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- PDB-8aas: Crystal structure of the Pyrococcus abyssi RPA trimerization core... -

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Basic information

Entry
Database: PDB / ID: 8aas
TitleCrystal structure of the Pyrococcus abyssi RPA trimerization core bound to poly-dT20 ssDNA
Components
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination
  • RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
  • Replication factor A
KeywordsDNA BINDING PROTEIN / Replication protein A / ssDNA-Binding protein
Function / homology
Function and homology information


macromolecule metabolic process / intracellular organelle / primary metabolic process / : / nucleic acid binding / DNA binding
Similarity search - Function
Replication factor A protein-like / Replication factor A, C-terminal / Replication factor-A C terminal domain / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / Winged helix-like DNA-binding domain superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / Replication factor A / RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination / RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
Similarity search - Component
Biological speciesPyrococcus abyssi GE5 (archaea)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.2 Å
AuthorsMadru, C. / Legrand, P. / Sauguet, L.
Funding support France, 2items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-10-JCJC-1501 ARCHPOL France
Agence Nationale de la Recherche (ANR)ANR-20-CE11-0003 ARCHAPRIM France
CitationJournal: Nat Commun / Year: 2023
Title: DNA-binding mechanism and evolution of replication protein A.
Authors: Clément Madru / Markel Martínez-Carranza / Sébastien Laurent / Alessandra C Alberti / Maelenn Chevreuil / Bertrand Raynal / Ahmed Haouz / Rémy A Le Meur / Marc Delarue / Ghislaine ...Authors: Clément Madru / Markel Martínez-Carranza / Sébastien Laurent / Alessandra C Alberti / Maelenn Chevreuil / Bertrand Raynal / Ahmed Haouz / Rémy A Le Meur / Marc Delarue / Ghislaine Henneke / Didier Flament / Mart Krupovic / Pierre Legrand / Ludovic Sauguet /
Abstract: Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair. Little is known about the structure of RPA in ...Replication Protein A (RPA) is a heterotrimeric single stranded DNA-binding protein with essential roles in DNA replication, recombination and repair. Little is known about the structure of RPA in Archaea, the third domain of life. By using an integrative structural, biochemical and biophysical approach, we extensively characterize RPA from Pyrococcus abyssi in the presence and absence of DNA. The obtained X-ray and cryo-EM structures reveal that the trimerization core and interactions promoting RPA clustering on ssDNA are shared between archaea and eukaryotes. However, we also identified a helical domain named AROD (Acidic Rpa1 OB-binding Domain), and showed that, in Archaea, RPA forms an unanticipated tetrameric supercomplex in the absence of DNA. The four RPA molecules clustered within the tetramer could efficiently coat and protect stretches of ssDNA created by the advancing replisome. Finally, our results provide insights into the evolution of this primordial replication factor in eukaryotes.
History
DepositionJul 1, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 3, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Replication factor A
B: RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination
C: RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination
F: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)75,34810
Polymers75,0824
Non-polymers2666
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area9240 Å2
ΔGint-82 kcal/mol
Surface area24560 Å2
MethodPISA
Unit cell
Length a, b, c (Å)75.234, 75.234, 215.487
Angle α, β, γ (deg.)90, 90, 120
Int Tables number154
Space group name H-MP3221

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Components

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Protein , 3 types, 3 molecules ABC

#1: Protein Replication factor A


Mass: 33756.676 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Strain: GE5 / Orsay / Gene: PAB2163 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G8ZHS0
#2: Protein RPA32 subunit of the hetero-oligomeric complex involved in homologous recombination


Mass: 21277.865 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Insertion of SER after first M from reference sequence NCBI Reference Sequence: WP_048146526.1 to improve expression
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Strain: GE5 / Orsay / Gene: PAB2165 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V1Z1
#3: Protein RPA14 subunit of the hetero-oligomeric complex involved in homologous recombination


Mass: 14008.925 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus abyssi GE5 (archaea) / Strain: GE5 / Orsay / Gene: PAB2164 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9V1Z0

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DNA chain , 1 types, 1 molecules F

#4: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 6038.899 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 6 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca

