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- PDB-5i7m: Crystal structure of Y345F mutant of human primase p58 iron-sulfu... -

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Basic information

Entry
Database: PDB / ID: 5i7m
TitleCrystal structure of Y345F mutant of human primase p58 iron-sulfur cluster domain
ComponentsDNA primase large subunitPrimase
KeywordsREPLICATION / DNA replication / primase / [4Fe-4S] / p58C
Function / homology
Function and homology information


positive regulation of DNA primase activity / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / alpha DNA polymerase:primase complex / Polymerase switching / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere ...positive regulation of DNA primase activity / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / alpha DNA polymerase:primase complex / Polymerase switching / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere / DNA replication, synthesis of primer / Activation of the pre-replicative complex / DNA replication initiation / Defective pyroptosis / 4 iron, 4 sulfur cluster binding / DNA binding / nucleoplasm / metal ion binding
Similarity search - Function
DNA primase, large subunit, eukaryotic / DNA primase large subunit, eukaryotic/archaeal / Eukaryotic and archaeal DNA primase, large subunit
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / DNA primase large subunit
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.93 Å
AuthorsSalay, L.E. / Thompson, M.K. / Chazin, W.J.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM61077 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM120087 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM65484 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM118089 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM07616 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM08230 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA009582 United States
CitationJournal: Science / Year: 2017
Title: The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport.
Authors: O'Brien, E. / Holt, M.E. / Thompson, M.K. / Salay, L.E. / Ehlinger, A.C. / Chazin, W.J. / Barton, J.K.
History
DepositionFeb 17, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 1, 2017Provider: repository / Type: Initial release
Revision 1.1Mar 8, 2017Group: Database references
Revision 1.2Sep 13, 2017Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Nov 27, 2019Group: Author supporting evidence / Category: pdbx_audit_support
Revision 1.4Sep 27, 2023Group: 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 primase large subunit
B: DNA primase large subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)44,2195
Polymers43,4202
Non-polymers7993
Water1,856103
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2380 Å2
ΔGint-65 kcal/mol
Surface area16940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)109.963, 52.624, 89.195
Angle α, β, γ (deg.)90.000, 115.270, 90.000
Int Tables number5
Space group name H-MC121

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Components

#1: Protein DNA primase large subunit / Primase / Primase regulatory subunit / DNA primase 58 kDa subunit / p58


Mass: 21709.867 Da / Num. of mol.: 2 / Fragment: iron-sulfur cluster domain (UNP residues 272-457) / Mutation: Y345F
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM2, PRIM2A / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P49643, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe4S4
#3: Chemical ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 103 / 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: 2.75 Å3/Da / Density % sol: 55.3 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 75 mg/mL p58C Y345F in 20 mM MES, pH 6.5, 50 mM sodium chloride, reservoir solution: 100 mM Tris, pH 8.5, 150 mM lithium sulfate, 18% PEG3350

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-G / Wavelength: 0.987 Å
DetectorType: MARMOSAIC 300 mm CCD / Detector: CCD / Date: Feb 6, 2016
RadiationMonochromator: diamond(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.987 Å / Relative weight: 1
ReflectionResolution: 1.93→80.66 Å / Num. obs: 34870 / % possible obs: 99.9 % / Redundancy: 4.2 % / Rmerge(I) obs: 0.087 / Rpim(I) all: 0.048 / Rrim(I) all: 0.099 / Χ2: 1.475 / Net I/av σ(I): 18.692 / Net I/σ(I): 6.9 / Num. measured all: 144740
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique allCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.93-1.9640.58417600.8860.3330.6750.85999.8
1.96-24.10.44817070.8950.2510.5150.71599.9
2-2.044.10.39117510.9240.2180.4490.772100
2.04-2.084.10.41316900.7490.240.481.26199.8
2.08-2.124.20.31317420.9580.1740.3590.812100
2.12-2.174.20.26317440.970.1450.3010.836100
2.17-2.234.20.2317200.9710.1260.2630.906100
2.23-2.2940.30617550.790.1790.3551.52899.5
2.29-2.364.20.18517210.9780.1020.2121.021100
2.36-2.434.20.16717210.980.0910.1911.067100
2.43-2.524.20.15817400.9780.0860.181.075100
2.52-2.624.20.1317560.9850.0710.1481.162100
2.62-2.744.20.12717250.9870.0690.1451.39199.9
2.74-2.884.20.117490.9880.0550.1141.254100
2.88-3.064.20.08217420.9920.0450.0941.479100
3.06-3.34.20.07117520.9940.0390.0811.681100
3.3-3.634.20.06317370.9950.0350.0721.86100
3.63-4.164.10.05417560.9940.030.0622.09999.7
4.16-5.244.10.04217810.9960.0240.0482.001100
5.24-503.90.04618210.9950.0270.0545.79699.6

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation1.93 Å49.7 Å
Translation6.45 Å49.7 Å

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Processing

Software
NameVersionClassification
REFMAC5.8.0135refinement
HKL-2000data collection
HKL-2000data scaling
PHASER2.5.7phasing
PDB_EXTRACT3.2data extraction
HKL-2000data reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3L9Q

3l9q


Resolution: 1.93→80.66 Å / Cor.coef. Fo:Fc: 0.95 / Cor.coef. Fo:Fc free: 0.934 / SU B: 0.003 / SU ML: 0 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.12 / ESU R Free: 0.142 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2498 1764 5.1 %RANDOM
Rwork0.2111 ---
obs0.2131 33095 99.75 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 69.72 Å2 / Biso mean: 29.392 Å2 / Biso min: 14.98 Å2
Baniso -1Baniso -2Baniso -3
1--1.51 Å20 Å2-1.56 Å2
2--5.31 Å2-0 Å2
3----1.6 Å2
Refinement stepCycle: final / Resolution: 1.93→80.66 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2623 0 21 104 2748
Biso mean--25.7 27.27 -
Num. residues----329
LS refinement shellResolution: 1.93→1.98 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.355 125 -
Rwork0.313 2394 -
all-2519 -
obs--98.28 %

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