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- PDB-8dh1: T7 RNA polymerase elongation complex with unnatural base dDs-PaTP pair -

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Basic information

Entry
Database: PDB / ID: 8dh1
TitleT7 RNA polymerase elongation complex with unnatural base dDs-PaTP pair
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
Chem-S8L / 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: 2.65 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

Downloads & links

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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: T7 RNA polymerase
M: Template strand DNA
N: RNA
O: Non-template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)442,60221
Polymers441,81015
Non-polymers7926
Water1,13563
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)111,8199
Polymers111,1194
Non-polymers7005
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
E: T7 RNA polymerase
F: Template strand DNA
G: RNA
H: Non-template strand DNA


Theoretical massNumber of molelcules
Total (without water)111,1194
Polymers111,1194
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
I: T7 RNA polymerase
J: Template strand DNA
K: RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)108,5444
Polymers108,4523
Non-polymers921
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
L: T7 RNA polymerase
M: Template strand DNA
N: RNA
O: Non-template strand DNA


Theoretical massNumber of molelcules
Total (without water)111,1194
Polymers111,1194
Non-polymers00
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)78.866, 86.156, 201.676
Angle α, β, γ (deg.)89.85, 85.09, 69.56
Int Tables number1
Space group name H-MP1

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Components

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DNA chain , 2 types, 7 molecules AFJMDHO

#2: DNA chain
Template strand DNA


Mass: 5616.727 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.: 3 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Protein / RNA chain , 2 types, 8 molecules BEILCGKN

#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 , 4 types, 69 molecules

#5: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#6: Chemical ChemComp-S8L / 1-{5-O-[(S)-hydroxy{[(R)-hydroxy(phosphonooxy)phosphoryl]oxy}phosphoryl]-beta-D-ribofuranosyl}-1H-pyrrole-2-carbaldehyde


Mass: 467.154 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16NO14P3 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 63 / Source method: isolated from a natural source / Formula: H2O

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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: 2.89 Å3/Da / Density % sol: 57.51 % / Mosaicity: 0.14 °
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: SSRL / Beamline: BL12-1 / Wavelength: 1 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 16, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.65→39.34 Å / Num. obs: 130161 / % possible obs: 90.3 % / Redundancy: 3.9 % / CC1/2: 0.994 / Rmerge(I) obs: 0.121 / Rpim(I) all: 0.071 / Rrim(I) all: 0.14 / Χ2: 0.94 / Net I/σ(I): 8.1 / Num. measured all: 509307
Reflection shellResolution: 2.65→2.69 Å / % possible obs: 89.9 % / Redundancy: 3.9 % / Rmerge(I) obs: 2.619 / Num. measured all: 25182 / Num. unique obs: 6486 / CC1/2: 0.315 / Rpim(I) all: 1.533 / Rrim(I) all: 3.036 / Χ2: 0.92 / Net I/σ(I) obs: 0.7

