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- EMDB-35671: Structure of Full-Length AsfvPrimPol in Complex-Form -

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Basic information

Entry
Database: EMDB / ID: EMD-35671
TitleStructure of Full-Length AsfvPrimPol in Complex-Form
Map data
Sample
  • Complex: Full-Length AsfvPrimPol in complex with DNA and AMPPNP
    • Complex: Full-Length AsfvPrimPol
      • Protein or peptide: Putative primase C962R
    • Complex: DNA
      • DNA: DNA (32-MER)
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
Keywordspolymerase / primase / PrimPol / Helicase / DNA BINDING PROTEIN
Function / homology
Function and homology information


hydrolase activity, acting on acid anhydrides / helicase activity / DNA replication / ATP binding / metal ion binding
Similarity search - Function
Primase, C-terminal 2 / Primase C terminal 2 (PriCT-2) / Domain of unknown function DUF5906 / Family of unknown function (DUF5906) / Bacteriophage/plasmid primase, P4, C-terminal / D5 N terminal like / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Putative primase C962R
Similarity search - Component
Biological speciesAfrican swine fever virus BA71V / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsShao ZW / Su SC / Gan JH
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structures and implications of the C962R protein of African swine fever virus.
Authors: Zhiwei Shao / Shichen Su / Jie Yang / Weizhen Zhang / Yanqing Gao / Xin Zhao / Yixi Zhang / Qiyuan Shao / Chulei Cao / Huili Li / Hehua Liu / Jinru Zhang / Jinzhong Lin / Jinbiao Ma / Jianhua Gan /
Abstract: African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. Although it has been extensively studied in the past, no vaccine or other useful treatment against ASFV is ...African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. Although it has been extensively studied in the past, no vaccine or other useful treatment against ASFV is available. The genome of ASFV encodes more than 170 proteins, but the structures and functions for the majority of the proteins remain elusive, which hindered our understanding on the life cycle of ASFV and the development of ASFV-specific inhibitors. Here, we report the structural and biochemical studies of the highly conserved C962R protein of ASFV, showing that C962R is a multidomain protein. The N-terminal AEP domain is responsible for the DNA polymerization activity, whereas the DNA unwinding activity is catalyzed by the central SF3 helicase domain. The middle PriCT2 and D5_N domains and the C-terminal Tail domain all contribute to the DNA unwinding activity of C962R. C962R preferentially works on forked DNA, and likely functions in Base-excision repair (BER) or other repair pathway in ASFV. Although it is not essential for the replication of ASFV, C962R can serve as a model and provide mechanistic insight into the replicative primase proteins from many other species, such as nitratiruptor phage NrS-1, vaccinia virus (VACV) and other viruses.
History
DepositionMar 16, 2023-
Header (metadata) releaseJul 26, 2023-
Map releaseJul 26, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35671.png

Downloads & links

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Map

FileDownload / File: emd_35671.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.1 Å
Density
Contour LevelBy AUTHOR: 0.002
Minimum - Maximum-0.018027961 - 0.047165
Average (Standard dev.)0.00015000174 (±0.0014758043)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_35671_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_35671_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Full-Length AsfvPrimPol in complex with DNA and AMPPNP

EntireName: Full-Length AsfvPrimPol in complex with DNA and AMPPNP
Components
  • Complex: Full-Length AsfvPrimPol in complex with DNA and AMPPNP
    • Complex: Full-Length AsfvPrimPol
      • Protein or peptide: Putative primase C962R
    • Complex: DNA
      • DNA: DNA (32-MER)
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Full-Length AsfvPrimPol in complex with DNA and AMPPNP

SupramoleculeName: Full-Length AsfvPrimPol in complex with DNA and AMPPNP
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: ASFV ORF C962R
Molecular weightTheoretical: 660 KDa

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Supramolecule #2: Full-Length AsfvPrimPol

SupramoleculeName: Full-Length AsfvPrimPol / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: African swine fever virus BA71V

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2

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Macromolecule #1: Putative primase C962R

MacromoleculeName: Putative primase C962R / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: African swine fever virus BA71V
Molecular weightTheoretical: 111.706273 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSMREESWEE HDTIQLTAQR KYLAEVQALE TLLARELSVF LTEPGSKKTN IINRITGKTY ALPSTELLRF YEHLEQCRKQ GALMYFLER QGTYSGLMLD YDLKLNTNAA PSLESSVLSR LCHRIFVHIK NSSVLPEGSH KIHFFFTLKP EAVQGKYGFH V LIPGLKMA ...String:
GSMREESWEE HDTIQLTAQR KYLAEVQALE TLLARELSVF LTEPGSKKTN IINRITGKTY ALPSTELLRF YEHLEQCRKQ GALMYFLER QGTYSGLMLD YDLKLNTNAA PSLESSVLSR LCHRIFVHIK NSSVLPEGSH KIHFFFTLKP EAVQGKYGFH V LIPGLKMA ASTKKSIIAS LQHDATVQKI LHEQGVANPE SCLDPHSASV PSLLYGSSKL NHRPYQLKTG FELVFDSSDP DY IPIHQIK NIESYNLVSE LSLTNEQGSL VRPVYCAADI AAEKEEEIPA DDHSLSILML HDPEARYLHK ILNLLPPEYY VEY PLWSNV VFALANTSAN YRPLAEWFSQ KCPEKWNTGG KEKLEKLWND ASRHTEKKIT KRSIMYWAHK HAPQQYKEIV EQGY FSILA EYVYSYNGTL EHYMIAKVIY AMMGNKFVVD VDSNGKYVWF EFVLPGQPMN QGEIWKWRKE VNPDELHIYI SENFS RVMD RITEHIKYHL SQPHETNILN YYKKLLKAFE RSKSKIFNDS FKKGVIRQAE FLFRQRSFIQ TLDTNPYLLG VGNGVL SIE TIPAKLINHF HEHPIHQYTH ICYEPFNPEN PWTKLLLNAL QDIIPELDAR LWIMFYLSTA IFRGLKEALM LLWLGGG CN GKTFLMRLVA MVLGDHYASK LNISLLTSYR ETAEKPNSAF MRLKGRGYGY FEETNKSEIL NTSRLKEMVN PGDVTARE L NQKQESFQMT ATMVAASNYN FIIDTTDHGT WRRLRHYRSK VKFCHNPDPN NSYEKKEDPR FIHEYIMDPN CQNAFFSIL VYFWEKLQKE YNGQIKKVFC PTIESETEAY RKSQDTLHRF ITERVVESPS AETVYNLSEV VTAYAEWYNA NINVKRHIAL ELSQELENS VLEKYLQWSP NKTRILKGCR ILHKFETLQP GESYIGVSST GTLLNTPICE PKNKWWEWSP NPSAPPEKEA S APTP

UniProtKB: Putative primase C962R

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Macromolecule #2: DNA (32-MER)

MacromoleculeName: DNA (32-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.689213 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)

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Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 6 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration1.2 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 64000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionNumber classes used: 5 / Resolution.type: BY AUTHOR / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 589492
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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