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- EMDB-28784: EsN-dhsU36mm2 local map (map 2) -

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Basic information

Entry
Database: EMDB / ID: EMD-28784
TitleEsN-dhsU36mm2 local map (map 2)
Map dataRaw map
Sample
  • Complex: EsN-dhsU36mm2
    • Complex: RNA polymerase complex
    • Complex: DNA
Keywordspromoter-bound / initiation / TRANSCRIPTION / TRANSCRIPTION-DNA complex
Biological speciesEscherichia coli (E. coli) / Aquifex aeolicus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsMueller AU / Chen J / Darst SA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM118130 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2023
Title: A general mechanism for transcription bubble nucleation in bacteria.
Authors: Andreas U Mueller / James Chen / Mengyu Wu / Courtney Chiu / B Tracy Nixon / Elizabeth A Campbell / Seth A Darst /
Abstract: Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ, nucleates DNA melting via recognition of conserved bases ...Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ, nucleates DNA melting via recognition of conserved bases of the promoter -10 motif, which are unstacked and captured in pockets of σ. By contrast, the mechanism of transcription bubble nucleation and formation during the unrelated σ-mediated transcription initiation is poorly understood. Herein, we combine structural and biochemical approaches to establish that σ, like σ, captures a flipped, unstacked base in a pocket formed between its N-terminal region I (RI) and extra-long helix features. Strikingly, RI inserts into the nascent bubble to stabilize the nucleated bubble prior to engagement of the obligate ATPase activator. Our data suggest a general paradigm of transcription initiation that requires σ factors to nucleate an early melted intermediate prior to productive RNA synthesis.
History
DepositionNov 4, 2022-
Header (metadata) releaseApr 5, 2023-
Map releaseApr 5, 2023-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28784.png

Downloads & links

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Map

FileDownload / File: emd_28784.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRaw map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-0.81943244 - 2.4642658
Average (Standard dev.)0.0030964683 (±0.058263578)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28784_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: B-factor sharpened map (-100.6)

Fileemd_28784_additional_1.map
AnnotationB-factor sharpened map (-100.6)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Local resolution map (blocres)

Fileemd_28784_additional_2.map
AnnotationLocal resolution map (blocres)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Locally filtered map (blocfilt)

Fileemd_28784_additional_3.map
AnnotationLocally filtered map (blocfilt)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_28784_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_28784_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 : EsN-dhsU36mm2

EntireName: EsN-dhsU36mm2
Components
  • Complex: EsN-dhsU36mm2
    • Complex: RNA polymerase complex
    • Complex: DNA

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Supramolecule #1: EsN-dhsU36mm2

SupramoleculeName: EsN-dhsU36mm2 / type: complex / ID: 1 / Parent: 0

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Supramolecule #2: RNA polymerase complex

SupramoleculeName: RNA polymerase complex / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Escherichia coli (E. coli)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Aquifex aeolicus (bacteria) / Synthetically produced: Yes

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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
Component:
ConcentrationNameFormula
20.0 mMHEPES
100.0 mMpotassium acetateKCH3COO
10.0 mMmagnesium chlorideMgCl2
2.0 mMdithiothreitol
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: 295 K / Instrument: FEI VITROBOT MARK IV
Details: Octyl beta-D-glucopyranoside (beta-OG) was added to the sample to 0.1%w/v final concentration (from 10x stock) just prior to plunge vitrification.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Cs corrector installed
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 7101 / Average exposure time: 2.5 sec. / Average electron dose: 64.3 e/Å2
Details: dose-fractionation with 0.05 seconds per frame (=50 frames)
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 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: ab initio generated map
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.1.0) / Software - details: Local refinement / Number images used: 198385
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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