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- PDB-6xb8: Adeno-Associated Virus Origin Binding Domain in complex with ssDNA -

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Basic information

Entry
Database: PDB / ID: 6xb8
TitleAdeno-Associated Virus Origin Binding Domain in complex with ssDNA
Components
  • DNA (5'-D(*GP*CP*TP*CP*TP*T)-3')
  • Protein Rep68
KeywordsVIRAL PROTEIN / AAV / Protein-DNA / AAA+ / SF3 / HUH
Function / homology
Function and homology information


symbiont-mediated arrest of host cell cycle during G2/M transition / symbiont entry into host cell via permeabilization of host membrane / viral DNA genome replication / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / endonuclease activity / DNA helicase / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity ...symbiont-mediated arrest of host cell cycle during G2/M transition / symbiont entry into host cell via permeabilization of host membrane / viral DNA genome replication / symbiont-mediated perturbation of host cell cycle G1/S transition checkpoint / endonuclease activity / DNA helicase / forked DNA-dependent helicase activity / single-stranded 3'-5' DNA helicase activity / four-way junction helicase activity / double-stranded DNA helicase activity / DNA replication / host cell nucleus / ATP hydrolysis activity / DNA binding / ATP binding / metal ion binding
Similarity search - Function
Rep protein catalytic-like / Rep protein catalytic domain like / : / Parvovirus (PV) NS1 nuclease (NS1-Nuc) domain profile. / Parvovirus non-structural protein 1, helicase domain / Parvovirus non-structural protein NS1 / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesAdeno-associated virus 2
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.3 Å
AuthorsEscalante, C.R. / Musayev, F.N.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM124204 United States
CitationJournal: Nucleic Acids Res / Year: 2020
Title: The Cryo-EM structure of AAV2 Rep68 in complex with ssDNA reveals a malleable AAA+ machine that can switch between oligomeric states.
Authors: Vishaka Santosh / Faik N Musayev / Rahul Jaiswal / Francisco Zárate-Pérez / Bram Vandewinkel / Caroline Dierckx / Molly Endicott / Kamyar Sharifi / Kelly Dryden / Els Henckaerts / Carlos R Escalante /
Abstract: The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and ...The adeno-associated virus (AAV) non-structural Rep proteins catalyze all the DNA transactions required for virus viability including, DNA replication, transcription regulation, genome packaging, and during the latent phase, site-specific integration. Rep proteins contain two multifunctional domains: an Origin Binding Domain (OBD) and a SF3 helicase domain (HD). Studies have shown that Rep proteins have a dynamic oligomeric behavior where the nature of the DNA substrate molecule modulates its oligomeric state. In the presence of ssDNA, Rep68 forms a large double-octameric ring complex. To understand the mechanisms underlying AAV Rep function, we investigated the cryo-EM and X-ray structures of Rep68-ssDNA complexes. Surprisingly, Rep68 generates hybrid ring structures where the OBD forms octameric rings while the HD forms heptamers. Moreover, the binding to ATPγS promotes a large conformational change in the entire AAA+ domain that leads the HD to form both heptamer and hexamers. The HD oligomerization is driven by an interdomain linker region that acts as a latch to 'catch' the neighboring HD subunit and is flexible enough to permit the formation of different stoichiometric ring structures. Overall, our studies show the structural basis of AAV Rep's structural flexibility required to fulfill its multifunctional role during the AAV life cycle.
History
DepositionJun 5, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 9, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 6, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein Rep68
B: Protein Rep68
C: Protein Rep68
D: Protein Rep68
E: DNA (5'-D(*GP*CP*TP*CP*TP*T)-3')
F: DNA (5'-D(*GP*CP*TP*CP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)99,42910
Polymers99,1676
Non-polymers2624
Water00
1
A: Protein Rep68
D: Protein Rep68
E: DNA (5'-D(*GP*CP*TP*CP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,7145
Polymers49,5843
Non-polymers1312
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Protein Rep68
C: Protein Rep68
F: DNA (5'-D(*GP*CP*TP*CP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,7145
Polymers49,5843
Non-polymers1312
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)170.180, 173.220, 173.450
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number23
Space group name H-MI222
Space group name HallI22

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Components

#1: Protein
Protein Rep68


Mass: 23904.166 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Adeno-associated virus 2 (isolate Srivastava/1982)
Strain: isolate Srivastava/1982 / Gene: Rep68 / Plasmid: PET-15b / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS / References: UniProt: P03132, DNA helicase
#2: DNA chain DNA (5'-D(*GP*CP*TP*CP*TP*T)-3')


Mass: 1775.190 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
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: 6.51 Å3/Da / Density % sol: 80.91 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 10mM Na cacodylate, pH 6.5, 30% PEG400 and 0.2M LiCl

