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- PDB-7r08: Abortive infection DNA polymerase Abi-P2 -

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Basic information

Entry
Database: PDB / ID: 7r08
TitleAbortive infection DNA polymerase Abi-P2
ComponentsReverse transcriptase
KeywordsANTIVIRAL PROTEIN / DNA polymerase / protein-primed DNA synthesis / template-independent DNA synthesis / abortive infection
Function / homologyReverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / RNA-directed DNA polymerase activity / Reverse transcriptase
Function and homology information
Biological speciesEscherichia phage P2 (virus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.1 Å
AuthorsGapinska, M.A. / Figiel, M. / Czarnocki Cieciura, M. / Nowotny, M. / Zajko, W.
Funding support Poland, European Union, 2items
OrganizationGrant numberCountry
Foundation for Polish SciencePOIR.04.04.00-00-20E7/16-00 Poland
European Regional Development FundPOIG.02.02.00-14-024/08-00European Union
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases.
Authors: Małgorzata Figiel / Marta Gapińska / Mariusz Czarnocki-Cieciura / Weronika Zajko / Małgorzata Sroka / Krzysztof Skowronek / Marcin Nowotny
Abstract: Abortive infection (Abi) is a bacterial antiphage defense strategy involving suicide of the infected cell. Some Abi pathways involve polymerases that are related to reverse transcriptases. They are ...Abortive infection (Abi) is a bacterial antiphage defense strategy involving suicide of the infected cell. Some Abi pathways involve polymerases that are related to reverse transcriptases. They are unique in the way they combine the ability to synthesize DNA in a template-independent manner with protein priming. Here, we report crystal and cryo-electron microscopy structures of two Abi polymerases: AbiK and Abi-P2. Both proteins adopt a bilobal structure with an RT-like domain that comprises palm and fingers subdomains and a unique helical domain. AbiK and Abi-P2 adopt a hexameric and trimeric configuration, respectively, which is unprecedented for reverse transcriptases. Biochemical experiments showed that the formation of these oligomers is required for the DNA polymerization activity. The structure of the AbiK-DNA covalent adduct visualized interactions between the 3' end of DNA and the active site and covalent attachment of the 5' end of DNA to a tyrosine residue used for protein priming. Our data reveal a structural basis of the mechanism of highly unusual template-independent protein-priming polymerases.
History
DepositionFeb 1, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 7, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 28, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 5, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3May 1, 2024Group: 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
A: Reverse transcriptase
B: Reverse transcriptase
C: Reverse transcriptase
D: Reverse transcriptase
E: Reverse transcriptase
F: Reverse transcriptase


Theoretical massNumber of molelcules
Total (without water)379,3076
Polymers379,3076
Non-polymers00
Water00
1
A: Reverse transcriptase
C: Reverse transcriptase
D: Reverse transcriptase


Theoretical massNumber of molelcules
Total (without water)189,6533
Polymers189,6533
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5290 Å2
ΔGint-34 kcal/mol
Surface area60700 Å2
MethodPISA
2
B: Reverse transcriptase
E: Reverse transcriptase
F: Reverse transcriptase


Theoretical massNumber of molelcules
Total (without water)189,6533
Polymers189,6533
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5610 Å2
ΔGint-31 kcal/mol
Surface area60830 Å2
MethodPISA
Unit cell
Length a, b, c (Å)134.334, 88.359, 161.094
Angle α, β, γ (deg.)90.000, 103.180, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Reverse transcriptase


Mass: 63217.785 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage P2 (virus) / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q2P9X6

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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: 2.45 Å3/Da / Density % sol: 49.88 %
Crystal growTemperature: 277 K / Method: vapor diffusion, sitting drop / pH: 4.5
Details: Well condition: 6% (v/v) 2-propanol, 26% PEG MME 550, 0.1 M sodium acetate trihydrate (pH 4.5) Protein concentration: 6 mg/mL

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, DESY / Beamline: P11 / Wavelength: 1.0332 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Nov 24, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 3.1→47.91 Å / Num. obs: 67114 / % possible obs: 99.3 % / Redundancy: 4.76 % / Rrim(I) all: 0.106 / Net I/σ(I): 12.22
Reflection shellResolution: 3.1→3.28 Å / Num. unique obs: 10560 / Rrim(I) all: 1.24 / % possible all: 97.5

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Processing

Software
NameVersionClassification
XDSdata reduction
XSCALEdata scaling
PHENIX1.18.2_3874refinement
PDB_EXTRACT3.27data extraction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: CryoEM model

