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- PDB-6b6h: The cryo-EM structure of a bacterial class I transcription activa... -

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Entry
Database: PDB / ID: 6b6h
TitleThe cryo-EM structure of a bacterial class I transcription activation complex
Components
  • (DNA-directed RNA polymerase subunit ...) x 5
  • NASCENT RNA 3-MER
  • RNA polymerase sigma factor RpoD
  • SYNTHETIC NONTEMPLATE STRAND DNA (88-MER)
  • SYNTHETIC TEMPLATE STRAND DNA (88-MER)
  • cAMP-activated global transcriptional regulator CRP
KeywordsTRANSCRIPTION/TRANSFERASE/DNA/RNA / transcription / RNA polymerase / catabolite activator protein / cAMP / TRANSCRIPTION-TRANSFERASE-DNA-RNA complex
Function / homology
Function and homology information


sigma factor antagonist complex / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / cAMP binding / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase ...sigma factor antagonist complex / cytosolic DNA-directed RNA polymerase complex / regulation of DNA-templated transcription initiation / sigma factor activity / cAMP binding / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / response to heat / protein dimerization activity / DNA-binding transcription factor activity / negative regulation of DNA-templated transcription / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
: / helix_turn_helix, cAMP Regulatory protein / Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / Crp-like helix-turn-helix domain / Crp-type HTH domain profile. / Crp-type HTH domain / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 ...: / helix_turn_helix, cAMP Regulatory protein / Transcription regulator HTH, Crp-type, conserved site / Crp-type HTH domain signature. / Crp-like helix-turn-helix domain / Crp-type HTH domain profile. / Crp-type HTH domain / RNA polymerase sigma factor 70, non-essential domain / Sigma-70, non-essential region / RNA polymerase sigma factor 70, region 1.1 / Sigma-70 factor, region 1.1 superfamily / Sigma-70 factor, region 1.1 / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / Cyclic nucleotide-binding domain signature 2. / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / Cyclic nucleotide-binding domain signature 1. / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Cyclic nucleotide-binding, conserved site / Sigma-70 factors family signature 1. / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : / RNA polymerase sigma-70 region 1.2 / Sigma-70 factor, region 1.2 / RNA polymerase sigma-70 region 3 / Sigma-70 region 3 / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like / Gyrase A; domain 2 / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / Jelly Rolls / 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 / Beta Complex / RmlC-like jelly roll fold / Winged helix-like DNA-binding domain superfamily/Winged helix DNA-binding domain / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / 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, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / 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 / Arc Repressor Mutant, subunit A
Similarity search - Domain/homology
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta / RNA polymerase sigma factor RpoD / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta' / cAMP-activated global transcriptional regulator CRP
Similarity search - Component
Biological speciesEscherichia coli O157:H7 (bacteria)
Escherichia coli O45:K1 (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsLiu, B. / Hong, C. / Huang, R. / Yu, Z. / Steitz, T.A.
Funding support United States, 1items
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, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 15, 2017Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Dec 6, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.energyfilter_name
Revision 1.4Jan 1, 2020Group: Author supporting evidence / Data collection / Category: em_imaging_optics / pdbx_audit_support
Item: _em_imaging_optics.energyfilter_name / _pdbx_audit_support.funding_organization
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn / struct_conn_type
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn_type.id

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
E: DNA-directed RNA polymerase subunit omega
F: RNA polymerase sigma factor RpoD
G: cAMP-activated global transcriptional regulator CRP
H: cAMP-activated global transcriptional regulator CRP
I: DNA-directed RNA polymerase subunit alpha
1: SYNTHETIC NONTEMPLATE STRAND DNA (88-MER)
2: SYNTHETIC TEMPLATE STRAND DNA (88-MER)
3: NASCENT RNA 3-MER
hetero molecules


Theoretical massNumber of molelcules
Total (without water)573,75117
Polymers572,93812
Non-polymers8145
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area66660 Å2
ΔGint-356 kcal/mol
Surface area212670 Å2

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Components

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DNA-directed RNA polymerase subunit ... , 5 types, 6 molecules ABCDEI

#1: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 36558.680 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: rpoA, Z4665, ECs4160 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7Z6, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 150820.875 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O45:K1 (strain S88 / ExPEC) (bacteria)
Strain: S88 / ExPEC / Gene: rpoB, ECS88_4448 / Production host: Escherichia coli (E. coli) / References: UniProt: B7MIX3, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 155366.781 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: rpoC, Z5561, ECs4911 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A8T8, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 10249.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O45:K1 (bacteria) / Strain: S88 / ExPEC / Gene: rpoZ, ECS88_4064 / Production host: Escherichia coli (E. coli) / References: UniProt: B7MFL0, DNA-directed RNA polymerase
#7: Protein DNA-directed RNA polymerase subunit alpha / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 8346.699 Da / Num. of mol.: 1
Fragment: Alpha C-terminal domain (alpha-CTD) residues 250-324
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: rpoA, Z4665, ECs4160 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A7Z6, DNA-directed RNA polymerase

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Protein , 2 types, 3 molecules FGH

#5: Protein RNA polymerase sigma factor RpoD / Sigma-70


Mass: 72206.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: rpoD, alt, b3067, JW3039 / Production host: Escherichia coli (E. coli) / References: UniProt: P00579
#6: Protein cAMP-activated global transcriptional regulator CRP / Catabolite activator protein / CAP / Catabolite gene activator / cAMP receptor protein / CRP / cAMP ...Catabolite activator protein / CAP / Catabolite gene activator / cAMP receptor protein / CRP / cAMP regulatory protein


Mass: 23672.439 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli O157:H7 (bacteria) / Gene: crp, Z4718, ECs4208 / Production host: Escherichia coli (E. coli) / References: UniProt: P0ACK0

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DNA chain , 2 types, 2 molecules 12

#8: DNA chain SYNTHETIC NONTEMPLATE STRAND DNA (88-MER)


Mass: 27133.393 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#9: DNA chain SYNTHETIC TEMPLATE STRAND DNA (88-MER)


Mass: 27217.404 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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RNA chain , 1 types, 1 molecules 3

#10: RNA chain NASCENT RNA 3-MER / NASCENT RNA 3-MER


Mass: 1134.619 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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Non-polymers , 3 types, 5 molecules

#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#12: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#13: Chemical ChemComp-CMP / ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / CYCLIC AMP / CAMP


Mass: 329.206 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H12N5O6P

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: The complex of class-I bacterial transcription activation complex
Type: ORGANELLE OR CELLULAR COMPONENT
Details: A CAP dimer, a sigma70-RNA polymerase holoenzyme, an intact CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide
Entity ID: #1-#10 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.59 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Details: 20 mM TRIS pH 7.5, 50 mM sodium chloride, 0.1mM EDTA, 5 mM MgCl2, 5 mM DTT
Buffer component
IDConc.NameBuffer-ID
120 mMTris1
25 mMMgCl21
30.1 mMEDTA1
450 mMNaCl1
55 mMDTT1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3 second blotting

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Cs corrector
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Calibrated magnification: 37037 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Calibrated defocus min: 1200 nm / Calibrated defocus max: 2600 nm / Cs: 0.01 mm / C2 aperture diameter: 70 µm / Alignment procedure: OTHER
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K / Residual tilt: 15 mradians
Image recordingAverage exposure time: 0.25 sec. / Electron dose: 1.37 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2382
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansSampling size: 5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1EMAN2particle selection
2RELIONparticle selection
3SerialEMimage acquisition
5CTFFINDCTF correction
8UCSF Chimera1.11model fitting
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
14PHENIX1.12model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 835000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 161000 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: correlation coefficient

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