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- PDB-8dh2: T7 RNA polymerase elongation complex with unnatural base dDs-ATP ... -

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Basic information

Entry
Database: PDB / ID: 8dh2
TitleT7 RNA polymerase elongation complex with unnatural base dDs-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 / mitochondrial promoter sequence-specific DNA binding / DNA-directed RNA polymerase complex / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA-templated transcription
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
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: 2.9 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)443,21526
Polymers441,81015
Non-polymers1,40511
Water00
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)111,5868
Polymers111,1194
Non-polymers4664
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)111,4016
Polymers111,1194
Non-polymers2822
Water0
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)108,8276
Polymers108,4523
Non-polymers3743
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
L: T7 RNA polymerase
M: Template strand DNA
N: RNA
O: Non-template strand DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)111,4016
Polymers111,1194
Non-polymers2822
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)78.702, 85.047, 200.477
Angle α, β, γ (deg.)89.870, 85.380, 69.510
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 , 3 types, 11 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-3PO / TRIPHOSPHATE


Mass: 257.955 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: H5O10P3 / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / 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: 2.83 Å3/Da / Density % sol: 56.61 % / Mosaicity: 0.26 °
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: 2.89→48.55 Å / Num. obs: 106891 / % possible obs: 98.3 % / Redundancy: 3.5 % / Biso Wilson estimate: 77.48 Å2 / CC1/2: 0.995 / Rmerge(I) obs: 0.139 / Rpim(I) all: 0.087 / Rrim(I) all: 0.164 / Net I/σ(I): 5.5 / Num. measured all: 376957 / Scaling rejects: 3
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
2.89-2.943.52.0031895253540.2731.2512.370.598
15.83-48.553.70.02223366320.9990.0130.02531.895.5

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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

8dh0


Resolution: 2.9→47.37 Å / SU ML: 0.57 / Cross valid method: THROUGHOUT / σ(F): 1.96 / Phase error: 37.55 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.3075 1978 1.88 %
Rwork0.26 103376 -
obs0.2609 105354 97.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 316.4 Å2 / Biso mean: 123.9104 Å2 / Biso min: 30.12 Å2
Refinement stepCycle: final / Resolution: 2.9→47.37 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms25382 2350 74 0 27806
Biso mean--123.87 --
Num. residues----3384
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 (%)
2.9-2.970.45271430.41547167731096
2.97-3.050.38421400.35867420756098
3.05-3.140.38031480.32697460760898
3.14-3.240.35641360.30657343747998
3.24-3.360.33631410.2997426756798
3.36-3.490.35561460.31767324747098
3.49-3.650.37131320.33437326745897
3.65-3.850.38231440.3167302744697
3.85-4.090.31861310.26947382751398
4.09-4.40.31021450.22177417756299
4.4-4.840.27221440.20547448759299
4.84-5.540.3011400.22167482762299
5.55-6.980.27461450.25577439758499
6.98-47.370.23931430.21357440758399
Refinement TLS params.