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Processing

Software
NameVersionClassification
PHENIX1.19refinement
XDSdata reduction
Aimless0.7.4data scaling
PDB_EXTRACT3.27data extraction
PHENIX1.19phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 8DH0
Resolution: 2.65→39.34 Å / SU ML: 0.44 / Cross valid method: THROUGHOUT / σ(F): 1.96 / Phase error: 31.4 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2715 6387 4.95 %
Rwork0.2297 --
obs0.2318 128924 89.58 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.65→39.34 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms24720 2302 2 63 27087
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00427839
X-RAY DIFFRACTIONf_angle_d0.69738143
X-RAY DIFFRACTIONf_dihedral_angle_d13.7744580
X-RAY DIFFRACTIONf_chiral_restr0.0454202
X-RAY DIFFRACTIONf_plane_restr0.0094514
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.65-2.680.41711970.37823589X-RAY DIFFRACTION79
2.68-2.710.38542330.35764033X-RAY DIFFRACTION88
2.71-2.740.4062440.34093988X-RAY DIFFRACTION89
2.74-2.780.35982270.34354147X-RAY DIFFRACTION90
2.78-2.820.38522080.31894123X-RAY DIFFRACTION91
2.82-2.850.34611980.32264159X-RAY DIFFRACTION90
2.85-2.90.35522210.30564039X-RAY DIFFRACTION90
2.9-2.940.33782220.29594094X-RAY DIFFRACTION89
2.94-2.980.35662070.28454045X-RAY DIFFRACTION90
2.98-3.030.36181890.27814125X-RAY DIFFRACTION88
3.03-3.090.32992010.2983890X-RAY DIFFRACTION85
3.09-3.140.33421980.30353615X-RAY DIFFRACTION79
3.14-3.20.40142080.31514061X-RAY DIFFRACTION91
3.2-3.270.39252170.2984286X-RAY DIFFRACTION93
3.27-3.340.33082200.26694180X-RAY DIFFRACTION93
3.34-3.420.27782220.25614241X-RAY DIFFRACTION92
3.42-3.50.32220.23994216X-RAY DIFFRACTION93
3.5-3.60.29622160.23474204X-RAY DIFFRACTION91
3.6-3.70.2912050.22284139X-RAY DIFFRACTION92
3.7-3.820.24891830.21814155X-RAY DIFFRACTION91
3.82-3.960.26092310.214119X-RAY DIFFRACTION89
3.96-4.120.24271700.20423723X-RAY DIFFRACTION81
4.12-4.30.2332170.19554184X-RAY DIFFRACTION92
4.3-4.530.24822170.18684319X-RAY DIFFRACTION95
4.53-4.810.22782150.17794231X-RAY DIFFRACTION93
4.81-5.180.22292220.18664223X-RAY DIFFRACTION92
5.18-5.70.24822350.20874097X-RAY DIFFRACTION90
5.7-6.530.28321870.23133914X-RAY DIFFRACTION86
6.53-8.210.24212240.20824284X-RAY DIFFRACTION95
8.21-39.340.19762310.20194114X-RAY DIFFRACTION90
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)
12.5567-0.0399-0.42561.3159-0.30271.77120.14040.01861.28960.16760.05520.3201-0.4254-0.4621-0.17430.70090.02210.11060.5896-0.04261.1127-2.035145.2709-96.2395
22.76130.0546-0.250.8829-0.22871.47890.1848-0.3512-0.3432-0.2079-0.04760.27270.3733-0.0576-0.12910.8358-0.0579-0.16220.60230.00480.50828.46049.9241-91.8304
38.29824.6475-1.15176.0083-1.75232.06660.3108-0.531.698-0.40070.19171.98790.2034-0.9181-0.70550.791-0.1057-0.10450.84290.01860.94650.275123.0829-92.8794
44.3806-6.21061.5829.1518-3.4355.1940.5344-0.49860.5405-0.3764-0.56750.81750.1313-0.3766-0.03861.0223-0.1993-0.14930.6551-0.14270.97763.334222.1989-98.3779
51.77380.8219-0.21461.0635-1.13382.05410.0730.1642-0.36010.00920.3260.05220.6273-0.1522-0.30471.14790.037-0.23130.9467-0.08930.748110.0494-14.084353.4531
62.0561-0.04931.00630.85770.12242.21030.11070.40680.14410.0027-0.16450.0525-0.21750.22810.05050.95870.0317-0.05531.0449-0.07490.45322.040318.6449.2529
73.8003-3.60231.49396.9556-1.4813.25320.1313-0.2052-0.87750.6637-0.29611.8724-0.4868-0.0360.09731.00410.06130.14851.2673-0.24710.90793.912411.258353.4271
