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- EMDB-43627: Human DNA polymerase alpha/primase - 4 mM CHAPSO -

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Basic information

Entry
Database: EMDB / ID: EMD-43627
TitleHuman DNA polymerase alpha/primase - 4 mM CHAPSO
Map data
Sample
  • Complex: Human pola-primase
KeywordsPola-primase / CHAPSO / air-water-interface / REPLICATION
Function / homology
Function and homology information


: / DNA primase AEP / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / 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 / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / 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 / : / Removal of the Flap Intermediate / primosome complex / Polymerase switching on the C-strand of the telomere / DNA replication, synthesis of primer / lagging strand elongation / mitotic DNA replication initiation / DNA strand elongation involved in DNA replication / DNA synthesis involved in DNA repair / G1/S-Specific Transcription / leading strand elongation / 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 replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA repair / nucleotide binding / chromatin binding / chromatin / protein kinase binding / nucleolus / magnesium ion binding / DNA binding / zinc ion binding / nucleoplasm / membrane / nucleus / metal ion binding / 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 polymerase alpha catalytic subunit, N-terminal 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 polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / DNA primase small subunit / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA primase large subunit, eukaryotic/archaeal / Eukaryotic and archaeal DNA primase, large subunit / 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 polymerase family B signature. / 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 superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsAbe KM / Li G / He Q / Grant T / Lim C
Funding support United States, 3 items
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 5, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateDec 18, 2024-
Current statusDec 18, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43627.png

Downloads & links

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Map

FileDownload / File: emd_43627.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 320 pix.
= 340.48 Å		1.06 Å/pix.
x 320 pix.
= 340.48 Å		1.06 Å/pix.
x 320 pix.
= 340.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.064 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0712
Minimum - Maximum-0.8502967 - 1.4716502
Average (Standard dev.)-0.00014569434 (±0.021717194)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 340.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43627_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43627_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Human pola-primase

EntireName: Human pola-primase
Components
  • Complex: Human pola-primase

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Supramolecule #1: Human pola-primase

SupramoleculeName: Human pola-primase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
25.0 mMHEPESHEPES
150.0 mMNaClsodium chloride
4.0 mMCHAPSOCHAPSO
1.0 mMTCEPTCEP
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 674789
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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