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- PDB-6asg: Crystal structure of Thermus thermophilus RNA polymerase core enzyme -

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Basic information

Entry
Database: PDB / ID: 6asg
TitleCrystal structure of Thermus thermophilus RNA polymerase core enzyme
Components(DNA-directed RNA polymerase subunit ...Polymerase) x 4
KeywordsTRANSCRIPTION / Thermus thermophilus / RNA polymerase core enzyme
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / 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 / cytoplasm
Similarity search - Function
: / DNA-directed RNA polymerase subunit beta', hybrid domain / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Gyrase A; domain 2 / DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type ...: / DNA-directed RNA polymerase subunit beta', hybrid domain / RNA polymerase subunit, RPB6/omega / Eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, insert domain / RNA polymerase, RBP11-like subunit / RNA Polymerase Alpha Subunit; Chain A, domain 2 / Gyrase A; domain 2 / 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 / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / Alpha-Beta Complex / 2-Layer Sandwich / Mainly Beta / Alpha Beta
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
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.8 Å
AuthorsLiu, Y. / Lin, W. / Ying, R. / Ebright, R.H.
CitationJournal: Mol Cell / Year: 2018
Title: Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3).
Authors: Wei Lin / Kalyan Das / David Degen / Abhishek Mazumder / Diego Duchi / Dongye Wang / Yon W Ebright / Richard Y Ebright / Elena Sineva / Matthew Gigliotti / Aashish Srivastava / Sukhendu ...Authors: Wei Lin / Kalyan Das / David Degen / Abhishek Mazumder / Diego Duchi / Dongye Wang / Yon W Ebright / Richard Y Ebright / Elena Sineva / Matthew Gigliotti / Aashish Srivastava / Sukhendu Mandal / Yi Jiang / Yu Liu / Ruiheng Yin / Zhening Zhang / Edward T Eng / Dennis Thomas / Stefano Donadio / Haibo Zhang / Changsheng Zhang / Achillefs N Kapanidis / Richard H Ebright /
Abstract: Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial ...Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.
History
DepositionAug 24, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 11, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 18, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Oct 4, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: DNA-directed RNA polymerase subunit beta
D: DNA-directed RNA polymerase subunit beta'
A: DNA-directed RNA polymerase subunit alpha
B: DNA-directed RNA polymerase subunit alpha
E: DNA-directed RNA polymerase subunit omega
hetero molecules


Theoretical massNumber of molelcules
Total (without water)378,2358
Polymers378,0805
Non-polymers1553
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area33240 Å2
ΔGint-162 kcal/mol
Surface area110530 Å2
MethodPISA
Unit cell
Length a, b, c (Å)280.694, 280.694, 184.964
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number146
Space group name H-MH3

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Components

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

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


Mass: 125436.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / Gene: rpoB, TTHA1813 / Production host: Thermus thermophilus (bacteria) / References: UniProt: Q8RQE9, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 170997.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / Gene: rpoC, TTHA1812 / Production host: Thermus thermophilus (bacteria) / References: UniProt: Q8RQE8, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase subunit alpha / Polymerase / RNAP subunit alpha / RNA polymerase subunit alpha / Transcriptase subunit alpha


Mass: 35056.164 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / Gene: rpoA, TTHA1664 / Production host: Thermus thermophilus (bacteria) / References: UniProt: Q5SHR6, DNA-directed RNA polymerase
#4: Protein DNA-directed RNA polymerase subunit omega / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11533.316 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / Gene: rpoZ, TTHA1561 / Production host: Thermus thermophilus (bacteria) / References: UniProt: Q8RQE7, DNA-directed RNA polymerase

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Non-polymers , 2 types, 3 molecules

#5: Chemical ChemComp-ZN / ZINC ION


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


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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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: 3.71 Å3/Da / Density % sol: 66.84 %
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 7.5
Details: 0.02M Magnesium chloride hexahydrate, 0.1M HEPES pH 7.5, 22% Poly(acrylic acid sodium salt) 5,100