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Data collection

DiffractionMean temperature: 110 K / Ambient temp details: Oxford Cryosystem series 700 / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS / Beamline: X6A / Wavelength: 0.9795 Å
DetectorType: ADSC QUANTUM 210 / Detector: CCD / Date: Mar 10, 2011
RadiationMonochromator: Si(111) channel cut monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 3.3→50 Å / Num. obs: 38830 / % possible obs: 99.9 % / Redundancy: 10 % / Biso Wilson estimate: 75.78 Å2 / Rmerge(I) obs: 0.125 / Net I/σ(I): 15.5
Reflection shellResolution: 3.3→3.45 Å / Redundancy: 9.3 % / Rmerge(I) obs: 0.594 / Mean I/σ(I) obs: 5.42 / Num. unique obs: 4683 / Χ2: 1.04 / % possible all: 99.9

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Processing

Software
NameVersionClassification
PHENIX1.18rc2_3794refinement
pointlessdata scaling
PDB_EXTRACT3.25data extraction
MOSFLMdata reduction
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5BYG

5byg


Resolution: 3.3→40.86 Å / SU ML: 0.3044 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 21.9213
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2176 1937 4.99 %
Rwork0.201 36893 -
obs0.2019 38830 99.95 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 72.12 Å2
Refinement stepCycle: LAST / Resolution: 3.3→40.86 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6493 192 4 0 6689
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00546875
X-RAY DIFFRACTIONf_angle_d0.829396
X-RAY DIFFRACTIONf_chiral_restr0.04881035
X-RAY DIFFRACTIONf_plane_restr0.0051181
X-RAY DIFFRACTIONf_dihedral_angle_d14.0791959
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.3-3.380.3613940.31072651X-RAY DIFFRACTION99.96
3.38-3.470.28761270.26042628X-RAY DIFFRACTION100
3.47-3.580.28131540.25542554X-RAY DIFFRACTION100
3.58-3.690.23261280.2412618X-RAY DIFFRACTION100
3.69-3.820.25581640.22132620X-RAY DIFFRACTION99.93
3.82-3.980.24881480.21442570X-RAY DIFFRACTION100
3.98-4.160.21921660.20282609X-RAY DIFFRACTION99.93
4.16-4.380.21861590.17612582X-RAY DIFFRACTION100
4.38-4.650.18191530.16112623X-RAY DIFFRACTION100
4.65-5.010.17541070.15532648X-RAY DIFFRACTION100
5.01-5.510.1721270.17262676X-RAY DIFFRACTION100
5.51-6.310.19821170.20352650X-RAY DIFFRACTION100
6.31-7.930.21891370.20712697X-RAY DIFFRACTION100
7.94-40.860.19671560.18922767X-RAY DIFFRACTION99.49
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.188786106711.243987785261.221329262894.055169479020.4895566351041.749788479620.0555824726538-0.247287820279-0.1331775859290.0916258405367-0.133714558899-0.1383987809940.094617466747-0.05142075635790.06851447536810.320357119413-0.05856523552360.06604219334260.4340517928190.08543472533380.29381684341758.7013568754-21.482675899845.4320485581
23.841347729370.4203553842141.113915391874.022654219070.00419445678166.727821820490.208100955678-0.121642406919-0.5982038308780.159880623213-0.212280144390.07213051525810.513599823751-0.8953429780560.01331132035050.519042218314-0.130990292677-0.004017319002010.521371766554-0.00310430944410.45345379251334.2448866476-20.650682222511.8283350249
32.1756910388-0.2057942477410.7890199850894.048560042230.8245064873483.84227576785-0.0225385522863-0.1191639257420.20971455347-0.162191834267-0.1400959078020.0944328479484-0.128358644599-0.2163675684030.1066660381450.2451224652320.0558346068050.05110792991070.460017744456-0.1240019303090.43004870819638.016044712621.27745622129.7566566163
44.71873072083-0.3570788277440.8659323355493.18268928153-0.3962899260352.95176184076-0.0467296506494-0.6718722984380.6812007007770.228212434777-0.0937904252060.181916502624-0.481876886246-0.07720028949430.09645640787110.4642582258560.0227103049750.01295073321040.585399952175-0.2076954929130.54351284796471.104881360120.170560099452.7540247222
51.26783733856-0.415008186537-1.190706924973.00280926585-0.09335960707831.594100623230.210979416157-1.205600543590.6311201627442.208167353370.326818726765-1.924321616680.2067766227290.492855072993-0.5965234668550.7740337258140.0304944369619-0.22855816810.820421038339-0.1465058338041.162928513666.0961338445-0.43781676967454.1517738605
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11(chain A and resseq 1:205)AA1 - 2051 - 205
22(chain B and resseq 1:200)BB1 - 2001 - 200
33(chain C and resseq 1:206)CC1 - 2061 - 206
44(chain D and resseq 1:196)DD1 - 1961 - 196
55(chain E and resseq 5:10)EE5 - 10

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