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- PDB-7r07: Abortive infection DNA polymerase AbiK from Lactococcus lactis -

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Basic information

Entry
Database: PDB / ID: 7r07
TitleAbortive infection DNA polymerase AbiK from Lactococcus lactis
Components
  • AbiK
  • DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
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. / DNA/RNA polymerase superfamily / metal ion binding / DNA / DNA (> 10) / AbiK
Function and homology information
Biological speciesLactococcus lactis (lactic acid bacteria)
Escherichia coli BL21 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.1 Å
AuthorsFigiel, M. / Gapinska, M. / Czarnocki-Cieciura, M. / Zajko, W. / Nowotny, M.
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 / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
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: AbiK
B: AbiK
C: AbiK
D: AbiK
E: AbiK
F: AbiK
G: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
H: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
I: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
J: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
K: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
L: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)449,31421
Polymers449,09512
Non-polymers2199
Water28816
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)200.990, 200.990, 570.720
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number180
Space group name H-MP6222
Space group name HallP622(x,y,z+1/3)

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Components

#1: Protein
AbiK


Mass: 71713.086 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lactococcus lactis (lactic acid bacteria)
Gene: abiK / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): Star / References: UniProt: Q48614
#2: DNA chain
DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')


Mass: 3136.042 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Escherichia coli BL21 (bacteria) / Variant: Star
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Mg
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 16 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 3.68 Å3/Da / Density % sol: 66.54 %
Crystal growTemperature: 291 K / Method: vapor diffusion, sitting drop
Details: 30% Jeffamine M-600, 0.1 M MES (pH 6.5), and 0.05 M cesium chloride

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P14 (MX2) / Wavelength: 1.0064 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Nov 28, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0064 Å / Relative weight: 1
ReflectionResolution: 3.1→49.627 Å / Num. obs: 123156 / % possible obs: 99.4 % / Redundancy: 40.82 % / Biso Wilson estimate: 115.74 Å2 / CC1/2: 1 / Net I/σ(I): 19.7
Reflection shellResolution: 3.1→3.29 Å / Num. unique obs: 19473 / CC1/2: 0.508

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.13_2998refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: D_1292120632

