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- EMDB-14220: Abortive infection DNA polymerase AbiK from Lactococcus lactis -

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Basic information

Entry
Database: EMDB / ID: EMD-14220
TitleAbortive infection DNA polymerase AbiK from Lactococcus lactis
Map data
Sample
  • Complex: Homohexamer of AbiK with single-stranded DNA covalently attached to each of the subunits
    • Complex: Abortive infection protein AbiK
      • Protein or peptide: AbiK
    • Complex: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
      • DNA: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
Function / homologyReverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase domain / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily / metal ion binding / AbiK
Function and homology information
Biological speciesLactococcus lactis (lactic acid bacteria) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.27 Å
AuthorsFigiel M / Nowotny M / Gapinska M / Czarnocki-Cieciura M / Zajko W
Funding support Poland, European Union, 2 items
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, 2022-
Header (metadata) releaseSep 7, 2022-
Map releaseSep 7, 2022-
UpdateOct 5, 2022-
Current statusOct 5, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14220.png

Downloads & links

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Map

FileDownload / File: emd_14220.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.95 Å
Density
Contour LevelBy AUTHOR: 2.5
Minimum - Maximum-8.282538 - 12.547474
Average (Standard dev.)0.0061991303 (±0.30287468)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 323.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14220_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_14220_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_14220_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Homohexamer of AbiK with single-stranded DNA covalently attached ...

EntireName: Homohexamer of AbiK with single-stranded DNA covalently attached to each of the subunits
Components
  • Complex: Homohexamer of AbiK with single-stranded DNA covalently attached to each of the subunits
    • Complex: Abortive infection protein AbiK
      • Protein or peptide: AbiK
    • Complex: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')
      • DNA: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')

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Supramolecule #1: Homohexamer of AbiK with single-stranded DNA covalently attached ...

SupramoleculeName: Homohexamer of AbiK with single-stranded DNA covalently attached to each of the subunits
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Molecular weightExperimental: 420 KDa

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Supramolecule #2: Abortive infection protein AbiK

SupramoleculeName: Abortive infection protein AbiK / type: complex / Chimera: Yes / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Lactococcus lactis (lactic acid bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Supramolecule #3: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')

SupramoleculeName: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3') / type: complex / Chimera: Yes / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: AbiK

MacromoleculeName: AbiK / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Lactococcus lactis (lactic acid bacteria)
Molecular weightTheoretical: 71.713086 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSMKKEFTEL YDFIFDPIFL VRYGYYDRSI KNKKMNTAKV ELDNE(PTR)GKSD SFYFKVFNME SFADYLRSHD LKTHFN GKK PLSTDPVYFN IPKNIEARRQ YKMPNLYSYM ALNYYICDNK KEFIEVFIDN KFSTSKFFNQ LNFDYPKTQE ITQTLLY GG IKKLHLDLSN ...String:
GSMKKEFTEL YDFIFDPIFL VRYGYYDRSI KNKKMNTAKV ELDNE(PTR)GKSD SFYFKVFNME SFADYLRSHD LKTHFN GKK PLSTDPVYFN IPKNIEARRQ YKMPNLYSYM ALNYYICDNK KEFIEVFIDN KFSTSKFFNQ LNFDYPKTQE ITQTLLY GG IKKLHLDLSN FYHTLYTHSI PWMIDGKSAS KQNRKKGFSN TLDTLITACQ YDETHGIPTG NLLSRIITEL YMCHFDKQ M EYKKFVYSRY VDDFIFPFTF ENEKQEFLNE FNLICRENNL IINDNKTKVD NFPFVDKSSK SDIFSFFENI TSTNSNDKW IKEISNFIDY CVNEEHLGNK GAIKCIFPVI TNTLKQKKVD TKNIDNIFSK RNMVTNFNVF EKILDLSLKD SRLTNKFLTF FENINEFGF SSLSASNIVK KYFSNNSKGL KEKIDHYRKN NFNQELYQIL LYMVVFEIDD LLNQEELLNL IDLNIDDYSL I LGTILYLK NSSYKLEKLL KKIDQLFINT HANYDVKTSR MAEKLWLFRY FFYFLNCKNI FSQKEINSYC QSQNYNSGQN GY QTELNWN YIKGQGKDLR ANNFFNELIV KEVWLISCGE NEDFKYLN

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Macromolecule #2: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3')

MacromoleculeName: DNA (5'-D(*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*CP*C)-3') / type: dna / ID: 2 / Number of copies: 6 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 3.425224 KDa
SequenceString:
(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC)(DC) (DC)(DC)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
Component:
ConcentrationFormulaName
400.0 mMNaClsodium chloride
20.0 mMC8H18N2O4SHepes
1.0 mMC4H10O2S2dithiothreitol
0.5 mMC10H16N2O8ethylenediaminetetraacetic acid
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 150000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.27 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 227461
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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