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- PDB-6t7x: Crystal structure of PCNA from P. abyssi -

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Basic information

Entry
Database: PDB / ID: 6t7x
TitleCrystal structure of PCNA from P. abyssi
ComponentsDNA polymerase sliding clampDNA clamp
KeywordsREPLICATION / PCNA / Pyrococcus abyssi / sliding-clamp
Function / homology
Function and homology information


DNA polymerase processivity factor activity / regulation of DNA replication / DNA replication / DNA binding
Similarity search - Function
Box / Proliferating Cell Nuclear Antigen / Proliferating Cell Nuclear Antigen - #10 / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / 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 ...Box / Proliferating Cell Nuclear Antigen / Proliferating Cell Nuclear Antigen - #10 / Proliferating cell nuclear antigen, PCNA, conserved site / Proliferating cell nuclear antigen signature 1. / 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 / : / Alpha Beta
Similarity search - Domain/homology
DNA polymerase sliding clamp
Similarity search - Component
Biological speciesPyrococcus abyssi (archaea)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.3 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


Theoretical massNumber of molelcules
Total (without water)29,4721
Polymers29,4721
Non-polymers00
Water1,00956
1
A: DNA polymerase sliding clamp

A: DNA polymerase sliding clamp

A: DNA polymerase sliding clamp


Theoretical massNumber of molelcules
Total (without water)88,4153
Polymers88,4153
Non-polymers00
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_665-y+1,x-y+1,z1
crystal symmetry operation3_565-x+y,-x+1,z1
Buried area4040 Å2
ΔGint-11 kcal/mol
Surface area32110 Å2
MethodPISA
Unit cell
Length a, b, c (Å)91.902, 91.902, 64.188
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number173
Space group name H-MP63

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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: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 56 / 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: 2.66 Å3/Da / Density % sol: 53.67 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 5.5 / Details: 20% PEG 400 0.2 M CaCl2 0.1 M MES pH 5.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 2 / Wavelength: 0.9786 Å
DetectorType: DECTRIS EIGER2 X 9M / Detector: PIXEL / Date: Feb 21, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9786 Å / Relative weight: 1
ReflectionResolution: 2.3→20 Å / Num. obs: 13821 / % possible obs: 99.78 % / Redundancy: 14 % / CC1/2: 0.99 / Net I/σ(I): 14.02
Reflection shellResolution: 2.3→2.48 Å / Num. unique obs: 1387 / CC1/2: 0.32

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
PDB_EXTRACT3.25data extraction
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5auj

5auj


Resolution: 2.3→20 Å / Cor.coef. Fo:Fc: 0.95 / Cor.coef. Fo:Fc free: 0.931 / SU R Cruickshank DPI: 0.262 / Cross valid method: THROUGHOUT / σ(F): 0 / SU R Blow DPI: 0.256 / SU Rfree Blow DPI: 0.204 / SU Rfree Cruickshank DPI: 0.209
RfactorNum. reflection% reflectionSelection details
Rfree0.243 672 4.86 %RANDOM
Rwork0.202 ---
obs0.204 13820 100 %-
Displacement parametersBiso max: 159.36 Å2 / Biso mean: 77.23 Å2 / Biso min: 48.12 Å2
Baniso -1Baniso -2Baniso -3
1-1.9894 Å20 Å20 Å2
2--1.9894 Å20 Å2
3----3.9789 Å2
Refine analyzeLuzzati coordinate error obs: 0.33 Å
Refinement stepCycle: final / Resolution: 2.3→20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1877 0 0 56 1933
Biso mean---76.99 -
Num. residues----239
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d714SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes54HARMONIC2
X-RAY DIFFRACTIONt_gen_planes268HARMONIC5
X-RAY DIFFRACTIONt_it1909HARMONIC20
X-RAY DIFFRACTIONt_nbd
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion255SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact2091SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d1909HARMONIC20.01
X-RAY DIFFRACTIONt_angle_deg2564HARMONIC21.24
X-RAY DIFFRACTIONt_omega_torsion3.18
X-RAY DIFFRACTIONt_other_torsion19.43
LS refinement shellResolution: 2.3→2.48 Å / Rfactor Rfree error: 0 / Total num. of bins used: 7
RfactorNum. reflection% reflection
Rfree0.2326 149 5.23 %
Rwork0.2221 2700 -
all0.2227 2849 -
obs--99.96 %
Refinement TLS params.Method: refined / Origin x: -23.5583 Å / Origin y: 44.6413 Å / Origin z: -1.0469 Å
111213212223313233
T-0.1956 Å2-0.0707 Å20.0023 Å2--0.0845 Å20.079 Å2---0.1771 Å2
L2.8785 °2-1.6158 °2-0.3745 °2-4.8311 °21.0278 °2--1.4298 °2
S0.1571 Å °0.145 Å °0.0238 Å °-0.2091 Å °-0.053 Å °0.1686 Å °-0.0479 Å °-0.2435 Å °-0.1041 Å °
Refinement TLS groupSelection details: { A|* }

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