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- PDB-5x22: Crystal structure of Thermus thermophilus transcription initiatio... -

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Basic information

Entry
Database: PDB / ID: 5x22
TitleCrystal structure of Thermus thermophilus transcription initiation complex with GpA and CMPcPP
Components
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • (promoter DNA ...) x 2
  • RNA (5'-R(*GP*A)-3')
  • RNA polymerase sigma factor SigA
KeywordsTRANSFERASE/DNA / Transcription / RNA polymerase / primer-dependent initiation / 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 / RNA polymerase sigma-70 region 1.2 ...: / 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 / 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 / Winged helix-like DNA-binding domain superfamily / Alpha-Beta Complex / 2-Layer Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
Chem-2TM / 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: 3.35 Å
AuthorsZhang, Y. / Ebright, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI104660 United States
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 1.1Sep 20, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Nov 29, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Other / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_status / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_status.process_site / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_alt_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_alt_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_alt_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_ptnr1_label_alt_id / _struct_conn.pdbx_ptnr2_label_alt_id / _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')
K: DNA-directed RNA polymerase subunit alpha
L: DNA-directed RNA polymerase subunit alpha
M: DNA-directed RNA polymerase subunit beta
N: DNA-directed RNA polymerase subunit beta'
O: DNA-directed RNA polymerase subunit omega
P: RNA polymerase sigma factor SigA
Q: promoter DNA template strand
R: promoter DNA nontemplate strand
S: RNA (5'-R(*GP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)890,21636
Polymers888,70018
Non-polymers1,51618
Water0
1
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)445,10818
Polymers444,3509
Non-polymers7589
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area54700 Å2
ΔGint-247 kcal/mol
Surface area139400 Å2
MethodPISA
2
K: DNA-directed RNA polymerase subunit alpha
L: DNA-directed RNA polymerase subunit alpha
M: DNA-directed RNA polymerase subunit beta
N: DNA-directed RNA polymerase subunit beta'
O: DNA-directed RNA polymerase subunit omega
P: RNA polymerase sigma factor SigA
Q: promoter DNA template strand
R: promoter DNA nontemplate strand
S: RNA (5'-R(*GP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)445,10818
Polymers444,3509
Non-polymers7589
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area55020 Å2
ΔGint-248 kcal/mol
Surface area142970 Å2
MethodPISA
Unit cell
Length a, b, c (Å)186.410, 104.320, 297.290
Angle α, β, γ (deg.)90.000, 98.360, 90.000
Int Tables number4
Space group name H-MP1211

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Components

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DNA-directed RNA polymerase subunit ... , 4 types, 10 molecules ABKLCMDNEO

#1: 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.: 4
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 / Polymerase / RNAP subunit beta / RNA polymerase subunit beta / Transcriptase subunit beta


Mass: 125436.539 Da / Num. of mol.: 2 / 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' / Polymerase / RNAP subunit beta' / RNA polymerase subunit beta' / Transcriptase subunit beta'


Mass: 170997.391 Da / Num. of mol.: 2 / 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 / Polymerase / RNAP omega subunit / RNA polymerase omega subunit / Transcriptase subunit omega


Mass: 11533.316 Da / Num. of mol.: 2 / 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, 4 molecules GQHR

#6: DNA chain promoter DNA template strand


Mass: 6480.172 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Thermus thermophilus (bacteria)
#7: DNA chain promoter DNA nontemplate strand


Mass: 8421.432 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Thermus thermophilus (bacteria)

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Protein / RNA chain , 2 types, 4 molecules FPIS

#5: Protein RNA polymerase sigma factor SigA


Mass: 50739.371 Da / Num. of mol.: 2
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.: 2 / Source method: obtained synthetically / Source: (synth.) Thermus thermophilus (bacteria)

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Non-polymers , 3 types, 18 molecules

#9: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg
#10: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#11: Chemical ChemComp-2TM / 5'-O-[(S)-hydroxy{[(S)-hydroxy(phosphonooxy)phosphoryl]methyl}phosphoryl]cytidine / CMPcPP


Mass: 481.184 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H18N3O13P3

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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: 3.22 Å3/Da / Density % sol: 61.81 % / Description: rod
Crystal growTemperature: 295 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 100 mM Tris-HCl, pH 8.7, 200 mM potassium chloride, 50 mM magnesium chloride, 10% PEG4000