85.19540.7936-2.44963.3006-3.98635.24960.2628-0.14310.11110.912-0.06051.6216-0.4182-0.4237-0.0120.96850.0820.06571.3824-0.23210.939712.54048.447858.8741
99.82980.3611-3.2042.9601-3.82145.6985-0.81610.56560.36530.2781-0.3483-0.3440.2884-1.12821.05092.91510.82510.61382.29380.14581.7184-7.838325.849544.3783
102.11190.42850.07880.2821-0.20060.98780.11821.23250.6793-0.1447-0.0799-0.0883-0.03780.2076-0.04320.47760.09560.07621.00450.37140.829233.075253.1652-56.5292
111.8647-0.20350.37881.1763-0.18190.99880.13370.34250.035-0.0469-0.0945-0.1330.17120.1397-0.04790.2897-0.00860.02090.40950.02050.360718.965530.75-35.0979
122.54752.15010.0631.87040.32573.74490.17970.50260.0999-0.0617-0.0822-1.26020.50451.0054-0.25330.36350.10070.06270.87570.12420.767835.221533.954-45.861
138.38795.6877-3.90593.8675-2.68294.67450.1240.6767-0.4043-0.2085-0.3126-0.68580.07410.80450.10860.3812-0.0138-0.00450.63260.12570.527726.700443.169-40.0977
144.40031.1693-3.9040.7752-1.24973.5572-0.29431.2751-0.193-0.23330.3907-0.22590.3354-0.3282-0.15052.4203-0.03840.04043.07960.1342.521143.584419.7423-49.7479
151.95550.04990.46942.00630.0211.02740.0223-0.6755-0.72910.2582-0.0095-0.34720.33140.1014-0.01740.5480.00560.05970.8970.12870.798635.369956.2437.5874
162.5090.0529-0.28471.278-0.091.0658-0.0103-0.63460.07380.0228-0.1198-0.0026-0.07440.03240.1260.41110.0807-0.02480.4982-0.12470.40636.48381.7608-5.8194
172.02191.97971.99098.69758.85999.0291-0.35840.6306-1.193-0.43270.4172-3.1058-0.63411.78050.02631.92070.3547-0.06472.8112-0.08632.446872.930673.56072.4182
187.12793.6654-0.91695.5391.4447.6818-0.4864-0.8384-1.4621-0.47570.2982-0.65370.36232.69610.40010.81770.1283-0.05511.17760.11130.62250.006271.9199-1.3149
197.01293.1158-1.21961.74821.12928.0502-0.1166-0.7705-1.6437-0.2564-0.2212-1.13711.43570.79350.69480.66410.2050.11040.74820.16360.656233.01760.68963.3718
203.6836-3.7728-1.5134.4580.41932.776-0.647-1.3374-0.0321-0.11330.0715-1.18750.49891.0170.61810.7075-0.00350.05620.93670.13080.710936.57864.3095-2.1265
213.28052.2793-0.1974.38963.85195.6879-1.1167-1.8419-1.72510.40350.6133-1.4647-1.5917-0.45580.47691.67150.3294-0.16992.11370.1652.003365.502475.10861.9813
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'I' and (resid 6 through 263 )
2X-RAY DIFFRACTION2chain 'I' and (resid 264 through 883 )
3X-RAY DIFFRACTION3chain 'J' and (resid 7 through 18 )
4X-RAY DIFFRACTION4chain 'K' and (resid 1 through 8 )
5X-RAY DIFFRACTION5chain 'L' and (resid 4 through 301 )
6X-RAY DIFFRACTION6chain 'L' and (resid 302 through 882 )
7X-RAY DIFFRACTION7chain 'M' and (resid 5 through 18 )
8X-RAY DIFFRACTION8chain 'N' and (resid 1 through 8 )
9X-RAY DIFFRACTION9chain 'O' and (resid 2 through 6 )
10X-RAY DIFFRACTION10chain 'B' and (resid 2 through 331 )
11X-RAY DIFFRACTION11chain 'B' and (resid 332 through 883 )
12X-RAY DIFFRACTION12chain 'A' and (resid 3 through 18 )
13X-RAY DIFFRACTION13chain 'C' and (resid 1 through 8 )
14X-RAY DIFFRACTION14chain 'D' and (resid 3 through 8 )
15X-RAY DIFFRACTION15chain 'E' and (resid 18 through 407 )
16X-RAY DIFFRACTION16chain 'E' and (resid 408 through 883 )
17X-RAY DIFFRACTION17chain 'F' and (resid 2 through 6 )
18X-RAY DIFFRACTION18chain 'F' and (resid 7 through 12 )
19X-RAY DIFFRACTION19chain 'F' and (resid 13 through 18 )
20X-RAY DIFFRACTION20chain 'G' and (resid 1 through 8 )
21X-RAY DIFFRACTION21chain 'H' and (resid 2 through 9 )

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