Resolution: 3.1→49.627 Å / SU ML: 0.4982 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 24.9168 / Stereochemistry target values: GeoStd + Monomer Library
RfactorNum. reflection% reflection
Rfree0.2323 2094 1.71 %
Rwork0.1893 120558 -
obs0.19 122652 99.12 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 116.85 Å2
Refinement stepCycle: LAST / Resolution: 3.1→49.627 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms29699 920 9 16 30644
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.002331427
X-RAY DIFFRACTIONf_angle_d0.50942646
X-RAY DIFFRACTIONf_chiral_restr0.03834595
X-RAY DIFFRACTIONf_plane_restr0.0035332
X-RAY DIFFRACTIONf_dihedral_angle_d9.578518414
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.1-3.170.461320.46047474X-RAY DIFFRACTION94.08
3.17-3.250.37431380.35237934X-RAY DIFFRACTION99.3
3.25-3.340.30041370.2897922X-RAY DIFFRACTION99.29
3.34-3.440.27731380.25547922X-RAY DIFFRACTION99.37
3.44-3.550.27171380.2387961X-RAY DIFFRACTION99.48
3.55-3.680.2521370.23127926X-RAY DIFFRACTION99.04
3.68-3.820.25221390.21527990X-RAY DIFFRACTION99.51
3.82-40.24571390.19458022X-RAY DIFFRACTION99.55
4-4.210.24191390.18098016X-RAY DIFFRACTION99.65
4.21-4.470.20331400.16268076X-RAY DIFFRACTION99.66
4.47-4.820.19321400.15218064X-RAY DIFFRACTION99.68
4.82-5.30.20131410.15578144X-RAY DIFFRACTION99.83
5.3-6.070.21621420.1888153X-RAY DIFFRACTION99.46
6.07-7.640.25711440.20368296X-RAY DIFFRACTION99.89
7.64-49.6270.20831500.15948658X-RAY DIFFRACTION99
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)
18.186184681191.77780696848-1.973085466373.313974254820.9260247328641.57462198808-0.309322386651-0.82433392892-0.2494382539810.6064480854470.0868635029607-0.4357825383250.5341469237660.4224436878490.2619726070061.262377891780.124092507244-0.1384304236810.8041266530260.1821189822180.75049931666460.23173964266.8169268149272.2774540279
21.918657082060.920947814001-0.03029462738277.16062536805-2.307416272432.72370647875-0.1283728356220.139575913749-0.3862206241-0.3968599256490.273158386409-0.09401179490610.249228567665-0.0330516328341-0.2076547619840.9735079663910.04914493556780.05450526620270.801158464301-0.009530712031350.7306280736460.913633638311.167445454850.5225143496
31.4862584867-0.743950323759-1.340774021561.22530070114-0.6311356970064.036143114630.00642249889635-0.2175169394520.1665444272650.05966188653280.0275419002004-0.157493526454-0.4462953675540.548270294923-0.05788209049631.05004790817-0.217735913437-0.06107552005311.063800310770.01276653448070.72828992011650.58283482735.189439503676.4116296514
42.19983047077-0.138929007559-0.5398334486563.62368659445-0.1501399028312.58491918278-0.0647355938421-0.446667319806-0.6088537678720.209921100941-0.03138708786320.2163971156240.8124188127520.007774761700780.1317690079071.22282082543-0.1197642176190.001441985430940.8864838809630.1917895707620.77042511807440.41060043982.1732330169983.1088839765
53.26216812-0.582479765016-1.005763527965.76376270625-0.2767658771232.064536774940.113022048910.5746613561020.012761275887-0.737594601811-0.08498376923520.275910214752-0.283131612442-0.176711275443-0.1985170956491.98524693609-0.209748683655-0.1680971820380.9066669049870.126181655870.79588590501440.315201794549.4444181568-4.28023411167
61.63125522635-0.246799755427-0.1065728344160.9715738758750.2084140164731.03340090425-0.1213631566160.2723326713850.184508053902-0.838977295580.07176681864150.136717557541-0.313932538831-0.1488554349550.04761659961611.73592205348-0.118343460459-0.2001956719840.7664010540520.001448216328080.75296342384237.16669907952.24837673516.09867989904
75.19341092009-0.9172024424680.8980759861057.2043203717-0.8869273694787.766322732360.111756900489-0.8477557163670.8809167990441.22383540397-0.00198754993045-0.0106344068047-0.8714570751060.926118889388-0.05304886829610.943713719946-0.1166872895710.09995457998030.828106703495-0.1186926212970.73304282995331.259964886364.723056457781.3569161164
