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- PDB-5exr: Crystal structure of human primosome -

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Basic information

Entry
Database: PDB / ID: 5exr
TitleCrystal structure of human primosome
Components
  • (DNA polymerase alpha ...) x 2
  • DNA primase large subunit
  • DNA primase small subunit
KeywordsREPLICATION / human primosome / complex / primase / DNA polymerase alpha / primer / DNA replication / DNA / RNA / replicase
Function / homology
Function and homology information


: / DNA primase AEP / ribonucleotide binding / DNA replication initiation / Telomere C-strand synthesis initiation / DNA/RNA hybrid binding / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production / alpha DNA polymerase:primase complex / Polymerase switching ...: / DNA primase AEP / ribonucleotide binding / DNA replication initiation / Telomere C-strand synthesis initiation / DNA/RNA hybrid binding / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / regulation of type I interferon production / alpha DNA polymerase:primase complex / Polymerase switching / : / Processive synthesis on the lagging strand / DNA replication, synthesis of primer / Removal of the Flap Intermediate / lagging strand elongation / Polymerase switching on the C-strand of the telomere / mitotic DNA replication initiation / DNA synthesis involved in DNA repair / DNA strand elongation involved in DNA replication / leading strand elongation / G1/S-Specific Transcription / DNA replication origin binding / Activation of the pre-replicative complex / DNA replication initiation / Defective pyroptosis / double-strand break repair via nonhomologous end joining / nuclear matrix / protein import into nucleus / nuclear envelope / single-stranded DNA binding / 4 iron, 4 sulfur cluster binding / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA replication / ciliary basal body / DNA repair / nucleotide binding / intracellular membrane-bounded organelle / chromatin binding / protein kinase binding / chromatin / nucleolus / magnesium ion binding / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / membrane / cytosol
Similarity search - Function
Alpha-Beta Plaits - #2820 / DNA primase, PRIM domain / DNA primase, PRIM domain / DNA polymerase alpha, subunit B, N-terminal domain superfamily / : / DNA polymerase alpha subunit B, OB domain / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal ...Alpha-Beta Plaits - #2820 / DNA primase, PRIM domain / DNA primase, PRIM domain / DNA polymerase alpha, subunit B, N-terminal domain superfamily / : / DNA polymerase alpha subunit B, OB domain / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / Eukaryotic and archaeal DNA primase, large subunit / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA polymerase alpha catalytic subunit, catalytic domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H-like superfamily/Ribonuclease H / Nucleotidyltransferase; domain 5 / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Alpha-Beta Plaits / DNA/RNA polymerase superfamily / Alpha-Beta Complex / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / DNA polymerase alpha catalytic subunit / DNA primase small subunit / DNA primase large subunit / DNA polymerase alpha subunit B
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.6 Å
AuthorsTahirov, T.H. / Baranovskiy, A.G. / Babayeva, N.D.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM101167 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103403 United States
CitationJournal: J.Biol.Chem. / Year: 2016
Title: Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome.
Authors: Baranovskiy, A.G. / Babayeva, N.D. / Zhang, Y. / Gu, J. / Suwa, Y. / Pavlov, Y.I. / Tahirov, T.H.
History
DepositionNov 24, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 23, 2016Provider: repository / Type: Initial release
Revision 1.1Mar 30, 2016Group: Database references
Revision 1.2Jun 1, 2016Group: Database references
Revision 1.3Sep 20, 2017Group: Author supporting evidence / Database references / Derived calculations
Category: citation / pdbx_audit_support / pdbx_struct_oper_list
Item: _citation.journal_id_CSD / _pdbx_audit_support.funding_organization / _pdbx_struct_oper_list.symmetry_operation
Revision 1.4Nov 1, 2017Group: Author supporting evidence / Category: pdbx_struct_assembly_auth_evidence
Revision 1.5Dec 25, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Sep 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
Revision 1.7Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA primase small subunit
B: DNA primase large subunit
C: DNA polymerase alpha catalytic subunit
D: DNA polymerase alpha subunit B
E: DNA primase small subunit
F: DNA primase large subunit
G: DNA polymerase alpha catalytic subunit
H: DNA polymerase alpha subunit B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)609,22616
Polymers608,1308
Non-polymers1,0968
Water00
1
A: DNA primase small subunit
B: DNA primase large subunit
C: DNA polymerase alpha catalytic subunit
D: DNA polymerase alpha subunit B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)304,6138
Polymers304,0654
Non-polymers5484
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
E: DNA primase small subunit
F: DNA primase large subunit
G: DNA polymerase alpha catalytic subunit
H: DNA polymerase alpha subunit B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)304,6138
Polymers304,0654
Non-polymers5484
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)113.096, 210.164, 172.565
Angle α, β, γ (deg.)90.000, 93.560, 90.000
Int Tables number4
Space group name H-MP1211
Detailstetramer according to electrophoresis

