[English] 日本語
Yorodumi
- EMDB-42037: Human Polymerase alpha-Primase, polymerase alpha catalytic domain... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-42037
TitleHuman Polymerase alpha-Primase, polymerase alpha catalytic domain deletion mutant (Conformation 2)
Map data
Sample
  • Complex: Polymerase alpha-primase, pol alpha catalytic domain deletion mutant
    • Protein or peptide: DNA polymerase alpha subunit B
    • Protein or peptide: DNA polymerase alpha catalytic subunit
    • Protein or peptide: DNA primase large subunit
    • Protein or peptide: DNA primase small subunit
KeywordsPolymerase / Primase / Polymerase Alpha / RNA priming / REPLICATION
Function / homology
Function and homology information


positive regulation of DNA primase activity / 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 / Polymerase switching / alpha DNA polymerase:primase complex ...positive regulation of DNA primase activity / 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 / Polymerase switching / alpha DNA polymerase:primase complex / Processive synthesis on the lagging strand / DNA primase activity / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere / primosome complex / 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 / 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 / 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 / nucleolus / protein kinase binding / 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 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 / 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 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 / negative staining / Resolution: 10.96 Å
AuthorsCordoba JJ / Chazin WJ
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)2T32GM008320-31 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118089 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM136401 United States
CitationJournal: J Mol Biol / Year: 2023
Title: Flexibility and Distributive Synthesis Regulate RNA Priming and Handoff in Human DNA Polymerase α-Primase.
Authors: John J Cordoba / Elwood A Mullins / Lauren E Salay / Brandt F Eichman / Walter J Chazin /
Abstract: DNA replication in eukaryotes relies on the synthesis of a ∼30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of ...DNA replication in eukaryotes relies on the synthesis of a ∼30-nucleotide RNA/DNA primer strand through the dual action of the heterotetrameric polymerase α-primase (pol-prim) enzyme. Synthesis of the 7-10-nucleotide RNA primer is regulated by the C-terminal domain of the primase regulatory subunit (PRIM2C) and is followed by intramolecular handoff of the primer to pol α for extension by ∼20 nucleotides of DNA. Here, we provide evidence that RNA primer synthesis is governed by a combination of the high affinity and flexible linkage of the PRIM2C domain and the surprisingly low affinity of the primase catalytic domain (PRIM1) for substrate. Using a combination of small angle X-ray scattering and electron microscopy, we found significant variability in the organization of PRIM2C and PRIM1 in the absence and presence of substrate, and that the population of structures with both PRIM2C and PRIM1 in a configuration aligned for synthesis is low. Crosslinking was used to visualize the orientation of PRIM2C and PRIM1 when engaged by substrate as observed by electron microscopy. Microscale thermophoresis was used to measure substrate affinities for a series of pol-prim constructs, which showed that the PRIM1 catalytic domain does not bind the template or emergent RNA-primed templates with appreciable affinity. Together, these findings support a model of RNA primer synthesis in which generation of the nascent RNA strand and handoff of the RNA-primed template from primase to polymerase α is mediated by the high degree of inter-domain flexibility of pol-prim, the ready dissociation of PRIM1 from its substrate, and the much higher affinity of the POLA1cat domain of polymerase α for full-length RNA-primed templates.
History
DepositionSep 19, 2023-
Header (metadata) releaseNov 29, 2023-
Map releaseNov 29, 2023-
UpdateNov 29, 2023-
Current statusNov 29, 2023Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_42037.png

Downloads & links

-
Map

FileDownload / File: emd_42037.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.79 Å/pix.
x 256 pix.
= 458.278 Å		1.79 Å/pix.
x 256 pix.
= 458.278 Å		1.79 Å/pix.
x 256 pix.
= 458.278 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.79015 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.83
Minimum - Maximum-0.30851015 - 4.624917
Average (Standard dev.)0.0024374912 (±0.24218652)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 458.2784 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: #1

Fileemd_42037_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: #2

Fileemd_42037_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : Polymerase alpha-primase, pol alpha catalytic domain deletion mutant

