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- PDB-3e3j: Crystal Structure of an Intermediate Complex of T7 RNAP and 8nt of RNA -

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Basic information

Entry
Database: PDB / ID: 3e3j
TitleCrystal Structure of an Intermediate Complex of T7 RNAP and 8nt of RNA
Components
  • DNA (28-MER)
  • DNA (5'-D(*DTP*DAP*DAP*DTP*DAP*DCP*DGP*DAP*DCP*DTP*DCP*DAP*DCP*DTP*DAP*DTP*DAP*DTP*DTP*DTP*DCP*DTP*DGP*DCP*DCP*DAP*DAP*DAP*DCP*DGP*DGP*DC)-3')
  • DNA-directed RNA polymerasePolymerase
  • RNA (5'-R(*GP*GP*GP*AP*GP*UP*AP*A)-3')
KeywordsTransferase/RNA/DNA / T7 RNA Polymerase / DNA-directed RNA Polymerase / Transcription / Nucleotidyltransferase / Transferase / Transferase-RNA-DNA COMPLEX
Function / homology
Function and homology information


DNA-templated viral transcription / DNA-directed RNA polymerase complex / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-templated transcription / DNA binding
Similarity search - Function
DNA-directed RNA polymerase, helix hairpin domain superfamily / DNA-directed RNA polymerase, N-terminal / DNA-directed RNA polymerase, N-terminal domain superfamily / DNA-directed RNA polymerase N-terminal / Bacteriophage-type RNA polymerase family active site signature 1. / DNA-directed RNA polymerase N-terminal / DNA-directed RNA polymerase, phage-type / : / DNA-dependent RNA polymerase / Bacteriophage-type RNA polymerase family active site signature 2. / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / T7 RNA polymerase
Similarity search - Component
Biological speciesBacteriophage T7 (virus)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / Resolution: 6.7 Å
AuthorsDurniak, K.J. / Bailey, S. / Steitz, T.A.
CitationJournal: Science / Year: 2008
Title: The structure of a transcribing t7 RNA polymerase in transition from initiation to elongation
Authors: Durniak, K.J. / Bailey, S. / Steitz, T.A.
History
DepositionAug 7, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 4, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 25, 2017Group: Refinement description / Source and taxonomy / Category: entity_src_gen / pdbx_entity_src_syn / software / Item: _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id
Revision 1.3Oct 20, 2021Group: Database references / Category: database_2 / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details
Revision 1.4Aug 30, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: DNA-directed RNA polymerase
T: DNA (28-MER)
N: DNA (5'-D(*DTP*DAP*DAP*DTP*DAP*DCP*DGP*DAP*DCP*DTP*DCP*DAP*DCP*DTP*DAP*DTP*DAP*DTP*DTP*DTP*DCP*DTP*DGP*DCP*DCP*DAP*DAP*DAP*DCP*DGP*DGP*DC)-3')
B: DNA-directed RNA polymerase
X: DNA (28-MER)
Y: DNA (5'-D(*DTP*DAP*DAP*DTP*DAP*DCP*DGP*DAP*DCP*DTP*DCP*DAP*DCP*DTP*DAP*DTP*DAP*DTP*DTP*DTP*DCP*DTP*DGP*DCP*DCP*DAP*DAP*DAP*DCP*DGP*DGP*DC)-3')
R: RNA (5'-R(*GP*GP*GP*AP*GP*UP*AP*A)-3')
Z: RNA (5'-R(*GP*GP*GP*AP*GP*UP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)243,9838
Polymers243,9838
Non-polymers00
Water0
1
B: DNA-directed RNA polymerase
X: DNA (28-MER)
Y: DNA (5'-D(*DTP*DAP*DAP*DTP*DAP*DCP*DGP*DAP*DCP*DTP*DCP*DAP*DCP*DTP*DAP*DTP*DAP*DTP*DTP*DTP*DCP*DTP*DGP*DCP*DCP*DAP*DAP*DAP*DCP*DGP*DGP*DC)-3')
Z: RNA (5'-R(*GP*GP*GP*AP*GP*UP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)121,9914
Polymers121,9914
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8040 Å2
ΔGint-30.6 kcal/mol
Surface area46070 Å2
MethodPISA
2
C: DNA-directed RNA polymerase
T: DNA (28-MER)
N: DNA (5'-D(*DTP*DAP*DAP*DTP*DAP*DCP*DGP*DAP*DCP*DTP*DCP*DAP*DCP*DTP*DAP*DTP*DAP*DTP*DTP*DTP*DCP*DTP*DGP*DCP*DCP*DAP*DAP*DAP*DCP*DGP*DGP*DC)-3')
R: RNA (5'-R(*GP*GP*GP*AP*GP*UP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)121,9914
Polymers121,9914
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8400 Å2
ΔGint-13.4 kcal/mol
Surface area45930 Å2
MethodPISA
Unit cell
Length a, b, c (Å)99.216, 180.602, 198.619
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein DNA-directed RNA polymerase / Polymerase