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Components

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Protein , 2 types, 4 molecules AEBF

#1: Protein DNA primase small subunit / DNA primase 49 kDa subunit / p49


Mass: 49981.012 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM1 / Production host: Escherichia coli (E. coli)
References: UniProt: P49642, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: Protein DNA primase large subunit / DNA primase 58 kDa subunit / p58


Mass: 58890.918 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM2, PRIM2A / Production host: Escherichia coli (E. coli)
References: UniProt: P49643, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases

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DNA polymerase alpha ... , 2 types, 4 molecules CGDH

#3: Protein DNA polymerase alpha catalytic subunit / DNA polymerase alpha catalytic subunit p180


Mass: 129308.773 Da / Num. of mol.: 2 / Mutation: V516A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLA1, POLA / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P09884, DNA-directed DNA polymerase
#4: Protein DNA polymerase alpha subunit B / DNA polymerase alpha 70 kDa subunit


Mass: 65884.344 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLA2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q14181

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Non-polymers , 2 types, 8 molecules

#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn
#6: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe4S4

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Details

Has protein modificationY

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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: 3.37 Å3/Da / Density % sol: 63.46 % / Description: thin plate in form of parallelogram
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 0.2 M lithium sulphate, 50 mM TRIS HCl pH 8.5, 2 mM TCEP pH 7.5, 11.2% w/v PEG 4,000, 3% v/v ethanol, 0.5% v/v polypropylene glycol P400 and 0.2 mM EDTA

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Mar 1, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 3.6→50 Å / Num. obs: 74238 / % possible obs: 80.5 % / Observed criterion σ(I): -1 / Redundancy: 2.6 % / Rmerge(I) obs: 0.063 / Χ2: 2.353 / Net I/av σ(I): 8 / Net I/σ(I): 12.2 / Num. measured all: 236349
Reflection shellResolution: 3.6→3.66 Å / Redundancy: 1.9 % / Rmerge(I) obs: 0.364 / Mean I/σ(I) obs: 1.93 / Num. unique all: 3348 / Χ2: 0.764 / Rejects: 0 / % possible all: 72.7

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
HKL-3000data reduction
HKL-3000data scaling
CNS1.1phasing
CNS1.1refinement
PDB_EXTRACT3.15data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4QCL, 4Q5V, 4RR2, 4Y97

4qcl

4q5v

4rr2

4y97


Resolution: 3.6→39.94 Å / Data cutoff high absF: 6416908 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 1
RfactorNum. reflection% reflectionSelection details
Rfree0.326 3215 5 %RANDOM
Rwork0.268 ---
obs-64105 68.9 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 161.81 Å2 / ksol: 0.4022 e/Å3
Displacement parametersBiso max: 145.71 Å2 / Biso mean: 61.2 Å2 / Biso min: 1.1 Å2
Baniso -1Baniso -2Baniso -3
1-1.91 Å20 Å22.94 Å2
2---5.74 Å20 Å2
3---3.83 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.63 Å0.53 Å
Luzzati d res low-5 Å
Luzzati sigma a1.19 Å1.15 Å
Refinement stepCycle: final / Resolution: 3.6→39.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms37642 0 22 0 37664
Biso mean--19.57 --
Num. residues----4648
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.012
X-RAY DIFFRACTIONc_angle_deg1.8
X-RAY DIFFRACTIONc_dihedral_angle_d23.6
X-RAY DIFFRACTIONc_improper_angle_d1.19
X-RAY DIFFRACTIONc_mcbond_it3.971.5
X-RAY DIFFRACTIONc_mcangle_it6.672
X-RAY DIFFRACTIONc_scbond_it4.762
X-RAY DIFFRACTIONc_scangle_it7.412.5
LS refinement shellResolution: 3.6→3.83 Å / Rfactor Rfree error: 0.02 / Total num. of bins used: 6
RfactorNum. reflection% reflection
Rfree0.391 389 4.8 %
Rwork0.377 7661 -
all-8050 -
obs--52.1 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1protein_rep.paramsf4:protein.top
X-RAY DIFFRACTION2water_rep.paramwater.top
X-RAY DIFFRACTION3ion.paramion.top
X-RAY DIFFRACTION4sf4.parsf4.top

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