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- PDB-8d9d: Human DNA polymerase-alpha/primase elongation complex II bound to... -

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Basic information

Entry
Database: PDB / ID: 8d9d
TitleHuman DNA polymerase-alpha/primase elongation complex II bound to primer/template
Components
  • (DNA polymerase alpha ...) x 2
  • DNA (5'-D(*AP*TP*GP*GP*TP*CP*GP*TP*GP*CP*CP*GP*CP*CP*AP*AP*TP*AP*A)-3')
  • DNA primase large subunit
  • DNA primase small subunit
  • DNA/RNA (5'-D(*(GTP))-R(P*GP*CP*GP*GP*CP*AP*CP*G)-D(P*AP*CP*C)-3')
KeywordsREPLICATION / DNA replication / human DNA polymerase alpha/primase / human primosome / elongation complex
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 / Processive synthesis on the lagging strand ...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 / lagging strand elongation / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere / mitotic DNA replication initiation / DNA replication, synthesis of primer / DNA strand elongation involved in DNA replication / DNA synthesis involved in DNA repair / leading strand elongation / G1/S-Specific Transcription / DNA replication origin binding / DNA replication initiation / Activation of the pre-replicative complex / Defective pyroptosis / double-strand break repair via nonhomologous end joining / nuclear matrix / protein import into nucleus / DNA-directed RNA polymerase activity / 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
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 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 C-terminal domain / 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-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B signature. / 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 superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
2'-DEOXYADENOSINE 5'-TRIPHOSPHATE / IRON/SULFUR CLUSTER / DNA / DNA (> 10) / DNA/RNA hybrid / DNA/RNA hybrid (> 10) / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.59 Å
AuthorsHe, Q. / Baranovskiy, A. / Lim, C. / Tahirov, T.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM131023 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM127085 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Structures of human primosome elongation complexes.
Authors: Qixiang He / Andrey G Baranovskiy / Lucia M Morstadt / Alisa E Lisova / Nigar D Babayeva / Benjamin L Lusk / Ci Ji Lim / Tahir H Tahirov /
Abstract: The synthesis of RNA-DNA primer by primosome requires coordination between primase and DNA polymerase α subunits, which is accompanied by unknown architectural rearrangements of multiple domains. ...The synthesis of RNA-DNA primer by primosome requires coordination between primase and DNA polymerase α subunits, which is accompanied by unknown architectural rearrangements of multiple domains. Using cryogenic electron microscopy, we solved a 3.6 Å human primosome structure caught at an early stage of RNA primer elongation with deoxynucleotides. The structure confirms a long-standing role of primase large subunit and reveals new insights into how primosome is limited to synthesizing short RNA-DNA primers.
History
DepositionJun 9, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 19, 2023Provider: repository / Type: Initial release
Revision 1.1May 3, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2May 31, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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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/RNA (5'-D(*(GTP))-R(P*GP*CP*GP*GP*CP*AP*CP*G)-D(P*AP*CP*C)-3')
F: DNA (5'-D(*AP*TP*GP*GP*TP*CP*GP*TP*GP*CP*CP*GP*CP*CP*AP*AP*TP*AP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)334,97213
Polymers333,8856
Non-polymers1,0887
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 2 molecules AB

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


Mass: 49981.012 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM1
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P49642, DNA primase AEP
#2: Protein DNA primase large subunit / DNA primase 58 kDa subunit / p58


Mass: 58890.918 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM2, PRIM2A
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: P49643

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DNA polymerase alpha ... , 2 types, 2 molecules CD

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


Mass: 166131.094 Da / Num. of mol.: 1
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: 49074.590 Da / Num. of mol.: 1
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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DNA/RNA hybrid / DNA chain , 2 types, 2 molecules EF

#5: DNA/RNA hybrid DNA/RNA (5'-D(*(GTP))-R(P*GP*CP*GP*GP*CP*AP*CP*G)-D(P*AP*CP*C)-3')


Mass: 3977.352 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: DNA chain DNA (5'-D(*AP*TP*GP*GP*TP*CP*GP*TP*GP*CP*CP*GP*CP*CP*AP*AP*TP*AP*A)-3')


Mass: 5829.786 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 4 types, 7 molecules

#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
#8: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4
#9: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#10: Chemical ChemComp-DTP / 2'-DEOXYADENOSINE 5'-TRIPHOSPHATE


Mass: 491.182 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O12P3

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Elongation complex II of human DNA polymerase alpha/primase bound to RNA-DNA primer and DNA template
Type: COMPLEX
Details: Larger elongation complex solved using cryo-EM single-particle analysis
Entity ID: #1-#6 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.340 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Cell: combined with SF21 cells
Buffer solutionpH: 7.5
Details: CHAPSO is made fresh at 80 mM before added to the sample at a final concentration of 4-8 mM immediately before vitrification.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris buffer pH 7.7TRIS-HCl1
2100 mMPotassium ChlorideKCl1
31 mMTCEPTCEP1
44 mMCHAPSOCHAPSO1
51 percentGlycerolGlycerol1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 13243

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameCategory
2Latitudeimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2522283
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.59 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 199286 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
14QCL

4qcl

4QCL1
25F0Q

5f0q

5F0Q2
35EXR

5exr

A5EXR3
45EXR

5exr

D5EXR3
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00419941
ELECTRON MICROSCOPYf_angle_d0.64727106
ELECTRON MICROSCOPYf_dihedral_angle_d9.6562844
ELECTRON MICROSCOPYf_chiral_restr0.0422976
ELECTRON MICROSCOPYf_plane_restr0.0063354

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