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- EMDB-7060: Focused map on the aCTD-CAP region of the class-I bacterial trans... -

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Basic information

Entry
Database: EMDB / ID: EMD-7060
TitleFocused map on the aCTD-CAP region of the class-I bacterial transcription activation complex
Map dataFocused map of class I transcription activation complex
Sample
  • Complex: The aCTD-CAP region of the class-I bacterial transcription activation complex
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.0 Å
AuthorsLiu B / Hong C / Huang R / Yu Z / Steitz TA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM22778 United States
CitationJournal: Science / Year: 2017
Title: Structural basis of bacterial transcription activation.
Authors: Bin Liu / Chuan Hong / Rick K Huang / Zhiheng Yu / Thomas A Steitz /
Abstract: In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic ...In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact class I TAC containing a CAP dimer, a σ-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.
History
DepositionOct 2, 2017-
Header (metadata) releaseOct 18, 2017-
Map releaseNov 15, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6b6h
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7060.png

Downloads & links

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Map

FileDownload / File: emd_7060.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused map of class I transcription activation complex
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.055
Minimum - Maximum-0.014565958 - 0.14903507
Average (Standard dev.)0.00044035586 (±0.005036362)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0150.1490.000

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Supplemental data

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Sample components

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Entire : The aCTD-CAP region of the class-I bacterial transcription activa...

EntireName: The aCTD-CAP region of the class-I bacterial transcription activation complex
Components
  • Complex: The aCTD-CAP region of the class-I bacterial transcription activation complex

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Supramolecule #1: The aCTD-CAP region of the class-I bacterial transcription activa...

SupramoleculeName: The aCTD-CAP region of the class-I bacterial transcription activation complex
type: complex / ID: 1 / Parent: 0 / Details: Focused map only on the aCTD-CAP region
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 590 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

Concentration3 mg/mL
BufferpH: 7.5
Details: 20 mM TRIS pH 7.5, 50 mM sodium chloride, 0.1mM EDTA, 5 mM MgCl2
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 3 seconds blotting.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.6 µm / Calibrated defocus min: 1.2 µm / Calibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 98.0 K / Max: 98.0 K
DetailsCs Corrector
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 2382 / Average exposure time: 0.25 sec. / Average electron dose: 1.37 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 835000
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5ipl

Initial angle assignmentType: OTHER / Software - Name: RELION / Details: Relion
Final 3D classificationNumber classes: 4 / Software - Name: RELION
Final angle assignmentType: OTHER / Software - Name: RELION / Details: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 10800

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient

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