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- EMDB-23249: Cryo-EM structure of PCV2 Replicase bound to ssDNA -

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Basic information

Entry
Database: EMDB / ID: EMD-23249
TitleCryo-EM structure of PCV2 Replicase bound to ssDNA
Map dataCryo-EM map of PCV2 Replicase at 3.8 Angstrom resolution
Sample
  • Complex: PCV2 Replicase in complex with ssDNA
    • Protein or peptide: ATP-dependent helicase Rep
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*T)-3')
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsRolling circle replication / CRESS-DNA virus / SF3 helicase / Porcine Circovirus / PCV2 / REPLICATION / HYDROLASE-DNA complex
Function / homology
Function and homology information


endodeoxyribonuclease activity, producing 5'-phosphomonoesters / nucleotidyltransferase activity / DNA replication / RNA helicase activity / RNA binding
Similarity search - Function
Putative viral replication protein / : / CRESS-DNA virus replication initiator protein (Rep) endonuclease domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Replication-associated protein
Similarity search - Component
Biological speciesPorcine circovirus 2 / unidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsKhayat R
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)SC1AI114843 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5G12MD007603-30 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
Simons Foundation349247 United States
CitationJournal: mBio / Year: 2021
Title: Mechanism of DNA Interaction and Translocation by the Replicase of a Circular Rep-Encoding Single-Stranded DNA Virus.
Authors: Elvira Tarasova / Sonali Dhindwal / Matthew Popp / Sakeenah Hussain / Reza Khayat /
Abstract: Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses infect members from all three domains of life (, , and ). The replicase (Rep) from these viruses is responsible for initiating rolling ...Circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses infect members from all three domains of life (, , and ). The replicase (Rep) from these viruses is responsible for initiating rolling circle replication (RCR) of their genomes. Rep is a multifunctional enzyme responsible for nicking and ligating ssDNA and unwinding double-stranded DNA (dsDNA). We report the structure of porcine circovirus 2 (PCV2) Rep bound to ADP and single-stranded DNA (ssDNA), and Rep bound to ADP and double-stranded DNA (dsDNA). The structures demonstrate Rep to be a member of the superfamily 3 (SF3) of ATPases Associated with diverse cellular Activities (AAA) superfamily clade 4. At the Rep N terminus is an endonuclease domain () that is responsible for ssDNA nicking and ligation, in the center of Rep is an oligomerization domain () responsible for hexamerization, and at the C terminus is an ATPase domain () responsible for ssDNA/dsDNA interaction and translocation. The Rep binds to DNA such that the faces the replication fork. The six spiral around the DNA to interact with the backbone phosphates from four consecutive nucleotides. Three of the six are able to sense the backbone phosphates from the second strand of dsDNA. Heterogeneous classification of the data demonstrates the and to be mobile. Furthermore, we demonstrate that Rep exhibits basal nucleoside triphosphatase (NTPase) activity. CRESS-DNA viruses encompass a significant portion of the biosphere's virome. However, little is known about the structure of Rep responsible for initiating the RCR of CRESS-DNA viruses. We use cryo-electron microscopy (cryo-EM) to determine the structure of PCV2 Rep in complex with ADP and ss/dsDNA. Our structures demonstrate CRESS-DNA Reps to be SF3 members (clade 4) of the AAA+ superfamily. The structures further provide the mechanism by which CRESS-DNA virus Reps recognize DNA and translocate DNA for genome replication. Our structures also demonstrate the and of PCV2 Rep to be highly mobile. We propose the mobile nature of these domains to be necessary for proper functioning of Reps. We further demonstrate that Reps exhibit basal NTPase activity. Our studies also provide initial insight into the mechanism of RCR.
History
DepositionJan 6, 2021-
Header (metadata) releaseAug 25, 2021-
Map releaseAug 25, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.65
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.65
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lar
  • Surface level: 0.65
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23249.png

Downloads & links

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Map

FileDownload / File: emd_23249.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of PCV2 Replicase at 3.8 Angstrom resolution
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.65 / Movie #1: 0.65
Minimum - Maximum-2.6459107 - 4.0768757
Average (Standard dev.)0.007404869 (±0.17653611)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 212.992 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8320.8320.832
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z212.992212.992212.992
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.6464.0770.007

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Supplemental data

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Sample components

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Entire : PCV2 Replicase in complex with ssDNA

EntireName: PCV2 Replicase in complex with ssDNA
Components
  • Complex: PCV2 Replicase in complex with ssDNA
    • Protein or peptide: ATP-dependent helicase Rep
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*TP*T)-3')
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: PCV2 Replicase in complex with ssDNA

SupramoleculeName: PCV2 Replicase in complex with ssDNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Porcine circovirus 2
Molecular weightTheoretical: 216 KDa

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Macromolecule #1: ATP-dependent helicase Rep

MacromoleculeName: ATP-dependent helicase Rep / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Porcine circovirus 2
Molecular weightTheoretical: 35.8765 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPSKKNGRSG PQPHKRWVFT LNNPSEDERK KIRELPISLF DYFIVGEEGN EEGRTPHLQG FANFVKKQTF NKVKWYFGAR CHIEKAKGT DQQNKEYCSK EGNLLMECGA PRSQGQRSDL STAVSTLLES GSLVTVAEQH PVTFVRNFRG LAELLKVSGK M QKRDWKTN ...String:
MPSKKNGRSG PQPHKRWVFT LNNPSEDERK KIRELPISLF DYFIVGEEGN EEGRTPHLQG FANFVKKQTF NKVKWYFGAR CHIEKAKGT DQQNKEYCSK EGNLLMECGA PRSQGQRSDL STAVSTLLES GSLVTVAEQH PVTFVRNFRG LAELLKVSGK M QKRDWKTN VHVIVGPPGC GKSKWAANFA DPETTYWKPP RNKWWDGYHG EEVVVIDDFY GWLPWDDLLR LCDRYPLTVE TK GGTVPFL ARSILITSNQ TPLEWYSSTA VPAVEALYRR ITSLVFWKNA TEQSTEEGGQ FVTLSPPCPE FPYEINY

UniProtKB: Replication-associated protein

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Macromolecule #2: DNA (5'-D(P*TP*TP*TP*TP*TP*T)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*TP*T)-3') / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 1.780199 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*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

BufferpH: 8.2
Details: 20 mM HEPES, pH 8.2, 500 mM NaCl, 0.2 mM TCEP, 10 mM MgCl2
GridModel: UltrAuFoil / Material: GOLD / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 4 uL samples were applied to the grids for 3 seconds and blotted using a blot force of 2, a blot time of 4 seconds, and a drain time of 0 seconds..
DetailsSample was monodisperse according to size exclusion chromatography

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average exposure time: 6.0 sec. / Average electron dose: 71.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Ab initio determined by cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14) / Details: Resolution is based on 3DFSC global value / Number images used: 169830
Initial angle assignmentType: COMMON LINE / Software - Name: cryoSPARC (ver. 2.14)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 2.14) / Details: Non uniform refinement
Final 3D classificationNumber classes: 4

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 65.3 / Target criteria: Correlation coefficient
Output model

PDB-7lar:
Cryo-EM structure of PCV2 Replicase bound to ssDNA

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