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- PDB-8vy3: Human DNA polymerase alpha/primase - AavLEA1 (1:40 molar ratio) -

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Basic information

Entry
Database: PDB / ID: 8vy3
TitleHuman DNA polymerase alpha/primase - AavLEA1 (1:40 molar ratio)
Components
  • (DNA polymerase alpha ...) x 2
  • DNA primase large subunit
  • DNA primase small subunit
KeywordsREPLICATION / Human DNA poly alpha/primase / AavLEA1 / air-water-interface
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
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsAbe, K.M. / Li, G. / Grant, T. / Lim, C.J.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM130550 United States
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)DP2GM150023 United States
CitationJournal: Nat Commun / Year: 2024
Title: Small LEA proteins mitigate air-water interface damage to fragile cryo-EM samples during plunge freezing.
Authors: Kaitlyn M Abe / Gan Li / Qixiang He / Timothy Grant / Ci Ji Lim /
Abstract: Air-water interface (AWI) interactions during cryo-electron microscopy (cryo-EM) sample preparation cause significant sample loss, hindering structural biology research. Organisms like nematodes and ...Air-water interface (AWI) interactions during cryo-electron microscopy (cryo-EM) sample preparation cause significant sample loss, hindering structural biology research. Organisms like nematodes and tardigrades produce Late Embryogenesis Abundant (LEA) proteins to withstand desiccation stress. Here we show that these LEA proteins, when used as additives during plunge freezing, effectively mitigate AWI damage to fragile multi-subunit molecular samples. The resulting high-resolution cryo-EM maps are comparable to or better than those obtained using existing AWI damage mitigation methods. Cryogenic electron tomography reveals that particles are localized at specific interfaces, suggesting LEA proteins form a barrier at the AWI. This interaction may explain the observed sample-dependent preferred orientation of particles. LEA proteins offer a simple, cost-effective, and adaptable approach for cryo-EM structural biologists to overcome AWI-related sample damage, potentially revitalizing challenging projects and advancing the field of structural biology.
History
DepositionFeb 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2024Group: Database references / Structure summary / Category: citation / citation_author / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _pdbx_entry_details.has_protein_modification
Revision 1.2Jun 4, 2025Group: Data collection / Category: em_software / Item: _em_software.name

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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)277,6258
Polymers277,0774
Non-polymers5484
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
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: 49016.941 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P49642, DNA primase AEP
#2: Protein DNA primase large subunit / DNA primase 58 kDa subunit / p58


Mass: 50538.652 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM2, PRIM2A / Production host: Trichoplusia ni (cabbage looper) / 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: 128446.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLA1, POLA / Production host: Trichoplusia ni (cabbage looper) / 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q14181

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-ZN / ZINC ION


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


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4

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Details

Has ligand of interestN
Has protein modificationN

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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: DNA Polymerase alpha/primase / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESHEPES1
2150 mMsodium chlorideNaCl1
31 mMTCEPTCEP1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
2EPUimage acquisition
7PHENIXmodel fitting
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 856205 / Symmetry type: POINT
Atomic model buildingPDB-ID: 5EXR

5exr


Accession code: 5EXR / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00319254
ELECTRON MICROSCOPYf_angle_d0.62426025
ELECTRON MICROSCOPYf_dihedral_angle_d4.6492529
ELECTRON MICROSCOPYf_chiral_restr0.0432854
ELECTRON MICROSCOPYf_plane_restr0.0063341

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