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- PDB-8dh5: T7 RNA polymerase elongation complex with unnatural base dPa-ATP ... -

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Basic information

Entry
Database: PDB / ID: 8dh5
TitleT7 RNA polymerase elongation complex with unnatural base dPa-ATP mismatch
Components
  • Non-template strand DNA
  • RNA
  • T7 RNA polymerase
  • Template strand DNA
KeywordsTransferase/DNA/RNA / T7 RNA polymerase / elongation complex / unnatural base / Ds-Pa / synthetic DNA / transcription / Transferase-DNA-RNA 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
ADENOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / RNA (> 10) / T7 RNA polymerase
Similarity search - Component
Biological speciesEscherichia phage T7 (virus)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.85 Å
Model detailsT7 RNA polymerase, elongation complex, unnatural base pair, synthetic DNA
AuthorsOh, J. / Wang, D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM102362 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of transcription recognition of a hydrophobic unnatural base pair by T7 RNA polymerase.
Authors: Oh, J. / Kimoto, M. / Xu, H. / Chong, J. / Hirao, I. / Wang, D.
History
DepositionJun 24, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 25, 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
B: T7 RNA polymerase
A: Template strand DNA
C: RNA
D: Non-template strand DNA
E: T7 RNA polymerase
F: Template strand DNA
G: RNA
H: Non-template strand DNA
I: T7 RNA polymerase
J: Template strand DNA
K: RNA
L: Non-template strand DNA
M: T7 RNA polymerase
N: Template strand DNA
O: RNA
P: Non-template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)446,12922
Polymers444,05216
Non-polymers2,0776
Water0
1
B: T7 RNA polymerase
A: Template strand DNA
C: RNA
D: Non-template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,5446
Polymers111,0134
Non-polymers5312
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
E: T7 RNA polymerase
F: Template strand DNA
G: RNA
H: Non-template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,5446
Polymers111,0134
Non-polymers5312
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
I: T7 RNA polymerase
J: Template strand DNA
K: RNA
L: Non-template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,5205
Polymers111,0134
Non-polymers5071
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
M: T7 RNA polymerase
N: Template strand DNA
O: RNA
P: Non-template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,5205
Polymers111,0134
Non-polymers5071
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)78.544, 86.305, 201.199
Angle α, β, γ (deg.)89.790, 85.390, 69.490
Int Tables number1
Space group name H-MP1

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Components

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DNA chain , 2 types, 8 molecules AFJNDHLP

#2: DNA chain
Template strand DNA


Mass: 5510.578 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain
Non-template strand DNA


Mass: 2666.761 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 8 molecules BEIMCGKO

#1: Protein
T7 RNA polymerase / / DNA-directed RNA polymerase


Mass: 98984.227 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Escherichia phage T7 (virus) / References: UniProt: P00573, DNA-directed RNA polymerase
#3: RNA chain
RNA /


Mass: 3851.360 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 6 molecules

#5: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE / Adenosine triphosphate


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestY

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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: 2.87 Å3/Da / Density % sol: 57.09 % / Mosaicity: 0.33 °
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8.1
Details: 10% PEG 8000, 8% glycerol, 5 mM B-mercaptoethanol, 100 mM Tris pH 8.1

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 5.0.3 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 315r / Detector: CCD / Date: Apr 21, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.83→47.43 Å / Num. obs: 115889 / % possible obs: 98.5 % / Redundancy: 3.5 % / Biso Wilson estimate: 76.73 Å2 / CC1/2: 0.997 / Rmerge(I) obs: 0.118 / Rpim(I) all: 0.073 / Rrim(I) all: 0.139 / Net I/σ(I): 6.1 / Num. measured all: 408951
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
2.83-2.883.31.7891921357360.2971.1692.1490.598
15.5-47.433.60.02724576790.9980.0170.03228.195.8

