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- EMDB-28797: EsN-dhsU36mm1 composite map -

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Basic information

Entry
Database: EMDB / ID: EMD-28797
TitleEsN-dhsU36mm1 composite map
Map dataComposite of raw full and local maps
Sample
  • Complex: EsN-dhsU36mm1
    • Complex: RNA polymerase complex
    • Complex: DNA
Keywordspromoter-bound / initiation / TRANSCRIPTION
Function / homology
Function and homology information


arginine metabolic process / DNA-binding transcription activator activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / sigma factor activity / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex ...arginine metabolic process / DNA-binding transcription activator activity / RNA polymerase complex / submerged biofilm formation / cellular response to cell envelope stress / sigma factor activity / regulation of DNA-templated transcription initiation / bacterial-type flagellum assembly / bacterial-type RNA polymerase core enzyme binding / cytosolic DNA-directed RNA polymerase complex / bacterial-type flagellum-dependent cell motility / nitrate assimilation / nucleotidyltransferase activity / transcription elongation factor complex / regulation of DNA-templated transcription elongation / transcription antitermination / DNA-templated transcription initiation / cell motility / protein-DNA complex / ribonucleoside binding / DNA-directed RNA polymerase / DNA-directed RNA polymerase activity / response to heat / protein-containing complex assembly / intracellular iron ion homeostasis / transcription cis-regulatory region binding / protein dimerization activity / response to antibiotic / regulation of DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / membrane / cytosol / cytoplasm
Similarity search - Function
Sigma-54 factors family signature 1. / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase sigma factor 54, DNA-binding / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / Sigma-54, DNA binding domain / Sigma-54 factor, core binding domain / Sigma-54 factors family signature 2. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54 ...Sigma-54 factors family signature 1. / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase sigma factor 54, DNA-binding / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / Sigma-54, DNA binding domain / Sigma-54 factor, core binding domain / Sigma-54 factors family signature 2. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54 / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, RBP11-like subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / RNA polymerase sigma-54 factor
Similarity search - Component
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

Downloads & links

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Map

FileDownload / File: emd_28797.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite of raw full and local maps
Voxel sizeX=Y=Z: 1.083 Å
Density
Contour LevelBy AUTHOR: 6.0
Minimum - Maximum-7.041057 - 23.28416
Average (Standard dev.)0.005527685 (±1.0563028)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 277.248 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : EsN-dhsU36mm1

EntireName: EsN-dhsU36mm1
Components
  • Complex: EsN-dhsU36mm1
    • Complex: RNA polymerase complex
    • Complex: DNA

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Supramolecule #1: EsN-dhsU36mm1

SupramoleculeName: EsN-dhsU36mm1 / 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 opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 12294 / Average exposure time: 2.0 sec. / Average electron dose: 51.0 e/Å2
Details: dose-fractionation with 0.05 seconds per frame (=40 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: PHENIX / Software - details: phenix.combine_focused_maps
Details: Particle numbers and FSC resolution estimate of the full map given
Number images used: 602287
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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