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- PDB-5x21: Crystal structure of Thermus thermophilus transcription initiatio... -

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Basic information

Entry
Database: PDB / ID: 5x21
TitleCrystal structure of Thermus thermophilus transcription initiation complex with GpA and pseudouridimycin (PUM)
Components
  • (DNA-directed RNA polymerase subunit ...) x 4
  • (promoter DNA ...) x 2
  • RNA (5'-R(*GP*A)-3')
  • RNA polymerase sigma factor SigA
KeywordsTRANSFERASE/DNA / Transcription / RNA polymerase / transcription inhibition / nucleoside-analog inhibitor / pseudouridimycin / TRANSFERASE-DNA 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 / 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 / RNA polymerase sigma factor RpoD, C-terminal / RNA polymerase sigma factor RpoD / : ...: / 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 / 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 / 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 / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / 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 / Alpha-Beta Complex / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Chem-PUM / DNA / DNA (> 10) / RNA / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit omega / RNA polymerase sigma factor SigA / DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit alpha
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.323 Å
AuthorsZhang, Y. / Ebright, R.
CitationJournal: Cell / Year: 2017
Title: Antibacterial Nucleoside-Analog Inhibitor of Bacterial RNA Polymerase.
Authors: Maffioli, S.I. / Zhang, Y. / Degen, D. / Carzaniga, T. / Del Gatto, G. / Serina, S. / Monciardini, P. / Mazzetti, C. / Guglierame, P. / Candiani, G. / Chiriac, A.I. / Facchetti, G. / ...Authors: Maffioli, S.I. / Zhang, Y. / Degen, D. / Carzaniga, T. / Del Gatto, G. / Serina, S. / Monciardini, P. / Mazzetti, C. / Guglierame, P. / Candiani, G. / Chiriac, A.I. / Facchetti, G. / Kaltofen, P. / Sahl, H.G. / Deho, G. / Donadio, S. / Ebright, R.H.
History
DepositionJan 29, 2017Deposition site: PDBJ / Processing site: RCSB
Revision 1.0Jul 5, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2018Group: Author supporting evidence / Category: pdbx_audit_support
Revision 1.2Oct 12, 2022Group: Database references / Derived calculations / Other / Category: database_2 / pdbx_database_status / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.deposit_site / _struct_conn.pdbx_dist_value / _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

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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: promoter DNA template strand
H: promoter DNA nontemplate strand
I: RNA (5'-R(*GP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)445,09718
Polymers444,3349
Non-polymers7639
Water63135
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area54920 Å2
ΔGint-244 kcal/mol
Surface area141560 Å2
MethodPISA
Unit cell
Length a, b, c (Å)186.790, 103.090, 296.230
Angle α, β, γ (deg.)90.00, 98.71, 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 genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (bacteria) / Gene: rpoA / Production host: Thermus thermophilus (bacteria)
References: UniProt: Q9Z9H6, UniProt: Q5SHR6*PLUS, 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 (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / 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 (strain HB8 / ATCC 27634 / DSM 579) (bacteria)
Strain: HB8 / ATCC 27634 / DSM 579 / 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 (strain HB27 / ATCC BAA-163 / DSM 7039) (bacteria)
Strain: HB27 / ATCC BAA-163 / DSM 7039 / References: UniProt: Q72ID6, DNA-directed RNA polymerase

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Promoter DNA ... , 2 types, 2 molecules GH

#6: DNA chain promoter DNA template strand


Mass: 6464.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermus thermophilus (bacteria)
#7: DNA chain promoter DNA nontemplate strand


Mass: 8421.432 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermus thermophilus (bacteria)

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Protein / RNA chain , 2 types, 2 molecules FI

#5: Protein RNA polymerase sigma factor SigA


Mass: 50739.371 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (strain HB27 / ATCC BAA-163 / DSM 7039) (bacteria)
Strain: HB27 / ATCC BAA-163 / DSM 7039 / Gene: sigA, rpoD, TT_C0164 / Plasmid: pET28a / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 (DE3) / References: UniProt: Q72L95
#8: RNA chain RNA (5'-R(*GP*A)-3')