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Processing

Software
NameVersionClassification
XDSdata reduction
Aimless0.7.4data scaling
PHENIX1.19refinement
PDB_EXTRACT3.27data extraction
PHENIX1.19phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 8DH0
Resolution: 2.85→47.43 Å / SU ML: 0.51 / Cross valid method: THROUGHOUT / σ(F): 1.96 / Phase error: 33.24 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2672 1951 1.72 %
Rwork0.2335 111411 -
obs0.2341 113362 98.44 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 398.81 Å2 / Biso mean: 108.6907 Å2 / Biso min: 28.22 Å2
Refinement stepCycle: final / Resolution: 2.85→47.43 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms25216 2652 126 0 27994
Biso mean--107.66 --
Num. residues----3367
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.85-2.920.43351200.40547876799697
2.92-30.38461530.35117900805398
3-3.090.34931460.31987931807798
3.09-3.190.35991370.30477951808898
3.19-3.30.3291310.29557953808498
3.3-3.430.3351510.29577940809198
3.43-3.590.32181370.25357985812298
3.59-3.780.30241260.22767961808799
3.78-4.020.30431670.22667886805398
4.02-4.330.27481240.21128045816999
4.33-4.760.22771360.19517967810399
4.76-5.450.261460.2078009815599
5.45-6.860.25991380.23338001813999
6.86-47.430.17431390.19158006814599
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
11.88880.37830.20880.3657-0.12331.03360.08010.98490.5108-0.1606-0.1132-0.1497-0.04180.30080.04340.50380.11290.08360.88690.32730.733932.976353.8532-56.361
21.5612-0.2398-0.02641.38210.06660.98220.0135-0.10010.17590.2127-0.0294-0.0242-0.0254-0.04140.0450.2675-0.00520.0150.4068-0.00570.37669.406441.769-28.4599
31.9684-0.1669-0.05511.3143-0.19691.70970.18790.6136-0.1365-0.1476-0.1563-0.2170.25160.38680.03350.3020.04870.05690.416-0.00450.374425.629423.8563-39.5297
42.55812.1780.12781.93340.3993.2280.17680.4331-0.0891-0.6777-0.1278-1.19970.39781.1667-0.23650.41780.12930.11680.90610.11940.828534.841934.7931-46.251
54.05233.9898-4.30054.2555-4.46594.7318-0.09422.00970.2421-1.35830.0703-0.54260.60320.36870.00780.5420.01710.09990.83650.05550.577127.203944.0904-41.3088
63.43051.0818-0.9660.5319-0.15670.4021-0.16480.7002-0.1748-0.9037-0.3347-0.41110.5817-0.68860.56052.87140.9491-0.86092.1008-0.49722.290243.070221.6452-47.7102
71.94010.18480.35232.34870.23120.94070.052-0.7532-0.67850.40530.0047-0.42870.3451-0.14990.04480.6273-0.01310.06010.91710.15070.729732.256558.28718.8694
82.35-0.02180.19841.29490.0470.9604-0.0462-0.62730.0359-0.0176-0.0887-0.0638-0.0838-0.0470.13220.4690.11490.00950.5034-0.14280.430637.403782.9867-6.3315
91.1356-0.12721.50130.0460.04353.42220.5655-1.33881.09610.52040.534-3.9482-0.22143.844-0.84011.25810.20130.07923.1874-0.2932.937272.950875.68442.8103
101.6595-0.1868-3.51347.09982.28097.9431-0.3818-1.2902-0.63310.3091-0.3167-1.08840.05182.3140.4970.86580.2544-0.09671.28050.15930.825150.262372.7923-0.6416
111.96463.73640.72068.9768-0.05455.1742-0.0447-0.3541-1.7375-0.8787-0.0367-1.41361.27411.57330.32670.81470.25590.16240.97590.09920.938532.874761.32223.515
122.7875-1.8932-4.53572.13043.24847.7403-0.1742-0.8937-1.1629-0.5476-0.3871-0.77591.05511.60560.74230.78830.06580.07591.06180.06980.920636.668564.7524-1.9154
132.2423-0.295-0.97070.64070.59760.7852-0.9123-0.367-0.15250.0634-0.4532-0.8085-0.1797-0.64551.26262.1820.10860.211.8907-0.31922.438564.776775.81632.578
