by the heterodimeric transcription factor PafBC, a member of the WYL domain-containing protein family. It has been shown that PafBC induces ...The DNA damage response in mycobacteria is controlled by the heterodimeric transcription factor PafBC, a member of the WYL domain-containing protein family. It has been shown that PafBC induces transcription of its regulon by reprogramming the housekeeping RNA polymerase holoenzyme to recognize PafBC-dependent promoters through sigma adaptation. However, the mechanism by which DNA damage is sensed and translated into PafBC activation has remained unclear. Here, we demonstrate that the binding of single-stranded DNA (ssDNA) to the WYL domains of PafBC activates the transcription factor. Our cryo-electron microscopy structure of full-length PafBC in its active conformation, bound to the transcription initiation complex, reveals a previously unknown mode of interaction between the ssDNA and the WYL domains. Using biochemical experiments, we show that short ssDNA fragments bind to PafBC dynamically, resulting in deactivation as ssDNA levels decrease postrepair. Our findings shed light on the mechanism linking DNA damage to PafBC activation and expand our understanding of WYL domain-containing proteins.
History
DepositionJun 10, 2024-
Header (metadata) releaseFeb 12, 2025-
Map releaseFeb 12, 2025-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50590.png

Downloads & links

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Map

FileDownload / File: emd_50590.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.58 Å/pix.
x 256 pix.
= 403.2 Å		1.58 Å/pix.
x 256 pix.
= 403.2 Å		1.58 Å/pix.
x 256 pix.
= 403.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.575 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0113
Minimum - Maximum-0.08540109 - 0.115036316
Average (Standard dev.)0.000021831827 (±0.0009949618)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 403.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map.

Fileemd_50590_additional_1.map
AnnotationUnsharpened map.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Density amplified using OccuPy.

Fileemd_50590_additional_2.map
AnnotationDensity amplified using OccuPy.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A.

Fileemd_50590_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_50590_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 : Transcriptional activator PafBC bound to mycobacterial RNA polymerase

EntireName: Transcriptional activator PafBC bound to mycobacterial RNA polymerase
Components
  • Complex: Transcriptional activator PafBC bound to mycobacterial RNA polymerase
    • Complex: PafBC
      • Protein or peptide: Transcriptional regulator-like protein
      • Protein or peptide: PafC
    • Complex: DNA-directed RNA polymerase
    • Complex: RNAP polymerase sigma factor A and RNA polymerase-binding protein RbpA

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Supramolecule #1: Transcriptional activator PafBC bound to mycobacterial RNA polymerase

SupramoleculeName: Transcriptional activator PafBC bound to mycobacterial RNA polymerase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Supramolecule #2: PafBC

SupramoleculeName: PafBC / type: complex / ID: 2 / Parent: 1 / Macromolecule list: all
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Supramolecule #3: DNA-directed RNA polymerase

SupramoleculeName: DNA-directed RNA polymerase / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Supramolecule #4: RNAP polymerase sigma factor A and RNA polymerase-binding protein RbpA

SupramoleculeName: RNAP polymerase sigma factor A and RNA polymerase-binding protein RbpA
type: complex / ID: 4 / Parent: 1
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)

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Macromolecule #1: Transcriptional regulator-like protein

MacromoleculeName: Transcriptional regulator-like protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 36.207555 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GLSAVSKVER LMNLVIALLS TRTYLPAEKI RTTVAGYADS PSDEAFSRMF ERDKNELRDL GIPLETGRVS KWDSTEGYRI NRDSYALPP IGLTADEAAA VAVATQLWQS PELVTATQNA VLKLRAAGVD VDADGVGVAI ASTATLPGVR GSEEVLQSLL S AIDEGRAV ...String:
GLSAVSKVER LMNLVIALLS TRTYLPAEKI RTTVAGYADS PSDEAFSRMF ERDKNELRDL GIPLETGRVS KWDSTEGYRI NRDSYALPP IGLTADEAAA VAVATQLWQS PELVTATQNA VLKLRAAGVD VDADGVGVAI ASTATLPGVR GSEEVLQSLL S AIDEGRAV QFEHRPSRSA DYTTRTVEPW GVVTHRGRWY LVGHDRDRED TRTFRLSRIS AAARPIGPAG AVQKPQDVNL RD IVRRAVA EQPTGERARI WIAGGRATAL RRQAVTSTPR TIGGRAGEEI TVDIGTWDRL AREIASYGSD AVALEPSSLR DDV VERLRA HAAGGER

UniProtKB: Transcriptional regulator-like protein

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Macromolecule #2: PafC

MacromoleculeName: PafC / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria)
Molecular weightTheoretical: 34.044402 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSQVSTRLVR LLNMVPYFQA NPKVTRAEAA AALGVTGKQL DADLDQLWMC GLPGYSPGDL IDFDFVGDTI EVTFSAGVDH PLRLTSTEA TGILVALRAL VDVPGMVDPE AARSAIAKIE SAVGSQRAVV EGITEDTSAE PGAAATVRTA VRENRALTLE Y YSASRDSL ...String:
MSQVSTRLVR LLNMVPYFQA NPKVTRAEAA AALGVTGKQL DADLDQLWMC GLPGYSPGDL IDFDFVGDTI EVTFSAGVDH PLRLTSTEA TGILVALRAL VDVPGMVDPE AARSAIAKIE SAVGSQRAVV EGITEDTSAE PGAAATVRTA VRENRALTLE Y YSASRDSL ATRTVDPIRV VLVGDNSYLE AWCRSAEAVR LFRFDRIVDA QLLDDPAAPP PPAVAAGPDT SLFDADPSLP SA TLLIGAA AAWMFDYYPL RDITERPDGS CEATMTYASE DWMARFILGF GAEVQVLAPE SLATRVRQAA EAALQAYARC V

UniProtKB: Protein pafC

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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: 8
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 78.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 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: cryoSPARC Ab-Initio model
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Software - details: 3DFlex / Number images used: 154302
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

PDB-9fnd:
Transcriptional activator PafBC bound to mycobacterial RNA polymerase

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