Mass: 629.454 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermus thermophilus (bacteria)

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Non-polymers , 4 types, 44 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: Chemical ChemComp-PUM / (1S)-1,4-anhydro-5-[(N-carbamimidoylglycyl-N~2~-hydroxy-L-glutaminyl)amino]-5-deoxy-1-(2,4-dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)-D-ribitol / pseudouridimycin


Mass: 486.437 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H26N8O9
#12: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 35 / Source method: isolated from a natural source / Formula: H2O

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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: 3.17 Å3/Da / Density % sol: 61.23 % / Description: rod
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 100 mM Tris-HCl, pH 8.4, 200 mM potassium chloride, 50 mM magnesium chloride, 9.5% PEG4000, VAPOR DIFFUSION, HANGING DROP, temperature 295K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: CHESS / Beamline: F1 / Wavelength: 0.918 Å
DetectorType: ADSC QUANTUM 270 / Detector: CCD / Date: Apr 2, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.918 Å / Relative weight: 1
ReflectionResolution: 3.3→40 Å / Num. obs: 76928 / % possible obs: 92 % / Redundancy: 3.4 % / Biso Wilson estimate: 60.43 Å2 / Rmerge(I) obs: 0.138 / Χ2: 1.068 / Net I/av σ(I): 8.35 / Net I/σ(I): 7.5
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsΧ2Diffraction-ID% possible all
3.3-3.363.40.6330.954187.4
3.36-3.423.40.570.998187.9
3.42-3.483.40.5020.986186.5
3.48-3.553.40.4381.021188.8
3.55-3.633.40.3580.988187.9
3.63-3.723.40.3081.015186.8
3.72-3.813.40.2750.994188.3
3.81-3.913.40.2541.065187.8
3.91-4.033.40.2031.073187.1
4.03-4.163.40.1811.094187.7
4.16-4.313.40.1631.11188.6
4.31-4.483.40.1421.059190.9
4.48-4.683.30.1391.173192.8
4.68-4.933.30.1271.106195.7
4.93-5.243.40.1291.069198.4
5.24-5.643.40.1331.103199.5
5.64-6.23.60.131.083199.8
6.2-7.13.70.1241.168199.9
7.1-8.933.70.1081.191100
8.93-403.50.091.023197.5

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Processing

Software
NameVersionClassification
PHENIX1.9_1692refinement
SCALEPACKdata scaling
PDB_EXTRACT3.15data extraction
HKL-3000data reduction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.323→39.668 Å / SU ML: 0.56 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 30.69 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2802 1565 2.23 %
Rwork0.2319 --
obs0.233 70069 85.12 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODELf
Refinement stepCycle: LAST / Resolution: 3.323→39.668 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms27667 987 42 35 28731
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00229322
X-RAY DIFFRACTIONf_angle_d0.54239851
X-RAY DIFFRACTIONf_dihedral_angle_d12.94211375
X-RAY DIFFRACTIONf_chiral_restr0.0224484
X-RAY DIFFRACTIONf_plane_restr0.0035058
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.3232-3.43040.4006450.34241944X-RAY DIFFRACTION27
3.4304-3.5530.38381060.33964952X-RAY DIFFRACTION68
3.553-3.69510.36711580.3226374X-RAY DIFFRACTION88
3.6951-3.86310.36811430.30116427X-RAY DIFFRACTION89
3.8631-4.06660.35631360.26946397X-RAY DIFFRACTION88
4.0666-4.32110.3171550.24436495X-RAY DIFFRACTION89
4.3211-4.65430.28141550.22376759X-RAY DIFFRACTION92
4.6543-5.12180.24981610.2067077X-RAY DIFFRACTION97
5.1218-5.86090.2781640.20847324X-RAY DIFFRACTION100
5.8609-7.37630.22891720.20767351X-RAY DIFFRACTION100
7.3763-39.67070.1851700.15927404X-RAY DIFFRACTION98

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