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- EMDB-52933: Consensus map of bacterial transcription intermediate complex med... -

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Basic information

Entry
Database: EMDB / ID: EMD-52933
TitleConsensus map of bacterial transcription intermediate complex mediated by activator PspF containing nifH promoter DNA containing mismatch from -11 to -8 - conformation 6
Map data
Sample
  • Complex: Subtracted refinement map of conformation 1 of PspF(1-275)-sigma54 bound to nifH DNA (-28 to +35) containing mismatch from -11 to -8.
Keywordssigma factor / transcription / complex / AAA+ / DNA-binding protein
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.2 Å
AuthorsGao F / Zhang X
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Subunit specialization in AAA+ proteins and substrate unfolding during transcription complex remodeling.
Authors: Forson Gao / Fuzhou Ye / Martin Buck / Xiaodong Zhang /
Abstract: Bacterial RNA polymerase (RNAP) is a multisubunit enzyme that copies DNA into RNA in a process known as transcription. Bacteria use σ factors to recruit RNAP to promoter regions of genes that need ...Bacterial RNA polymerase (RNAP) is a multisubunit enzyme that copies DNA into RNA in a process known as transcription. Bacteria use σ factors to recruit RNAP to promoter regions of genes that need to be transcribed, with 60% bacteria containing at least one specialized σ factor, σ. σ recruits RNAP to promoters of genes associated with stress responses and forms a stable closed complex that does not spontaneously isomerize to the open state where promoter DNA is melted out and competent for transcription. The σ-mediated open complex formation requires specific AAA+ proteins (TPases ssociated with diverse cellular ctivities) known as bacterial enhancer-binding proteins (bEBPs). We have now obtained structures of new intermediate states of bEBP-bound complexes during transcription initiation, which elucidate the mechanism of DNA melting driven by ATPase activity of bEBPs and suggest a mechanistic model that couples the Adenosine triphosphate (ATP) hydrolysis cycle within the bEBP hexamer with σ unfolding. Our data reveal that bEBP forms a nonplanar hexamer with the hydrolysis-ready subunit located at the furthest/highest point of the spiral hexamer relative to the RNAP. ATP hydrolysis induces conformational changes in bEBP that drives a vectoral transiting of the regulatory N terminus of σ into the bEBP hexamer central pore causing the partial unfolding of σ, while forming specific bEBP contacts with promoter DNA. Furthermore, our data suggest a mechanism of the bEBP AAA+ protein that is distinct from the hand-over-hand mechanism proposed for many other AAA+ proteins, highlighting the versatile mechanisms utilized by the large protein family.
History
DepositionFeb 26, 2025-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateMay 7, 2025-
Current statusMay 7, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52933.png

Downloads & links

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Map

FileDownload / File: emd_52933.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 280 pix.
= 308. Å		1.1 Å/pix.
x 280 pix.
= 308. Å		1.1 Å/pix.
x 280 pix.
= 308. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.021
Minimum - Maximum-0.01435149 - 0.07582354
Average (Standard dev.)0.0008621548 (±0.0045507196)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 308.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_52933_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Half map: #2

Fileemd_52933_half_map_2.map
Projections & Slices
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Sample components

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Entire : Subtracted refinement map of conformation 1 of PspF(1-275)-sigma5...

EntireName: Subtracted refinement map of conformation 1 of PspF(1-275)-sigma54 bound to nifH DNA (-28 to +35) containing mismatch from -11 to -8.
Components
  • Complex: Subtracted refinement map of conformation 1 of PspF(1-275)-sigma54 bound to nifH DNA (-28 to +35) containing mismatch from -11 to -8.

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Supramolecule #1: Subtracted refinement map of conformation 1 of PspF(1-275)-sigma5...

SupramoleculeName: Subtracted refinement map of conformation 1 of PspF(1-275)-sigma54 bound to nifH DNA (-28 to +35) containing mismatch from -11 to -8.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#12
Source (natural)Organism: Escherichia coli K-12 (bacteria)

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS 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 / 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: EMDB MAP
EMDB ID:

14171

Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 7.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 22724
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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