[English] 日本語
Yorodumi
- PDB-7las: Cryo-EM structure of PCV2 Replicase bound to ssDNA -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7las
TitleCryo-EM structure of PCV2 Replicase bound to ssDNA
Components
  • ATP-dependent helicase Rep
  • DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*A)-3')
  • DNA (5'-D(P*TP*TP*TP*TP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')
KeywordsREPLICATION / 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 / 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
ADENOSINE-5'-DIPHOSPHATE / DNA / DNA (> 10) / Replication-associated protein
Similarity search - Component
Biological speciesPorcine circovirus 2
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsKhayat, R.
Funding support United States, 4items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 25, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 15, 2021Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-23250
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: ATP-dependent helicase Rep
B: ATP-dependent helicase Rep
C: ATP-dependent helicase Rep
D: ATP-dependent helicase Rep
E: ATP-dependent helicase Rep
F: ATP-dependent helicase Rep
G: DNA (5'-D(P*TP*TP*TP*TP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')
H: DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)224,37116
Polymers222,5658
Non-polymers1,8068
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

-
Components

#1: Protein
ATP-dependent helicase Rep / RepP / Replication-associated protein / PCV2 Replicase


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 DNA (5'-D(P*TP*TP*TP*TP*TP*CP*GP*AP*TP*CP*GP*AP*TP*C)-3')


Mass: 4236.762 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli)
#3: DNA chain DNA (5'-D(P*GP*AP*TP*CP*GP*AP*TP*CP*GP*A)-3')


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 / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
Sample preparation

ComponentName: PCV2 Replicase in complex with ssDNA / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.216 MDa / Experimental value: NO
Source (natural)Organism: Porcine circovirus 2
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8.2
Details: 20 mM HEPES, pH 8.2, 500 mM NaCl, 0.2 mM TCEP, 10 mM MgCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was monodisperse according to size exclusion chromatography
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD
Image recordingAverage 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

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4CTF correction
7Coot0.8.9.2model fitting
9PHENIX1.18.2-3874model refinement
10cryoSPARC2.14initial Euler assignment
11cryoSPARC2.14final Euler assignment
13cryoSPARC2.143D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 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 buildingB value: 65.3 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0099712
ELECTRON MICROSCOPYf_angle_d1.37413395
ELECTRON MICROSCOPYf_dihedral_angle_d21.3593477
ELECTRON MICROSCOPYf_chiral_restr0.061480
ELECTRON MICROSCOPYf_plane_restr0.0081577

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbjlvh1.pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more