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.97926 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Oct 13, 2014
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97926 Å / Relative weight: 1
ReflectionResolution: 3.8→50 Å / Num. obs: 61395 / % possible obs: 99.2 % / Redundancy: 4.8 % / Biso Wilson estimate: 25.39 Å2 / Rmerge(I) obs: 0.245 / Rpim(I) all: 0.124 / Rrim(I) all: 0.275 / Χ2: 0.832 / Net I/σ(I): 5.2 / Num. measured all: 295018
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
3.8-3.873.20.70428580.4180.430.830.64392.3
3.87-3.943.50.6629580.5040.3840.7670.68795.2
3.94-4.0140.61630330.630.3350.7040.65997.7
4.01-4.094.20.56130590.6660.3010.6390.70798.9
4.09-4.184.40.50730690.7650.2660.5740.71999.7
4.18-4.284.50.47430990.7870.2450.5350.75599.8
4.28-4.394.70.40630910.8570.2060.4560.791100
4.39-4.54.80.35831080.8840.1790.4020.83499.9
4.5-4.644.90.3230810.9140.1570.3580.867100
4.64-4.794.90.28730810.940.1410.320.886100
4.79-4.9650.27330840.9470.1310.3030.928100
4.96-5.165.10.26931330.9470.130.30.865100
5.16-5.395.10.26630670.9490.1270.2950.881100
5.39-5.675.20.25231140.9570.1190.280.874100
5.67-6.035.20.23630880.9670.1110.2610.844100
6.03-6.495.30.21130920.9710.0980.2330.86100
6.49-7.155.40.1731080.9720.0780.1880.893100
7.15-8.185.50.12330930.9910.0550.1350.924100
8.18-10.295.50.0830860.9930.0370.0880.931100
10.29-505.50.06530930.9890.030.0720.80699.7

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Processing

Software
NameVersionClassification
HKL-2000data scaling
PHENIX1.9_1692refinement
PDB_EXTRACT3.22data extraction
HKL-2000data reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4GZY

4gzy


Resolution: 3.8→42.492 Å / SU ML: 0.53 / Cross valid method: FREE R-VALUE / σ(F): 1.98 / Phase error: 25.69
RfactorNum. reflection% reflection
Rfree0.2782 2611 3.86 %
Rwork0.2293 --
obs0.2312 67584 63.16 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 231.4 Å2 / Biso mean: 82.6876 Å2 / Biso min: 0 Å2
Refinement stepCycle: final / Resolution: 3.8→42.492 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms23551 0 3 0 23554
Biso mean--127.61 --
Num. residues----2983
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00323974
X-RAY DIFFRACTIONf_angle_d0.65832438
X-RAY DIFFRACTIONf_chiral_restr0.0253662
X-RAY DIFFRACTIONf_plane_restr0.0034265
X-RAY DIFFRACTIONf_dihedral_angle_d12.2049251
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 19

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.8002-3.86930.3286460.27761225127123
3.8693-3.94370.3274840.26621943202736
3.9437-4.02410.3015990.27452310240943
4.0241-4.11150.2949990.26582588268748
4.1115-4.20710.27421250.26662650277549
4.2071-4.31220.35421190.26482665278450
4.3122-4.42870.27371120.25382714282650
4.4287-4.55890.3276990.25332705280450
4.5589-4.70580.29241140.23962713282750
4.7058-4.87380.3464960.24792695279150
4.8738-5.06860.3281220.24172715283750
5.0686-5.29890.30771220.23082935305754
5.2989-5.57770.26881250.21643628375367
5.5777-5.92630.28852090.22754727493688
5.9263-6.38240.27792120.22975321553398
6.3824-7.02210.27612310.220854015632100
7.0221-8.03230.24561940.20065375556999
8.0323-10.09740.18922000.17425401560199
10.0974-42.49480.22882030.19775262546597

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