Resolution: 3.1→47.91 Å / SU ML: 0.51 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 32.6 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2737 2095 3.13 %
Rwork0.2196 64811 -
obs0.2213 66906 99.65 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 258.37 Å2 / Biso mean: 106.2898 Å2 / Biso min: 47.2 Å2
Refinement stepCycle: final / Resolution: 3.1→47.91 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms22211 0 0 0 22211
Num. residues----2899
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 15

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.1-3.170.45151380.381142874425100
3.17-3.250.36331390.326342794418100
3.25-3.340.36911380.314442824420100
3.34-3.440.3311390.29054282442199
3.44-3.550.35161390.271143254464100
3.55-3.680.37451390.276442954434100
3.68-3.820.28511390.229243024441100
3.82-40.29561390.233142914430100
4-4.210.2591390.213543254464100
4.21-4.470.28291400.189243074447100
4.47-4.810.22071390.18743004439100
4.82-5.30.26071400.195143584498100
5.3-6.060.28131400.228643374477100
6.06-7.640.30171420.241343874529100
7.64-47.910.20071450.16894454459999
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)
14.72160.2343-3.33321.5547-0.70644.69960.0979-1.07860.44970.28090.1180.1054-0.38040.0684-0.230.83120.1179-0.03740.9835-0.17640.669114.642335.479488.9854
25.17080.604-2.37122.9668-1.13618.4828-0.1271-1.0488-0.12440.17920.13830.0331-0.01980.59010.010.60850.0597-0.03360.6597-0.11650.628327.842226.146978.5557
37.4951-1.0106-3.30233.7743-0.21746.93520.1216-0.26850.6301-0.2051-0.01170.088-1.1661-0.7013-0.12530.72560.0968-0.05010.622-0.12260.5684-4.85537.037962.1639
43.9384-1.96072.92642.8510.43284.60960.007-0.90110.02330.2117-0.0650.1109-0.5341-0.9235-0.01750.75020.04190.18661.3393-0.07710.7917-6.477535.316988.9726
51.67750.2194-0.84761.5062-0.17090.3968-0.39690.4137-0.3443-0.10610.1151-0.16630.2004-0.20810.18240.9962-0.09640.12581.2563-0.53560.991560.02951.812121.3571
62.3912-0.3042-1.90711.0331.1213.3701-0.05390.01120.0445-0.08680.3057-0.3096-0.53740.915-0.22160.6754-0.27390.11490.918-0.28450.846355.32315.811934.6922
74.32051.52632.44571.48943.02654.32640.2603-0.20310.14630.2802-0.1242-0.2556-0.30920.1221-0.20070.5064-0.04950.0340.50650.0180.650631.01956.571138.0429
88.0891-2.72461.0764.4475-1.65812.3875-0.0943-0.0247-0.83160.05070.35660.03160.33250.3218-0.26270.7786-0.03940.09870.5248-0.14110.782127.3673-5.703721.6496
97.33862.5018-0.4262.1581.06071.9854-0.81751.4687-0.6131-0.85440.9881-0.46950.35680.8802-0.02990.9732-0.10130.16731.2855-0.27480.856344.16090.85848.1284
100.91420.04230.64040.9465-0.58964.2020.0505-0.3637-0.12980.02350.22890.3272-0.272-0.7413-0.27750.8471-0.0152-0.11330.76770.25930.8419-28.346427.668631.5436
111.0707-0.4859-0.44021.0384-1.25442.60090.1044-0.0986-0.1271-0.35020.39790.32660.0707-0.7018-0.50220.7555-0.0641-0.13480.6310.18730.7932-15.340322.208441.3322
126.2361-1.2526-0.77893.1132-1.95218.26130.03810.0130.6933-0.4660.1392-0.15-0.4231-0.3446-0.14940.93110.0825-0.07830.3274-0.02230.76271.763744.465326.0901
138.86931.52822.33650.8025-0.66481.4673-0.36820.95580.7207-0.27080.40280.2911-0.281-0.4168-0.0480.97160.0879-0.03510.8040.1540.7332-16.548439.846512.5464
142.26851.8182-0.60823.44851.39193.0086-0.22380.26230.5237-0.37430.11180.0474-0.63740.77220.17931.068-0.37790.14980.8232-0.04961.03744.546549.271222.1114
154.0018-1.26010.37541.4030.96641.0633-0.16190.3208-0.2084-0.33610.0422-0.13840.01050.26320.14141.03-0.15830.10190.6060.04550.73931.732433.345816.5575
