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- EMDB-48581: de novo SigN RNA polymerase transcription initiation intermediate... -

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Basic information

Entry
Database: EMDB / ID: EMD-48581
Titlede novo SigN RNA polymerase transcription initiation intermediate with pre-catalytic bEBP state (RPi1 open ring), SigN focus map
Map dataunsharpened SigN focus map
Sample
  • Complex: EsNdhsUC1+ATP
Keywordssigma N / sigma 54 / ATPase / bacterial enhancer binding protein / transcription initiation / intermediate / TRANSCRIPTION
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsMueller AU / Darst SA
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118130 United States
Life Sciences Research FoundationAgouron Institute United States
CitationJournal: Nat Commun / Year: 2025
Title: Real-time capture of σ transcription initiation intermediates reveals mechanism of ATPase-driven activation by limited unfolding.
Authors: Andreas U Mueller / Nina Molina / B Tracy Nixon / Seth A Darst /
Abstract: Bacterial σ factors bind RNA polymerase (E) to form holoenzyme (Eσ), conferring promoter specificity to E and playing a key role in transcription bubble formation. σ is unique among σ factors in ...Bacterial σ factors bind RNA polymerase (E) to form holoenzyme (Eσ), conferring promoter specificity to E and playing a key role in transcription bubble formation. σ is unique among σ factors in its structure and functional mechanism, requiring activation by specialized AAA+ ATPases. Eσ forms an inactive promoter complex where the N-terminal σ region I (σ-RI) threads through a small DNA bubble. On the opposite side of the DNA, the ATPase engages σ-RI within the pore of its hexameric ring. Here, we perform kinetics-guided structural analysis of de novo formed Eσ initiation complexes and engineer a biochemical assay to measure ATPase-mediated σ-RI translocation during promoter melting. We show that the ATPase exerts mechanical action to translocate about 30 residues of σ-RI through the DNA bubble, disrupting inhibitory structures of σ to allow full transcription bubble formation. A local charge switch of σ-RI from positive to negative may help facilitate disengagement of the otherwise processive ATPase, allowing subsequent σ disentanglement from the DNA bubble.
History
DepositionJan 9, 2025-
Header (metadata) releaseAug 13, 2025-
Map releaseAug 13, 2025-
UpdateAug 13, 2025-
Current statusAug 13, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48581.png

Downloads & links

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Map

FileDownload / File: emd_48581.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened SigN focus map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 448 pix.
= 385.28 Å		0.86 Å/pix.
x 448 pix.
= 385.28 Å		0.86 Å/pix.
x 448 pix.
= 385.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.86 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.51646394 - 1.014284
Average (Standard dev.)0.0004539759 (±0.011185183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 385.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48581_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: sharpened SigN focus map (b-factor 74.2)

Fileemd_48581_additional_1.map
Annotationsharpened SigN focus map (b-factor 74.2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_48581_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map A

Fileemd_48581_half_map_2.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : EsNdhsUC1+ATP

EntireName: EsNdhsUC1+ATP
Components
  • Complex: EsNdhsUC1+ATP

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Supramolecule #1: EsNdhsUC1+ATP

SupramoleculeName: EsNdhsUC1+ATP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Details: E = RNAP sN = Sigma N dhsU = dhsU promoter DNA C1 = NtrC1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 180 KDa

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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
Details: 40 mM Tris-HCl, pH 8/RT, 200 mM KCl, 10 mM MgCl2, 1 mM DTT; fluorinated fos-choline-8 (FC8F) added to a final concentration of 1.5 mM during grid preparation
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 310 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 11520 pixel / Digitization - Dimensions - Height: 8184 pixel / Number grids imaged: 1 / Number real images: 17199 / Average exposure time: 1.4 sec. / Average electron dose: 42.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm
Sample stageCooling holder cryogen: NITROGEN
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: PDB ENTRY
PDB model - PDB ID:

8f1k


Details: 8F1K and 4LZZ were placed in the map to generate the final startup model before building
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1) / Number images used: 118561
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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