+Open data
-Basic information
Entry | Database: PDB / ID: 7las | |||||||||||||||
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Title | Cryo-EM structure of PCV2 Replicase bound to ssDNA | |||||||||||||||
Components |
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Keywords | REPLICATION / HYDROLASE/DNA / Rolling circle replication / CRESS-DNA virus / SF3 helicase / Porcine Circovirus / PCV2 / HYDROLASE-DNA complex | |||||||||||||||
Function / homology | Function and homology information endodeoxyribonuclease activity, producing 5'-phosphomonoesters / nucleotidyltransferase activity / DNA replication / RNA helicase activity / nucleotide binding / DNA binding / RNA binding / metal ion binding Similarity search - Function | |||||||||||||||
Biological species | Porcine circovirus 2 Escherichia coli (E. coli) | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å | |||||||||||||||
Authors | Khayat, R. | |||||||||||||||
Funding support | United States, 4items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 7las.cif.gz | 391.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7las.ent.gz | 317.1 KB | Display | PDB format |
PDBx/mmJSON format | 7las.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7las_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 7las_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 7las_validation.xml.gz | 37.4 KB | Display | |
Data in CIF | 7las_validation.cif.gz | 54.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/la/7las ftp://data.pdbj.org/pub/pdb/validation_reports/la/7las | HTTPS FTP |
-Related structure data
Related structure data | 23250MC 7larC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 35876.500 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Porcine circovirus 2 / Gene: rep, ORF1 / Production host: Escherichia coli (E. coli) References: UniProt: Q6TC59, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement #2: DNA chain | | Mass: 4236.762 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) #3: DNA chain | | Mass: 3069.030 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Porcine circovirus 2 / Production host: Escherichia coli (E. coli) #4: Chemical | ChemComp-ADP / #5: Chemical | ChemComp-MG / Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: PCV2 Replicase in complex with ssDNA / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT |
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Molecular weight | Value: 0.216 MDa / Experimental value: NO |
Source (natural) | Organism: Porcine circovirus 2 |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 8.2 Details: 20 mM HEPES, pH 8.2, 500 mM NaCl, 0.2 mM TCEP, 10 mM MgCl2 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Sample was monodisperse according to size exclusion chromatography |
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K 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. |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Average exposure time: 6 sec. / Electron dose: 71.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 |
-Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43282 / Details: Global resolution according to 3DFSC / Symmetry type: POINT | ||||||||||||||||||||||||||||
Atomic model building | B value: 65.3 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient | ||||||||||||||||||||||||||||
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