163.7962-1.19610.76122.3095-1.2862.70.0953-0.15850.5155-0.0687-0.0466-0.0268-0.72620.1613-0.07230.795-0.1933-0.01410.4314-0.07450.779728.633846.36152.1939
175.70890.5308-4.09334.56031.92336.0270.712-0.77991.36880.23760.0676-0.463-1.3711.5732-0.72391.2192-0.42750.04770.9696-0.01221.174351.229154.529840.1979
183.6642.1918-0.2712.19560.01873.1017-0.14820.2189-0.3475-0.30030.0843-0.00560.5818-0.83510.09850.8602-0.2157-0.06240.7536-0.05520.7373-15.4831-10.502818.2358
199.26163.2602-0.72192.1886-0.82481.7024-0.16370.14360.31920.02330.1702-0.0595-0.2445-0.3851-0.01130.8148-0.012-0.04240.4059-0.03730.5705-3.54476.317215.1381
206.9151-0.5968-0.43912.78170.38645.15570.0618-0.3092-0.6306-0.28650.18870.04880.6875-0.1342-0.19270.5601-0.11950.01190.4246-0.00430.69451.7481-12.054547.7512
218.8115-1.60534.38785.9126-1.64844.99330.149-0.6134-0.62930.38530.30630.52050.5266-1.214-0.41620.9021-0.31440.1031.1083-0.0960.675-21.215-18.498635.9738
225.95410.13252.23040.5517-0.55933.48260.0994-1.569-0.51150.2406-0.1166-0.17810.1420.4159-0.0180.8020.0161-0.07321.44710.31960.780811.2039-3.560382.8607
237.7011-0.5448-2.54960.2050.98942.6077-0.0458-0.4959-0.13570.19880.0163-0.07070.1954-0.19130.01030.5598-0.0816-0.09190.54690.10950.734612.3973.55160.9451
246.74632.08455.01164.1834-1.55738.8020.32590.498-0.9682-0.7549-0.1119-0.80010.743-0.0118-0.12850.52220.06110.00130.3640.01380.886336.855-5.818358.7685
253.08881.292-1.4936-0.9389-1.03457.29190.011-2.0617-1.120.1976-0.0845-0.30340.72061.21820.17120.8830.0434-0.06751.57050.48821.150439.542-5.550780.7217
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 32 through 183 )A32 - 183
2X-RAY DIFFRACTION2chain 'A' and (resid 184 through 331 )A184 - 331
3X-RAY DIFFRACTION3chain 'A' and (resid 332 through 443 )A332 - 443
4X-RAY DIFFRACTION4chain 'A' and (resid 444 through 540 )A444 - 540
5X-RAY DIFFRACTION5chain 'B' and (resid 32 through 76 )B32 - 76
6X-RAY DIFFRACTION6chain 'B' and (resid 77 through 309 )B77 - 309
7X-RAY DIFFRACTION7chain 'B' and (resid 310 through 363 )B310 - 363
8X-RAY DIFFRACTION8chain 'B' and (resid 364 through 423 )B364 - 423
9X-RAY DIFFRACTION9chain 'B' and (resid 424 through 540 )B424 - 540
10X-RAY DIFFRACTION10chain 'C' and (resid 32 through 223 )C32 - 223
11X-RAY DIFFRACTION11chain 'C' and (resid 224 through 363 )C224 - 363
12X-RAY DIFFRACTION12chain 'C' and (resid 364 through 423 )C364 - 423
13X-RAY DIFFRACTION13chain 'C' and (resid 424 through 540 )C424 - 540
14X-RAY DIFFRACTION14chain 'D' and (resid 32 through 172 )D32 - 172
15X-RAY DIFFRACTION15chain 'D' and (resid 173 through 331 )D173 - 331
16X-RAY DIFFRACTION16chain 'D' and (resid 332 through 443 )D332 - 443
17X-RAY DIFFRACTION17chain 'D' and (resid 444 through 539 )D444 - 539
18X-RAY DIFFRACTION18chain 'E' and (resid 32 through 172 )E32 - 172
19X-RAY DIFFRACTION19chain 'E' and (resid 173 through 331 )E173 - 331
20X-RAY DIFFRACTION20chain 'E' and (resid 332 through 443 )E332 - 443
21X-RAY DIFFRACTION21chain 'E' and (resid 444 through 539 )E444 - 539
22X-RAY DIFFRACTION22chain 'F' and (resid 32 through 292 )F32 - 292
23X-RAY DIFFRACTION23chain 'F' and (resid 293 through 363 )F293 - 363
24X-RAY DIFFRACTION24chain 'F' and (resid 364 through 422 )F364 - 422
25X-RAY DIFFRACTION25chain 'F' and (resid 423 through 539 )F423 - 539

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