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

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.1622-0.02810.0914-0.0191-0.06091.0260.27212.77891.73310.1511-0.3156-0.2180.08320.73860.07030.42310.07730.0872.25111.00531.64640.243156.9721-55.6833
22.4739-0.51960.38261.2171-0.26661.26730.13810.82290.2211-0.1327-0.1967-0.16970.14510.27060.04210.3587-0.02940.02470.56180.02790.472717.273932.8128-37.8406
30.11650.1990.4410.34970.79111.7854-0.32790.6003-0.7486-0.2358-0.0576-0.69341.0260.64430.30581.52530.47230.26713.27-1.00923.280452.352819.6059-52.2523
40.2724-0.18851.11613.644-1.56794.78630.07641.21770.8701-0.1305-0.1064-0.3371-0.35091.1019-0.14150.3992-0.04260.00240.81540.28330.910528.988539.123-43.9383
53.24011.35051.4551.8309-0.63812.43850.71021.3054-0.0138-0.5258-0.8133-0.50620.23381.3885-0.00080.37660.0270.17630.82390.22820.78426.39243.4923-39.9136
64.83040.33860.48595.69211.82624.1641-0.70410.59650.829-0.0056-0.5352-0.75530.16020.37331.10282.48560.3372-0.31362.51460.17792.088144.609620.9653-49.2124
72.19150.34210.15372.07-0.12681.2733-0.0482-0.5682-0.79850.0756-0.011-0.2820.35560.2354-0.08270.49960.0880.13420.63660.06660.832131.198759.46024.6164
82.4957-0.1459-0.08951.004-0.19191.0365-0.0663-0.7983-0.13550.045-0.0119-0.1391-0.0280.26310.08280.47490.0025-0.00090.4906-0.09490.512443.505982.3769-4.2317
90.7829-0.61-0.14110.5092-0.07643.32960.1959-0.72140.91850.31410.3269-0.4803-0.7680.8722-0.44672.00320.29090.26742.6395-0.50233.433971.904573.0623.1946
103.64220.2643-0.76781.7533-0.23944.57970.0838-0.4627-1.034-0.3849-0.5844-0.65590.64841.470.33850.73580.23690.08760.84850.15550.863941.095666.31230.8943
112.1377-2.96850.13118.3017-0.6448.03730.0785-0.27860.13470.0101-0.5157-1.13031.11882.13010.2710.59360.0741-0.04691.1710.08030.874535.997563.8914-2.1289
121.06630.4654-1.00432.38333.46498.1518-1.270.64940.5733-0.3655-0.55920.2781-1.773-0.8191.65592.06510.2137-0.36292.38820.09252.450963.772574.68851.6879
136.071-3.24391.77813.4879-1.61042.43570.036-0.22930.82450.5951-0.239-0.5483-0.22530.95690.41120.7083-0.28670.13371.3886-0.09320.67827.71337.8177-95.5689
141.3655-0.0654-0.27992.3949-0.18083.63440.3812-0.22840.43460.78780.31741.4629-0.8369-0.46740.0421.01310.27510.28521.1565-0.35761.9188-8.710350.6817-89.9779
152.8144-0.7539-0.43990.6959-0.35112.0710.1042-0.38040.5890.09740.23550.1843-0.5526-0.2979-0.3540.9135-0.08580.06951.1593-0.00471.0312-2.872241.5807-96.1034
162.21050.1291-0.11232.07330.08542.45560.5495-0.5936-0.1751-0.0871-0.08070.00870.98030.1426-0.37821.0510.0608-0.16221.3832-0.03470.511318.985716.2429-99.9296
172.53610.08610.21621.9956-0.05281.59930.4267-0.5283-1.1058-0.4058-0.10330.151.23850.108-0.45971.71780.048-0.4421.1812-0.02890.970519.5424-0.8525-103.1985
184.03380.1654-0.05862.34770.29872.490.542-0.7511-1.1529-0.0538-0.30541.94880.6576-1.1112-0.45952.1998-0.4228-0.77181.60690.15532.0554-14.3656-0.0712-95.4613
192.7657-0.0118-0.16792.1557-0.20682.15540.8515-0.676-1.0519-0.8592-0.06871.30740.5211-1.041-0.32411.6066-0.5926-0.71331.93040.41261.4937-10.17786.4061-91.402
201.73311.067-0.11211.2386-0.69412.6950.6005-1.6315-0.56440.3706-0.46210.40460.61370.215-0.18031.3446-0.343-0.15251.99370.1020.71838.189612.6136-77.2697
215.93934.2769-1.67175.2297-1.90395.51320.38740.16621.04580.2071-0.08320.84850.15440.3015-0.34820.9876-0.1785-0.12621.4610.13810.90661.01822.7479-92.6265
228.78940.75751.03083.7825-2.33358.8608-0.207-1.13981.53810.1979-0.33221.36170.7781-0.90790.52941.2526-0.2565-0.17481.4127-0.25251.00264.083122.3336-98.1589