Mass: 99829.156 Da / Num. of mol.: 2 / Mutation: P266L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacteriophage T7 (virus) / Gene: 1 / Plasmid: pBH161 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P00573, DNA-directed RNA polymerase
#2: DNA chain DNA (28-MER)


Mass: 9788.297 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: Template DNA / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (5'-D(*DTP*DAP*DAP*DTP*DAP*DCP*DGP*DAP*DCP*DTP*DCP*DAP*DCP*DTP*DAP*DTP*DAP*DTP*DTP*DTP*DCP*DTP*DGP*DCP*DCP*DAP*DAP*DAP*DCP*DGP*DGP*DC)-3')


Mass: 9744.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: Non-template DNA / Source: (synth.) synthetic construct (others)
#4: RNA chain RNA (5'-R(*GP*GP*GP*AP*GP*UP*AP*A)-3')


Mass: 2629.649 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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: 3.65 Å3/Da / Density % sol: 66.27 %
Crystal growTemperature: 285 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: PEG 6000, MgCl2, Tris ph8.5 at 285K, VAPOR DIFFUSION, SITTING DROP
Components of the solutions
IDNameCrystal-IDSol-ID
1PEG 600011
2PEG 600012
3MgCl211
4MgCl212
5Tris11
6Tris12

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Data collection

DiffractionMean temperature: 77.4 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.97949
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Dec 10, 2007
RadiationProtocol: SINGLE WAVELENGTH / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97949 Å / Relative weight: 1
ReflectionResolution: 6.7→50 Å / Num. obs: 6015 / % possible obs: 87.8 % / Redundancy: 3.5 % / Rmerge(I) obs: 0.152
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obs% possible all
6.7-6.941.70.60139.7
6.94-7.222.10.48963.1
7.22-7.542.50.45183.8
7.54-7.9430.36694.2
7.94-8.433.30.25497.3
8.43-9.083.50.20399.4
9.08-9.993.90.16899.1
9.99-11.424.10.15498.8
11.42-14.334.30.13699.9
14.33-504.30.12397.8

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
REFMACrefinement
PDB_EXTRACT3.006data extraction
RefinementStarting model: PDB entry 3E2E

3e2e


Resolution: 6.7→49.81 Å / Cor.coef. Fo:Fc: 0.756 / Cor.coef. Fo:Fc free: 0.632 / Occupancy max: 1 / Occupancy min: 1 / SU B: 528.565 / SU ML: 4.54 / Cross valid method: THROUGHOUT / ESU R: 4.568 / ESU R Free: 5.29 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.45419 274 4.6 %RANDOM
Rwork0.39222 ---
obs0.39486 5736 87.24 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 94.419 Å2
Baniso -1Baniso -2Baniso -3
1-4.67 Å20 Å20 Å2
2---3.87 Å20 Å2
3----0.8 Å2
Refinement stepCycle: LAST / Resolution: 6.7→49.81 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13112 2294 0 0 15406
LS refinement shellResolution: 6.7→6.859 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.437 6 -
Rwork0.426 157 -
obs--33.82 %

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