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Details

Has ligand of interestY

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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: 2.4 Å3/Da / Density % sol: 48.82 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 10% w/v PEG 8K 0.1M imidazole pH 8 0.2 M calcium acetate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.978565 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Dec 9, 2021 / Details: KB Mirrors
RadiationMonochromator: Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.978565 Å / Relative weight: 1
ReflectionResolution: 3.2→48.26 Å / Num. obs: 10041 / % possible obs: 80.7 % / Redundancy: 27.2 % / Biso Wilson estimate: 163.2 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.067 / Rpim(I) all: 0.013 / Rrim(I) all: 0.068 / Net I/σ(I): 27.1
Reflection shellResolution: 3.2→3.28 Å / Redundancy: 26.8 % / Rmerge(I) obs: 4.189 / Mean I/σ(I) obs: 1 / Num. unique obs: 204 / CC1/2: 0.454 / Rpim(I) all: 0.816 / Rrim(I) all: 4.27 / % possible all: 21.2

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Processing

Software
NameVersionClassification
BUSTER2.10.4 (3-FEB-2022)refinement
XDSVERSION Feb 5, 2021data reduction
STARANISO2.3.77data scaling
MOLREP11.7.03phasing
MxCuBE2.1data collection
Coot0.9.8.1model building
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Working model of the full apo-RPA

Resolution: 3.2→48.26 Å / Cor.coef. Fo:Fc: 0.934 / Cor.coef. Fo:Fc free: 0.926 / Cross valid method: THROUGHOUT / SU Rfree Blow DPI: 0.634
RfactorNum. reflection% reflectionSelection details
Rfree0.2933 494 -RANDOM
Rwork0.2641 ---
obs0.2655 10041 81.5 %-
Displacement parametersBiso mean: 173.43 Å2
Baniso -1Baniso -2Baniso -3
1-1.688 Å20 Å20 Å2
2--1.688 Å20 Å2
3----3.3761 Å2
Refine analyzeLuzzati coordinate error obs: 0.59 Å
Refinement stepCycle: LAST / Resolution: 3.2→48.26 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3771 280 6 0 4057
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.0084135HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.855620HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d1503SINUSOIDAL2
X-RAY DIFFRACTIONt_gen_planes658HARMONIC5
X-RAY DIFFRACTIONt_it4135HARMONIC10
X-RAY DIFFRACTIONt_chiral_improper_torsion538SEMIHARMONIC5
X-RAY DIFFRACTIONt_utility_distance4HARMONIC1
X-RAY DIFFRACTIONt_utility_angle6HARMONIC1
X-RAY DIFFRACTIONt_ideal_dist_contact2765SEMIHARMONIC4
X-RAY DIFFRACTIONt_omega_torsion2.41
X-RAY DIFFRACTIONt_other_torsion16.91
LS refinement shellResolution: 3.2→3.35 Å
RfactorNum. reflection% reflection
Rfree0.4203 29 -
Rwork0.3586 --
obs0.3621 419 26.53 %
Refinement TLS params.

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)
11.5322-0.5018-0.41013.4984-1.40843.37860.2194-0.5789-0.5516-0.57890.0193-0.133-0.5516-0.133-0.23870.21150.22890.2076-0.2115-0.0556-0.2115-17.78698.5961-12.0859
27.62211.3032.0616-0.60382.258.3155-0.0798-0.1827-0.058-0.18270.1435-0.3144-0.058-0.3144-0.06370.14170.129-0.17040.34370.0771-0.304-36.50293.7573-37.5422
31.9616-0.758-3.97579.55811.06538.3155-0.2478-0.27750.3017-0.2775-0.20270.51380.30170.51380.45050.27120.18970.2056-0.2548-0.0522-0.1596-8.797118.7354-26.3414
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|* C|* B|1 - B|148 }A186 - 358
2X-RAY DIFFRACTION1{ A|* C|* B|1 - B|148 }A500
3X-RAY DIFFRACTION1{ A|* C|* B|1 - B|148 }B3 - 148
4X-RAY DIFFRACTION1{ A|* C|* B|1 - B|148 }C5 - 117
5X-RAY DIFFRACTION2{ B|149 - B|187 }B149 - 180
6X-RAY DIFFRACTION3{ F|* }F1 - 14

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