234.22391.5374-4.71372.9634-1.45045.1634-0.02540.9578-1.0769-0.8506-0.0959-0.0240.95720.4069-0.19181.32660.2228-0.25031.1122-0.36730.922225.7776-14.338350.4927
242.18520.8758-0.92890.8939-0.18260.3862-0.13350.13150.2748-0.218-0.21820.550.2711-0.6667-0.4110.8491-0.1935-0.19861.69020.42621.2738-9.924-13.669559.1038
251.54630.7755-0.76410.435-0.48120.4491-0.34330.65190.0357-0.15330.68020.24820.2113-0.122-0.32191.28450.0905-0.34091.16250.02861.1807-5.9112-19.253559.1578
262.0090.54131.08891.3920.1142.65670.19750.35480.06010.1676-0.091-0.06180.08160.6799-0.14820.97630.158-0.09361.3703-0.17050.549631.412810.293354.9969
271.71290.44241.13291.00570.14452.6282-0.042-0.06610.60540.1125-0.14610.1151-0.69410.36880.23241.17030.1695-0.03211.0003-0.20420.706417.175932.093751.8182
282.68840.28371.17621.3927-0.09472.9351-0.05920.5560.37590.0262-0.15480.2363-0.53520.17250.06571.09890.2145-0.07181.2563-0.09170.57512.887121.893539.6192
293.78651.2162-0.49281.44290.5120.9228-0.4676-0.2073-0.3632-0.07530.15041.93060.0488-0.23610.4561.20880.35110.03441.6828-0.06371.04853.970212.280952.7622
302.7859-0.8378-2.77615.626-3.40917.83170.3280.1904-0.22560.3348-0.01871.6086-0.42420.5546-0.21051.04080.07220.031.4062-0.32651.006912.45759.289758.2568
310.3360.8648-0.07142.2733-0.19950.0199-0.15150.29110.6413-0.3234-0.1402-0.47130.0993-0.39530.22922.98250.92150.53621.86980.0272.0688-7.68226.750744.3664
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'B' and (resid 1 through 247 )B1 - 247
2X-RAY DIFFRACTION2chain 'B' and (resid 248 through 883 )B248 - 883
3X-RAY DIFFRACTION3chain 'A' and (resid 2 through 6 )A2 - 6
4X-RAY DIFFRACTION4chain 'A' and (resid 7 through 18 )A7 - 18
5X-RAY DIFFRACTION5chain 'C' and (resid 1 through 8 )C1 - 8
6X-RAY DIFFRACTION6chain 'D' and (resid 2 through 9 )D2 - 9
7X-RAY DIFFRACTION7chain 'E' and (resid 4 through 527 )E4 - 527
8X-RAY DIFFRACTION8chain 'E' and (resid 528 through 883 )E528 - 883
9X-RAY DIFFRACTION9chain 'F' and (resid 2 through 6 )F2 - 6
10X-RAY DIFFRACTION10chain 'F' and (resid 7 through 18 )F7 - 18
11X-RAY DIFFRACTION11chain 'G' and (resid 1 through 8 )G1 - 8
12X-RAY DIFFRACTION12chain 'H' and (resid 2 through 9 )H2 - 9
13X-RAY DIFFRACTION13chain 'I' and (resid 2 through 61 )I2 - 61
14X-RAY DIFFRACTION14chain 'I' and (resid 62 through 127 )I62 - 127
15X-RAY DIFFRACTION15chain 'I' and (resid 128 through 301 )I128 - 301
16X-RAY DIFFRACTION16chain 'I' and (resid 302 through 467 )I302 - 467
17X-RAY DIFFRACTION17chain 'I' and (resid 468 through 558 )I468 - 558
18X-RAY DIFFRACTION18chain 'I' and (resid 559 through 637 )I559 - 637
19X-RAY DIFFRACTION19chain 'I' and (resid 638 through 706 )I638 - 706
20X-RAY DIFFRACTION20chain 'I' and (resid 707 through 883 )I707 - 883
21X-RAY DIFFRACTION21chain 'J' and (resid 7 through 18 )J7 - 18
22X-RAY DIFFRACTION22chain 'K' and (resid 1 through 8 )K1 - 8
23X-RAY DIFFRACTION23chain 'L' and (resid 3 through 61 )L3 - 61
24X-RAY DIFFRACTION24chain 'L' and (resid 62 through 149 )L62 - 149
25X-RAY DIFFRACTION25chain 'L' and (resid 150 through 264 )L150 - 264
26X-RAY DIFFRACTION26chain 'L' and (resid 265 through 524 )L265 - 524
27X-RAY DIFFRACTION27chain 'L' and (resid 525 through 653 )L525 - 653
28X-RAY DIFFRACTION28chain 'L' and (resid 654 through 883 )L654 - 883
29X-RAY DIFFRACTION29chain 'M' and (resid 5 through 18 )M5 - 18
30X-RAY DIFFRACTION30chain 'N' and (resid 1 through 8 )N1 - 8
31X-RAY DIFFRACTION31chain 'O' and (resid 2 through 6 )O2 - 6

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