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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: 3.333→50 Å / Num. obs: 158249 / % possible obs: 95.6 % / Redundancy: 5.3 % / Biso Wilson estimate: 66.48 Å2 / Rmerge(I) obs: 0.197 / Χ2: 1.068 / Net I/av σ(I): 7.656 / Net I/σ(I): 7.3
Reflection shell
Resolution (Å)Redundancy (%)Χ2Diffraction-ID% possible allRmerge(I) obs
3.35-3.415.30.95189.1
3.41-3.475.30.975189.90.877
3.47-3.545.30.9791900.762
3.54-3.615.31189.80.649
3.61-3.695.31.0131910.538
3.69-3.775.31.016190.90.438
3.77-3.875.21.046192.40.402
3.87-3.975.21.076193.40.33
3.97-4.095.21.096194.60.277
4.09-4.225.21.1141960.241
4.22-4.375.11.119197.50.208
4.37-4.555.21.134198.80.188
4.55-4.755.31.161199.20.178
4.75-55.41.151199.50.17
5-5.325.51.119199.90.168
5.32-5.735.61.13211000.17
5.73-6.35.61.1211000.163
6.3-7.215.61.07411000.147
7.21-9.085.61.0211000.121
9.08-505.41.033199.40.12

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Processing

Software
NameVersionClassification
PHENIXrefinement
SCALEPACKdata scaling
PDB_EXTRACT3.15data extraction
HKL-3000data reduction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4G7H

4g7h


Resolution: 3.35→49.022 Å / SU ML: 0.41 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 26.39
RfactorNum. reflection% reflection
Rfree0.2503 7382 5 %
Rwork0.2079 --
obs0.21 147764 89.26 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 182.03 Å2 / Biso mean: 41.92 Å2 / Biso min: 3.79 Å2
Refinement stepCycle: final / Resolution: 3.35→49.022 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms54818 1969 218 0 57005
Biso mean--18.85 --
Num. residues----7024
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00558116
X-RAY DIFFRACTIONf_angle_d0.76178958
X-RAY DIFFRACTIONf_chiral_restr0.0588890
X-RAY DIFFRACTIONf_plane_restr0.00310008
X-RAY DIFFRACTIONf_dihedral_angle_d13.33922560
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.35-3.37090.3352290.281485X-RAY DIFFRACTION9
3.3709-3.41060.3292750.27781516X-RAY DIFFRACTION30
3.4106-3.45220.29421430.28822906X-RAY DIFFRACTION55
3.4522-3.49590.34262110.28693981X-RAY DIFFRACTION77
3.4959-3.54190.35482490.27284671X-RAY DIFFRACTION90
3.5419-3.59040.31832520.26024724X-RAY DIFFRACTION90
3.5904-3.64160.32062520.26154641X-RAY DIFFRACTION91
3.6416-3.6960.29952500.25244772X-RAY DIFFRACTION91
3.696-3.75370.27672550.25064768X-RAY DIFFRACTION91
3.7537-3.81520.30122440.25294809X-RAY DIFFRACTION92
3.8152-3.8810.30662530.23694824X-RAY DIFFRACTION92
3.881-3.95150.27952610.22944859X-RAY DIFFRACTION94
3.9515-4.02750.28472630.22034947X-RAY DIFFRACTION94
4.0275-4.10970.25962610.2144938X-RAY DIFFRACTION95
4.1097-4.1990.26522630.20825065X-RAY DIFFRACTION96
4.199-4.29660.22512660.20785061X-RAY DIFFRACTION97
4.2966-4.4040.25052690.19635139X-RAY DIFFRACTION98
4.404-4.5230.23812730.19195201X-RAY DIFFRACTION99
4.523-4.6560.23092710.18915182X-RAY DIFFRACTION99
4.656-4.80610.22352730.18995178X-RAY DIFFRACTION99
4.8061-4.97770.24562770.18635284X-RAY DIFFRACTION100
4.9777-5.17680.24252740.18785213X-RAY DIFFRACTION100
5.1768-5.41220.22132740.19015204X-RAY DIFFRACTION100
5.4122-5.69710.21952780.19735277X-RAY DIFFRACTION100
5.6971-6.05340.25952770.20075276X-RAY DIFFRACTION100
6.0534-6.51980.21672790.20025274X-RAY DIFFRACTION100
6.5198-7.17410.23862780.18985289X-RAY DIFFRACTION100
7.1741-8.20810.19322790.16855297X-RAY DIFFRACTION100
8.2081-10.32540.19542800.1565336X-RAY DIFFRACTION100
10.3254-49.0220.2252730.20535265X-RAY DIFFRACTION96

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