82.887681171111.155383391960.2372007595644.87543562791-0.857473348564.89811928234-0.00455170088573-0.2630816444270.3382334828040.43705623962-0.1279649888360.615723328686-0.3063010356110.0262644653257-0.1851833329690.855816618495-0.001170750834550.2138351598470.589620256582-0.1214201786290.92541059059520.391214878367.617832414673.4706339378
94.28896461416-1.91152771721-0.225407165324.907367915020.6836462311061.947037358170.156682810851-0.234832732056-0.0776028595029-0.185044867617-0.05776133795730.5774274586050.0674256128121-0.26011861076-0.0621498014590.990536564206-0.1761842270810.04991197102670.8101266349990.002813477681890.80840285595821.08408986646.415521193169.1944542265
101.565174303530.587979965652-0.3769175424893.106446738690.1542204811361.139556731690.0610912954518-0.297160178535-0.002786621379280.1820859815930.00675729190353-0.398481055577-0.1629724411880.3319877573450.06771549185041.13286742732-0.142321589729-0.01148966853740.790646817810.012402809410.84568509879551.667313826965.131766940961.442552326
112.41939066677-0.0163345202637-0.8190269047421.89943592853-0.6209553187911.173207996960.003518415700740.02756305829840.4528849463630.05827920220340.07898949501940.374403797239-0.379327084148-0.100942348468-0.1418542949811.09354198215-0.0426298056615-0.003642780593220.687816990244-0.06767769891270.96594859097822.844202591885.112446007660.7093315178
122.590818543530.4284276243170.127445744454.12378596125-2.02580893496.168913255610.2991490011460.486069370949-0.132207621552-0.193904605832-0.2005921222810.8541777815440.413228213533-0.407009213884-0.1003183147720.798845600487-0.0302302215421-0.08692466021550.951485482952-0.2000882802611.57395510414-9.0132411679644.768607645544.2319509346
133.88399826132-0.0624892207729-2.763341376882.005707759520.2086818617133.66678197231-0.0352012552234-0.1588307663250.275463511643-0.301875173943-0.195170388830.411526747226-0.51121419318-0.1529505633020.1546464109771.21483400571-0.0203909267389-0.2525226642110.785550651178-0.04308837887191.220738465028.4577603153257.065996245140.7543999985
142.020233348350.0920962477769-0.7341130522121.01374587275-0.8129052224550.904729511048-0.092910431240.623592229084-0.429270636626-0.9109052356010.172523524903-0.574662374491-0.173214675006-0.1182549724650.118960172541.06819236491-0.0698813888519-0.1273101400550.770331273737-0.06576495482220.84743939226515.127538122130.650675886930.2830278329
152.61886460953-0.933605084923-1.43633769192.027416453141.206774826552.85652146336-0.0557548516028-0.0956534853631-0.1615148319960.147857291869-0.10842460270.7720343620260.305403973662-0.5146304788980.196739318550.80567190098-0.206131786577-0.03380104682950.763271214549-0.08886342702551.19996477568-7.2035114284226.811279991854.2772046754
164.17567420789-0.111667966989-1.118190607321.78410744720.3830910464683.58210150239-0.1306658759450.301667311212-0.548392243033-0.632273750047-0.1548673957070.6429511531390.900377232255-0.2587945985680.1947953349851.49337947284-0.0424895218169-0.04231928337570.553444275857-0.05106806808240.93887043453732.3543001475-9.8899813532132.3253042984
173.49765270761-1.83873635496-0.3552018471093.875039241870.8714193456262.886149297360.0787673840242-0.396576131709-0.146818465130.232247264149-0.02701864084090.002153290231590.2781533782660.149667840029-0.0526681220871.01717145329-0.12801677809-0.031095365230.695517982107-0.004702722692890.74339809961128.8845221075.6453181129947.3356202248
181.84876565032-0.9945993511230.4555017724042.68528179236-1.01888426732.310648879360.02729738100360.0946001219609-0.271870314424-0.5119509428-0.07614006194680.05841602889920.2298455574640.2625476016460.05189800009711.16023630299-0.03642131956070.07022079670060.595760459737-0.1402155816760.68369262843845.7310163813-1.4036805870517.5573916702
193.03275971154-0.581683111794-0.7557103483251.416035256380.1790012152961.50313787442-0.02888426732730.1868781646530.110974907192-0.6004544984530.0611078102578-0.192804125526-0.2255771515820.344156448244-0.02400780062761.40033172886-0.2571879441020.1714121245280.836219197254-0.01789301939110.82314427986866.685910972462.930015521729.470656436
