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- PDB-6t7y: Structure of PCNA bound to cPIP motif of DP2 from P. abyssi -

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Basic information

Entry
Database: PDB / ID: 6t7y
TitleStructure of PCNA bound to cPIP motif of DP2 from P. abyssi
Components
  • DNA polymerase sliding clampDNA clamp
  • cPIP motif from the DP2 large subunit of PolD
KeywordsREPLICATION / PCNA / DP2 / PolD / PIP-box
Function / homology
Function and homology information


exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / intein-mediated protein splicing / DNA catabolic process / DNA polymerase processivity factor activity / regulation of DNA replication / DNA-templated DNA replication / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding
Similarity search - Function
DNA polymerase II large subunit DP2 / DNA polymerase II large subunit DP2, N-terminal / DNA polymerase II large subunit DP2 / Intein splicing domain / Box / Proliferating Cell Nuclear Antigen / Proliferating Cell Nuclear Antigen - #10 / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / Intein N-terminal splicing region ...DNA polymerase II large subunit DP2 / DNA polymerase II large subunit DP2, N-terminal / DNA polymerase II large subunit DP2 / Intein splicing domain / Box / Proliferating Cell Nuclear Antigen / Proliferating Cell Nuclear Antigen - #10 / Intein C-terminal splicing region / Intein C-terminal splicing motif profile. / Intein N-terminal splicing region / Intein N-terminal splicing motif profile. / Hint domain N-terminal / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / Hint (Hedgehog/Intein) domain N-terminal region / Proliferating cell nuclear antigen, PCNA / Proliferating cell nuclear antigen, PCNA, N-terminal / Proliferating cell nuclear antigen, PCNA, C-terminal / Proliferating cell nuclear antigen, N-terminal domain / Proliferating cell nuclear antigen, C-terminal domain / Hint domain superfamily / : / Alpha Beta
Similarity search - Domain/homology
DNA polymerase sliding clamp / DNA polymerase II large subunit
Similarity search - Component
Biological speciesPyrococcus abyssi (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.7 Å
AuthorsMadru, C. / Raia, P. / Hugonneau Beaufet, I. / Delarue, M. / Carroni, M. / Sauguet, L.
Funding support France, 1items
OrganizationGrant numberCountry
French National Research AgencyANR-17-CE11-0005-01 France
CitationJournal: Nat Commun / Year: 2020
Title: Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA.
Authors: Clément Madru / Ghislaine Henneke / Pierre Raia / Inès Hugonneau-Beaufet / Gérard Pehau-Arnaudet / Patrick England / Erik Lindahl / Marc Delarue / Marta Carroni / Ludovic Sauguet /
Abstract: Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced ...Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography, protein-protein interaction measurements, and activity assays - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication.
History
DepositionOct 23, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 4, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 1, 2020Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Apr 8, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Jan 24, 2024Group: 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
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DNA polymerase sliding clamp
B: cPIP motif from the DP2 large subunit of PolD


Theoretical massNumber of molelcules
Total (without water)30,9582
Polymers30,9582
Non-polymers00
Water25214
1
A: DNA polymerase sliding clamp
B: cPIP motif from the DP2 large subunit of PolD

A: DNA polymerase sliding clamp
B: cPIP motif from the DP2 large subunit of PolD

A: DNA polymerase sliding clamp
B: cPIP motif from the DP2 large subunit of PolD


Theoretical massNumber of molelcules
Total (without water)92,8736
Polymers92,8736
Non-polymers00
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area7170 Å2
ΔGint-45 kcal/mol
Surface area33270 Å2
MethodPISA
Unit cell
Length a, b, c (Å)140.736, 140.736, 38.521
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number146
Space group name H-MH3

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Components

#1: Protein DNA polymerase sliding clamp / DNA clamp / Proliferating cell nuclear antigen homolog / PCNA


Mass: 29471.764 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (strain GE5 / Orsay) (archaea)
Strain: GE5 / Orsay / Gene: pcn, PYRAB13790, PAB1465 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9UYX8
#2: Protein/peptide cPIP motif from the DP2 large subunit of PolD


Mass: 1485.746 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi (archaea) / Production host: synthetic construct (others)
References: UniProt: Q9V2F4*PLUS, DNA-directed DNA polymerase
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 14 / Source method: isolated from a natural source / Formula: H2O

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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: 2.37 Å3/Da / Density % sol: 48.13 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.1 / Details: 30% PEG 400 0.2 M MgCl2 0.1M BIS-TRIS pH7.1

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.9793 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Nov 11, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 2.7→70.37 Å / Num. obs: 6983 / % possible obs: 89.5 % / Redundancy: 5.2 % / CC1/2: 0.999 / Net I/σ(I): 15.8
Reflection shellResolution: 2.701→2.863 Å / Num. unique obs: 436 / CC1/2: 0.21

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHASERphasing
REFMAC5.8.0158refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
STARANISOdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5auj

5auj


Resolution: 2.7→70.37 Å / Cor.coef. Fo:Fc: 0.966 / Cor.coef. Fo:Fc free: 0.937 / SU B: 50.167 / SU ML: 0.427 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.424
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2621 660 9.5 %RANDOM
Rwork0.1857 ---
obs0.1929 6323 89.4 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 218.27 Å2 / Biso mean: 134.05 Å2 / Biso min: 84.66 Å2
Baniso -1Baniso -2Baniso -3
1-0.54 Å20.27 Å2-0 Å2
2--0.54 Å2-0 Å2
3----1.76 Å2
Refinement stepCycle: final / Resolution: 2.7→70.37 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1952 0 0 14 1966
Biso mean---112.02 -
Num. residues----247
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.020.0191975
X-RAY DIFFRACTIONr_bond_other_d0.0070.021952
X-RAY DIFFRACTIONr_angle_refined_deg2.1381.9972650
X-RAY DIFFRACTIONr_angle_other_deg1.1834533
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.5935244
X-RAY DIFFRACTIONr_dihedral_angle_2_deg37.63525.28787
X-RAY DIFFRACTIONr_dihedral_angle_3_deg19.58515394
X-RAY DIFFRACTIONr_dihedral_angle_4_deg18.6771511
X-RAY DIFFRACTIONr_chiral_restr0.1280.2310
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.022134
X-RAY DIFFRACTIONr_gen_planes_other0.0110.02369
LS refinement shellResolution: 2.701→2.771 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.379 10 -
Rwork0.279 49 -
all-59 -
obs--10.05 %
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)
14.9011-1.0620.0850.8941-0.01120.01110.0346-0.2556-0.27170.1025-0.03370.01890.041-0.0165-0.00090.1802-0.0631-0.00460.03940.02450.095-3.4653-24.4055-10.7278
210.38051.9453-4.15051.5922-2.7574.85180.47281.0612-0.10030.02520.08530.355-0.0741-0.2512-0.55810.1311-0.02920.01250.1844-0.07340.12726.6259-33.9656-26.4644
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A2 - 247
2X-RAY DIFFRACTION2B1256 - 1264

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