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- PDB-6wdz: Porcine circovirus 2 Rep domain complexed with a single-stranded ... -

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Basic information

Entry
Database: PDB / ID: 6wdz
TitlePorcine circovirus 2 Rep domain complexed with a single-stranded DNA 10-mer comprising the cleavage site
Components
  • ATP-dependent helicase Rep
  • DNA (5'-D(*TP*AP*GP*TP*AP*TP*TP*AP*CP*C)-3')
KeywordsREPLICATION/DNA / HUH-tag / HUH motif / Rep domain / viral protein / single stranded DNA / ssDNA / ssDNA binding / REPLICATION / DNA BINDING PROTEIN / REPLICATION-DNA complex
Function / homology
Function and homology information


DNA endonuclease activity, producing 5'-phosphomonoesters / rolling circle single-stranded viral DNA replication / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / nucleotidyltransferase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / endonuclease activity / DNA replication / RNA helicase activity / nucleotide binding ...DNA endonuclease activity, producing 5'-phosphomonoesters / rolling circle single-stranded viral DNA replication / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides / nucleotidyltransferase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / endonuclease activity / DNA replication / RNA helicase activity / nucleotide binding / host cell nucleus / ATP hydrolysis activity / DNA binding / RNA binding / ATP binding / metal ion 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
: / DNA / Replication-associated protein / Replication-associated protein
Similarity search - Component
Biological speciesPorcine circovirus 2
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.03 Å
AuthorsLitzau, L.A. / Tompkins, K. / Shi, K. / Nelson, A. / Evans III, R.L. / Gordon, W.R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM119483 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118047 United States
CitationJournal: Nucleic Acids Res. / Year: 2021
Title: Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering.
Authors: Tompkins, K.J. / Houtti, M. / Litzau, L.A. / Aird, E.J. / Everett, B.A. / Nelson, A.T. / Pornschloegl, L. / Limon-Swanson, L.K. / Evans, R.L. / Evans, K. / Shi, K. / Aihara, H. / Gordon, W.R.
History
DepositionApr 1, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 17, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Oct 18, 2023Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)52,14512
Polymers51,8567
Non-polymers2895
Water3,495194
1
C: DNA (5'-D(*TP*AP*GP*TP*AP*TP*TP*AP*CP*C)-3')
A: ATP-dependent helicase Rep
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,3343
Polymers16,2792
Non-polymers551
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2040 Å2
ΔGint-6 kcal/mol
Surface area6260 Å2
MethodPISA
2
D: ATP-dependent helicase Rep
F: DNA (5'-D(*TP*AP*GP*TP*AP*TP*TP*AP*CP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,3343
Polymers16,2792
Non-polymers551
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2020 Å2
ΔGint-8 kcal/mol
Surface area6410 Å2
MethodPISA
3
I: DNA (5'-D(*TP*AP*GP*TP*AP*TP*TP*AP*CP*C)-3')
G: ATP-dependent helicase Rep
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,4585
Polymers16,2792
Non-polymers1793
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2400 Å2
ΔGint-3 kcal/mol
Surface area6350 Å2
MethodPISA
4
J: DNA (5'-D(*TP*AP*GP*TP*AP*TP*TP*AP*CP*C)-3')


Theoretical massNumber of molelcules
Total (without water)3,0191
Polymers3,0191
Non-polymers00
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area1260 Å2
MethodPISA
Unit cell
Length a, b, c (Å)99.527, 99.527, 73.695
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number172
Space group name H-MP64
Components on special symmetry positions
IDModelComponents
11I-404-

HOH

21G-327-

HOH

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Components

#1: DNA chain
DNA (5'-D(*TP*AP*GP*TP*AP*TP*TP*AP*CP*C)-3')


Mass: 3019.004 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) Porcine circovirus 2
#2: Protein ATP-dependent helicase Rep / RepP / Replication-associated protein


Mass: 13259.991 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Porcine circovirus 2 / Gene: Rep / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A060L113, UniProt: Q8BB16*PLUS
#3: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mn
#4: Chemical ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 194 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.14 Å3/Da / Density % sol: 42.59 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop
Details: 0.1M Ammonium acetate, 25% Polyethylene glycol 3,350, 0.1M Bis-Tris pH 7 soaked in 25% glycerol for cryoprotectant

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9791 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Mar 5, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 2.03→43.1 Å / Num. obs: 26261 / % possible obs: 97.52 % / Redundancy: 2.9 % / Biso Wilson estimate: 25.87 Å2 / CC1/2: 0.996 / CC star: 0.999 / Rmerge(I) obs: 0.06745 / Rpim(I) all: 0.04609 / Rrim(I) all: 0.08209 / Net I/σ(I): 12.49
Reflection shellResolution: 2.03→2.103 Å / Rmerge(I) obs: 0.3619 / Mean I/σ(I) obs: 3.63 / Num. unique obs: 2625 / CC1/2: 0.812 / CC star: 0.947 / Rpim(I) all: 0.2475 / Rrim(I) all: 0.4406 / % possible all: 98.17

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Processing

Software
NameVersionClassification
PHENIX1.18.2_3874refinement
PDB_EXTRACT3.27data extraction
HKL-3000data reduction
HKL-3000data scaling
PHENIXphasing
Cootmodel building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5xor + modeled 10-mer

5xor


Resolution: 2.03→43.1 Å / SU ML: 0.29 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 31.82 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2802 2323 4.71 %
Rwork0.2292 46955 -
obs0.2317 26261 93 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 97.07 Å2 / Biso mean: 32.539 Å2 / Biso min: 11.52 Å2
Refinement stepCycle: final / Resolution: 2.03→43.1 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2447 679 11 194 3331
Biso mean--33.51 31.84 -
Num. residues----336
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 17

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.03-2.070.29051000.27252814291494
2.07-2.120.29981220.27142753287593
2.12-2.170.33571250.25412750287592
2.17-2.220.30231060.26912576268286
2.22-2.280.31341740.27172666284093
2.28-2.350.3041600.26562771293195
2.35-2.420.39581290.24762901303096
2.42-2.510.40721180.27562819293795
2.51-2.610.28891330.26612835296896
2.61-2.730.32431690.24662802297195
2.73-2.870.36691260.25592803292995
2.87-3.050.2871460.25082738288493
3.05-3.290.27011210.21862576269787
3.29-3.620.27781200.18962878299897
3.62-4.140.21441630.18762851301496
4.14-5.220.22791370.1982827296496
5.22-43.10.261740.22752595276989

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