142.18580.0037-0.20460.7922-0.10971.6817-0.0267-0.03860.87120.05770.15430.1627-0.4445-0.4694-0.12540.687-0.03080.07160.53780.01720.8992-2.148545.9218-95.4235
152.7201-0.00090.1241.5635-0.36311.79850.3582-0.3825-0.4404-0.2496-0.04410.4260.5667-0.282-0.26890.9122-0.2127-0.26170.66920.06770.57558.402710.3958-91.21
161.09130.92210.87974.245-0.35114.81711.0238-0.5511.31011.1088-0.07441.9466-0.5828-1.5488-0.82980.9556-0.18380.15521.32780.26351.2893-6.575523.2337-88.4125
176.4949-0.1827-0.96911.0673-2.47646.0340.60180.11490.4560.42440.12480.81441.0062-0.9601-0.77260.8495-0.114-0.14940.66540.01330.99313.148622.8695-97.8879
180.0153-0.0040.01670.00160.00220.0089-0.03290.9607-0.47620.6628-0.77660.6718-0.306-0.06860.70752.5959-0.4365-0.87633.56460.15883.5124-17.967517.2941-77.9789
194.79572.2107-2.34241.3049-1.34032.1906-0.0690.7558-0.1271-0.12830.43510.04490.9344-0.5119-0.46361.38440.1269-0.37071.1505-0.13160.89557.5068-14.062354.6877
202.14680.0571.19950.98590.26952.47610.15310.51170.1134-0.0779-0.25520.0913-0.15570.25680.11160.94070.0908-0.04830.9721-0.0870.458219.784719.325650.1212
211.3142-1.8218-2.18122.54963.04593.63981.27071.92161.2862-2.8311-0.39591.1688-1.0891-2.2977-0.74223.04451.2191-0.76123.1737-0.75112.8513-19.635424.069242.7763
229.9142-4.4851.70645.0230.77771.0942-0.5080.3256-0.75810.6809-0.14211.18490.3730.04780.60141.21020.09740.14511.33130.03310.59296.623310.030954.0153
235.50133.38125.32713.09953.42415.19661.0044-0.9520.18780.7622-0.44631.0607-0.2799-0.5787-0.42641.41420.0853-0.02351.3929-0.26290.780211.08579.098658.7422
243.61140.0866-2.08440.7567-0.74051.8281-0.14490.53820.10810.05990.72370.4447-1.1414-1.6441-0.60232.42960.63610.06172.6304-0.37242.5259-12.075522.902343.9059
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'B' and (resid 1 through 331 )B1 - 331
2X-RAY DIFFRACTION2chain 'B' and (resid 332 through 569 )B332 - 569
3X-RAY DIFFRACTION3chain 'B' and (resid 570 through 883 )B570 - 883
4X-RAY DIFFRACTION4chain 'A' and (resid 3 through 18 )A3 - 18
5X-RAY DIFFRACTION5chain 'C' and (resid 0 through 8 )C0 - 8
6X-RAY DIFFRACTION6chain 'D' and (resid 2 through 8 )D2 - 8
7X-RAY DIFFRACTION7chain 'E' and (resid 3 through 437 )E3 - 437
8X-RAY DIFFRACTION8chain 'E' and (resid 438 through 883 )E438 - 883
9X-RAY DIFFRACTION9chain 'F' and (resid 2 through 6 )F2 - 6
10X-RAY DIFFRACTION10chain 'F' and (resid 7 through 12 )F7 - 12
11X-RAY DIFFRACTION11chain 'F' and (resid 13 through 18 )F13 - 18
12X-RAY DIFFRACTION12chain 'G' and (resid 1 through 8 )G1 - 8
13X-RAY DIFFRACTION13chain 'H' and (resid 2 through 9 )H2 - 9
14X-RAY DIFFRACTION14chain 'I' and (resid 2 through 263 )I2 - 263
15X-RAY DIFFRACTION15chain 'I' and (resid 264 through 883 )I264 - 883
16X-RAY DIFFRACTION16chain 'J' and (resid 3 through 18 )J3 - 18
17X-RAY DIFFRACTION17chain 'K' and (resid 1 through 8 )K1 - 8
18X-RAY DIFFRACTION18chain 'L' and (resid 2 through 8 )L2 - 8
19X-RAY DIFFRACTION19chain 'M' and (resid 1 through 284 )M1 - 284
20X-RAY DIFFRACTION20chain 'M' and (resid 285 through 882 )M285 - 882
21X-RAY DIFFRACTION21chain 'N' and (resid 2 through 6 )N2 - 6
22X-RAY DIFFRACTION22chain 'N' and (resid 7 through 18 )N7 - 18
23X-RAY DIFFRACTION23chain 'O' and (resid 1 through 8 )O1 - 8
24X-RAY DIFFRACTION24chain 'P' and (resid 2 through 9 )P2 - 9

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