201.59327191527-0.1670368778410.05827841518391.68339500307-0.04609432908121.414362092520.06247864130720.006271755483910.0199177076866-0.4172098215230.0569713319884-0.492069118005-0.1369166475570.509843232361-0.1310481136710.918450340239-0.1520042480580.1751050368610.809587362772-0.1092382749540.81041511901380.945816414241.67215336234.2345849889
217.39377186573-2.76793552590.7687256651462.35537914635-3.76271727449.25387936830.4642339353750.274396922002-0.426773444133-0.357658327162-0.0766064393827-1.1872778536-1.337254406371.87496062984-0.04304819027891.22504548850.03779269500810.2182512980511.68983140082-0.1207507920241.3357412802564.477414284126.651532983269.3469550573
225.10130931651-3.57308273774-0.6591451451225.52358327539-2.45573961142.976893760050.5949118522631.81317831992-2.16844192208-1.732513005030.373906963454-0.78907596531.324914146261.5863358122-1.056781398732.17484698260.05524284295850.05922406062282.00002466139-0.3119026013261.4450629613628.361715835934.51932364312.47305613179
232.84985420098-3.23379391975-0.05605816022728.62931585447-5.450430900896.280190091380.262566706702-3.04380428622-0.8187858630261.59348550221-0.364569217451-1.004745953560.150604622022-1.539212118150.5196886201881.34586129472-0.2612040542090.1366469023881.60247941710.05983887230480.99275823112843.10221818757.422889174374.8306273332
243.62549524374-2.78452867008-0.4295371095254.33203633526-4.089798709178.97849597839-1.341758194190.7516257569721.27945238314-1.359903479130.3767408594951.2718427367-1.83304665808-0.9421906517451.135222812331.62491931174-0.0329455991461-0.6918104123911.28478192070.1116736897761.924231273190.81421507697636.851983816627.5100173903
259.202910183371.14815059631-7.211615133186.68252823775-0.3696810111816.03718993987-0.396647199206-0.593008492697-1.44106157835-0.5058505170420.3526524149610.308086774386-0.410946861012-1.668290423840.05365765115261.75033622918-0.278796507105-0.1600195131921.282068199660.1604988873391.3596824414422.06102720385.6294623471424.8966041147
266.65769436635-0.310991961436-5.704792365038.61335588117-4.451309319067.48218979569-0.3767258548540.07616667214261.17541649241-0.256651121745-0.330293979973-0.529281284306-3.245935282851.409465854770.2691062202162.17287571488-0.1268915777450.1770066037420.8847471968560.1837467398511.089985969370.970535061357.858069793648.8951671453
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 0 through 115 )
2X-RAY DIFFRACTION2chain 'A' and (resid 116 through 286 )
3X-RAY DIFFRACTION3chain 'A' and (resid 287 through 418 )
4X-RAY DIFFRACTION4chain 'A' and (resid 419 through 599 )
5X-RAY DIFFRACTION5chain 'B' and (resid 0 through 102 )
6X-RAY DIFFRACTION6chain 'B' and (resid 103 through 599 )
7X-RAY DIFFRACTION7chain 'C' and (resid 0 through 49 )
8X-RAY DIFFRACTION8chain 'C' and (resid 50 through 126 )
9X-RAY DIFFRACTION9chain 'C' and (resid 127 through 286 )
10X-RAY DIFFRACTION10chain 'C' and (resid 287 through 418 )
11X-RAY DIFFRACTION11chain 'C' and (resid 419 through 599 )
12X-RAY DIFFRACTION12chain 'D' and (resid 0 through 115 )
13X-RAY DIFFRACTION13chain 'D' and (resid 116 through 273 )
14X-RAY DIFFRACTION14chain 'D' and (resid 274 through 331 )
15X-RAY DIFFRACTION15chain 'D' and (resid 332 through 599 )
16X-RAY DIFFRACTION16chain 'E' and (resid 0 through 126 )
17X-RAY DIFFRACTION17chain 'E' and (resid 127 through 295 )
18X-RAY DIFFRACTION18chain 'E' and (resid 296 through 599 )
19X-RAY DIFFRACTION19chain 'F' and (resid 0 through 273 )
20X-RAY DIFFRACTION20chain 'F' and (resid 274 through 599 )
21X-RAY DIFFRACTION21chain 'G' and (resid 1 through 10 )
22X-RAY DIFFRACTION22chain 'H' and (resid 1 through 10 )
23X-RAY DIFFRACTION23chain 'I' and (resid 1 through 10 )
24X-RAY DIFFRACTION24chain 'J' and (resid 1 through 10 )
25X-RAY DIFFRACTION25chain 'K' and (resid 1 through 11 )
26X-RAY DIFFRACTION26chain 'L' and (resid 1 through 10 )

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