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- EMDB-14435: Abortive infection DNA polymerase AbiK from Lactococcus lactis, Y... -

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Basic information

Entry
Database: EMDB / ID: EMD-14435
TitleAbortive infection DNA polymerase AbiK from Lactococcus lactis, Y44F variant
Map data
Sample
  • Complex: Homohexamer of L. lactis AbiK Y44F mutant
    • Protein or peptide: AbiK
KeywordsDNA polymerase / protein-primed DNA synthesis / template-independent DNA synthesis / abortive infection / ANTIVIRAL PROTEIN
Function / homologyReverse transcriptase domain / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / DNA/RNA polymerase superfamily / metal ion binding / AbiK
Function and homology information
Biological speciesLactococcus lactis (lactic acid bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.68 Å
AuthorsFigiel M / Gapinska M / Czarnocki-Cieciura M / Zajko W / Nowotny M
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 24, 2022-
Header (metadata) releaseSep 7, 2022-
Map releaseSep 7, 2022-
UpdateJul 17, 2024-
Current statusJul 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14435.png

Downloads & links

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Map

FileDownload / File: emd_14435.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 384 pix.
= 314.88 Å		0.82 Å/pix.
x 384 pix.
= 314.88 Å		0.82 Å/pix.
x 384 pix.
= 314.88 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-2.2418349 - 3.0248108
Average (Standard dev.)0.0039890944 (±0.087507695)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 314.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14435_msk_1.map
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AxesZYX

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Half map: #2

Fileemd_14435_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_14435_half_map_2.map
Projections & Slices
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Sample components

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Entire : Homohexamer of L. lactis AbiK Y44F mutant

EntireName: Homohexamer of L. lactis AbiK Y44F mutant
Components
  • Complex: Homohexamer of L. lactis AbiK Y44F mutant
    • Protein or peptide: AbiK

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Supramolecule #1: Homohexamer of L. lactis AbiK Y44F mutant

SupramoleculeName: Homohexamer of L. lactis AbiK Y44F mutant / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Lactococcus lactis (lactic acid bacteria)
Molecular weightTheoretical: 420 KDa

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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: 71.617109 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GSMKKEFTEL YDFIFDPIFL VRYGYYDRSI KNKKMNTAKV ELDNEFGKSD SFYFKVFNME SFADYLRSHD LKTHFNGKKP LSTDPVYFN IPKNIEARRQ YKMPNLYSYM ALNYYICDNK KEFIEVFIDN KFSTSKFFNQ LNFDYPKTQE ITQTLLYGGI K KLHLDLSN ...String:
GSMKKEFTEL YDFIFDPIFL VRYGYYDRSI KNKKMNTAKV ELDNEFGKSD SFYFKVFNME SFADYLRSHD LKTHFNGKKP LSTDPVYFN IPKNIEARRQ YKMPNLYSYM ALNYYICDNK KEFIEVFIDN KFSTSKFFNQ LNFDYPKTQE ITQTLLYGGI K KLHLDLSN FYHTLYTHSI PWMIDGKSAS KQNRKKGFSN TLDTLITACQ YDETHGIPTG NLLSRIITEL YMCHFDKQME YK KFVYSRY VDDFIFPFTF ENEKQEFLNE FNLICRENNL IINDNKTKVD NFPFVDKSSK SDIFSFFENI TSTNSNDKWI KEI SNFIDY CVNEEHLGNK GAIKCIFPVI TNTLKQKKVD TKNIDNIFSK RNMVTNFNVF EKILDLSLKD SRLTNKFLTF FENI NEFGF SSLSASNIVK KYFSNNSKGL KEKIDHYRKN NFNQELYQIL LYMVVFEIDD LLNQEELLNL IDLNIDDYSL ILGTI LYLK NSSYKLEKLL KKIDQLFINT HANYDVKTSR MAEKLWLFRY FFYFLNCKNI FSQKEINSYC QSQNYNSGQN GYQTEL NWN YIKGQGKDLR ANNFFNELIV KEVWLISCGE NEDFKYLN

UniProtKB: AbiK

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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

Concentration1.74 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
400.0 mMNaClsodium chloride
20.0 mMC8H18N2O4SHepes
1.0 mMC4H10O2S2dithiothreitol
0.5 mMC10H16N2O8ethylenediaminetetraacetic acid
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2306347
Startup modelType of model: EMDB MAP
EMDB ID:

14220

Final reconstructionApplied symmetry - Point group: D3 (2x3 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.68 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.1) / Number images used: 50340
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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