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- PDB-8w8n: Thermus thermophilus initiation transcription complex in the pre-... -

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Basic information

Entry
Database: PDB / ID: 8w8n
TitleThermus thermophilus initiation transcription complex in the pre-translocated state
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • DNA (21-MER)
  • DNA (27-MER)
  • RNA (5'-D(*PP*PP*P)-R(*GP*GP*U)-3')
  • RNA polymerase sigma factor SigA
KeywordsTRANSCRIPTION/DNA/RNA / Thermus thermophilus / RNA polymerase / pre-translocation / transcription / TRANSCRIPTION-DNA-RNA complex
Function / homology
Function and homology information


sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding ...sigma factor activity / DNA-directed RNA polymerase complex / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / metal ion binding / cytoplasm
Similarity search - Function
: / DNA-directed RNA polymerase subunit beta', hybrid domain / 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. ...: / DNA-directed RNA polymerase subunit beta', hybrid domain / 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 / 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 / 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 / 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 / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / 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 / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit alpha / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.693 Å
AuthorsLi, L. / Zhang, Y.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31670067 China
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2024
Title: Nanopore tweezers show fractional-nucleotide translocation in sequence-dependent pausing by RNA polymerase.
Authors: Nova, I.C. / Craig, J.M. / Mazumder, A. / Laszlo, A.H. / Derrington, I.M. / Noakes, M.T. / Brinkerhoff, H. / Yang, S. / Vahedian-Movahed, H. / Li, L. / Zhang, Y. / Bowman, J.L. / Huang, J.R. ...Authors: Nova, I.C. / Craig, J.M. / Mazumder, A. / Laszlo, A.H. / Derrington, I.M. / Noakes, M.T. / Brinkerhoff, H. / Yang, S. / Vahedian-Movahed, H. / Li, L. / Zhang, Y. / Bowman, J.L. / Huang, J.R. / Mount, J.W. / Ebright, R.H. / Gundlach, J.H.
History
DepositionSep 4, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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 SigA
G: DNA (21-MER)
H: DNA (27-MER)
I: RNA (5'-D(*PP*PP*P)-R(*GP*GP*U)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)445,06817
Polymers444,7919
Non-polymers2778
Water2,522140
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area54240 Å2
ΔGint-256 kcal/mol
Surface area141570 Å2
MethodPISA
Unit cell
Length a, b, c (Å)184.422, 103.704, 295.444
Angle α, β, γ (deg.)90.00, 98.67, 90.00
Int Tables number5
Space group name H-MC121

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE

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


Mass: 35056.164 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q5SHR6, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 125436.539 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q8RQE9, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit beta' / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 170997.391 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q8RQE8, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11533.316 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Thermus thermophilus HB8 (bacteria) / References: UniProt: Q8RQE7, DNA-directed RNA polymerase

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DNA chain , 2 types, 2 molecules GH

#6: DNA chain DNA (21-MER)


Mass: 6409.137 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#7: DNA chain DNA (27-MER)


Mass: 8421.432 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 2 molecules FI

#5: Protein RNA polymerase sigma factor SigA


Mass: 50769.398 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Gene: sigA, TTHA0532 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q5SKW1
#8: RNA chain RNA (5'-D(*PP*PP*P)-R(*GP*GP*U)-3')


Mass: 1111.579 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 148 molecules

#9: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#10: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#11: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 140 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.14 Å3/Da / Density % sol: 60.78 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop
Details: 0.2M KCl, 0.05M MgCl2, 0.1M Tris-HCl pH7.8, 9.5% PEG 4000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.97853 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Nov 26, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97853 Å / Relative weight: 1
ReflectionResolution: 2.693→50 Å / Num. obs: 147743 / % possible obs: 97.2 % / Redundancy: 3.9 % / Rmerge(I) obs: 0.128 / Net I/σ(I): 10.818
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2CC starRpim(I) allRrim(I) allΧ2% possible all
2.7-2.752.50.89552850.6290.8790.5871.0750.96570.3
2.75-2.82.60.85863810.6660.8940.5591.0280.99583.9
2.8-2.852.90.84970530.6970.9060.5431.0120.97494
2.85-2.913.30.81974300.7470.9250.510.9680.9898
2.91-2.973.50.72775140.8230.950.4390.8520.98699.4
2.97-3.043.60.63975400.860.9620.3880.7510.98799.8
3.04-3.123.80.5876070.8870.970.3350.6720.99999.9
3.12-3.24.20.49975530.9190.9790.2730.571.007100
3.2-3.34.30.39875280.9450.9860.2150.4531.005100
3.3-3.44.30.31775890.960.990.1720.3620.991100
3.4-3.524.20.24775940.9660.9910.1350.2820.976100
3.52-3.664.10.19375680.9690.9920.1080.2220.966100
3.66-3.833.90.15175300.9740.9930.0870.1750.92599.8
3.83-4.034.30.12376060.9780.9940.0680.1410.9299.8
4.03-4.294.30.09975940.9850.9960.0550.1140.87799.9
4.29-4.624.30.08376310.9870.9970.0460.0950.772100
4.62-5.0840.07575940.9850.9960.0430.0870.734100
5.08-5.814.40.07676620.9870.9970.0410.0860.737100
5.81-7.324.10.06976740.9830.9960.0390.0790.714100
7.32-504.10.06478100.9850.9960.0360.0741.09299.4

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Processing

Software
NameVersionClassification
PHENIX(1.12_2829: ???)refinement
HKL-2000data scaling
HKL-2000data reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.693→48.863 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.48 / Phase error: 29.54 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2599 2002 1.75 %
Rwork0.2124 --
obs0.2133 114360 74.9 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.693→48.863 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms27603 877 8 140 28628
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0129112
X-RAY DIFFRACTIONf_angle_d0.56839526
X-RAY DIFFRACTIONf_dihedral_angle_d23.81711288
X-RAY DIFFRACTIONf_chiral_restr0.0434457
X-RAY DIFFRACTIONf_plane_restr0.0045032
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.693-2.76020.5212250.31861520X-RAY DIFFRACTION14
2.7602-2.83490.3504450.31722210X-RAY DIFFRACTION21
2.8349-2.91830.3331550.31463062X-RAY DIFFRACTION29
2.9183-3.01240.35680.31994295X-RAY DIFFRACTION40
3.0124-3.12010.38891200.30816336X-RAY DIFFRACTION59
3.1201-3.2450.34261690.27979284X-RAY DIFFRACTION87
3.245-3.39260.35761810.261510481X-RAY DIFFRACTION98
3.3926-3.57150.26751900.243910636X-RAY DIFFRACTION100
3.5715-3.79510.27131880.21910682X-RAY DIFFRACTION100
3.7951-4.0880.24251920.195910680X-RAY DIFFRACTION100
4.088-4.49920.2481950.17510710X-RAY DIFFRACTION100
4.4992-5.14960.22171910.173310749X-RAY DIFFRACTION100
5.1496-6.48560.23441860.201810800X-RAY DIFFRACTION100
6.4856-48.8630.19641970.176410913X-RAY DIFFRACTION99

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