EntireName: Polymerase alpha-primase, pol alpha catalytic domain deletion mutant
Components
  • Complex: Polymerase alpha-primase, pol alpha catalytic domain deletion mutant
    • Protein or peptide: DNA polymerase alpha subunit B
    • Protein or peptide: DNA polymerase alpha catalytic subunit
    • Protein or peptide: DNA primase large subunit
    • Protein or peptide: DNA primase small subunit

-
Supramolecule #1: Polymerase alpha-primase, pol alpha catalytic domain deletion mutant

SupramoleculeName: Polymerase alpha-primase, pol alpha catalytic domain deletion mutant
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Complex stabilized by BS3 crosslinking.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 180 KDa

-
Macromolecule #1: DNA polymerase alpha subunit B

MacromoleculeName: DNA polymerase alpha subunit B / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GSATPSQKYN SRSNRGEVVT SFGLAQGV S WSGRGGAGNI SLKVLGCPEA LTGSYKSMFQ KLPDIREVLT CKIEELGSEL KEHYKIEAF TPLLAPAQEP VTLLGQIGCD SNGKLNNKSV ILEGDREHSS GAQIPVDLSE LKEYSLFPGQ VVIMEGINT TGRKLVATKL ...String:
GSATPSQKYN SRSNRGEVVT SFGLAQGV S WSGRGGAGNI SLKVLGCPEA LTGSYKSMFQ KLPDIREVLT CKIEELGSEL KEHYKIEAF TPLLAPAQEP VTLLGQIGCD SNGKLNNKSV ILEGDREHSS GAQIPVDLSE LKEYSLFPGQ VVIMEGINT TGRKLVATKL YEGVPLPFYQ PTEEDADFEQ SMVLVACGPY TTSDSITYDP L LDLIAVIN HDRPDVCILF GPFLDAKHEQ VENCLLTSPF EDIFKQCLRT IIEGTRSSGS HL VFVPSLR DVHHEPVYPQ PPFSYSDLSR EDKKQVQFVS EPCSLSINGV IFGLTSTDLL FHL GAEEIS SSSGTSDRFS RILKHILTQR SYYPLYPPQE DMAIDYESFY VYAQLPVTPD VLII PSELR YFVKDVLGCV CVNPGRLTKG QVGGTFARLY LRRPAADGAE RQSPCIAVQV VRI

UniProtKB: DNA polymerase alpha subunit B

-
Macromolecule #2: DNA polymerase alpha catalytic subunit

MacromoleculeName: DNA polymerase alpha catalytic subunit / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MAQLTDEEKY RDCERFKCPC PTCGTENIYD NVFDGSGTDM EPSLYRCSNI DCKASPL TF TVQLSNKLIM DIRRFIKKYY DGWLICEEPT CRNRTRHLPL QFSRTGPLCP ACMKATLQ P EYSDKSLYTQ LCFYRYIFDA ECALEKLTTD HEKDKLKKQF FTPKVLQDYR ...String:
MAQLTDEEKY RDCERFKCPC PTCGTENIYD NVFDGSGTDM EPSLYRCSNI DCKASPL TF TVQLSNKLIM DIRRFIKKYY DGWLICEEPT CRNRTRHLPL QFSRTGPLCP ACMKATLQ P EYSDKSLYTQ LCFYRYIFDA ECALEKLTTD HEKDKLKKQF FTPKVLQDYR KLKNTAEQF LSRSGYSEVN LSKLFAGCAV KS

UniProtKB: DNA polymerase alpha catalytic subunit

-
Macromolecule #3: DNA primase large subunit

MacromoleculeName: DNA primase large subunit / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MEFSGRKWRK LRLAGDQRNA SYPHCLQFYL QPPSENISLI EFENLAIDRV KLLKSVENLG VSYVKGTEQ YQSKLESELR KLKFSYRENL EDEYEPRRRD HISHFILRLA YCQSEELRRW F IQQEMDLL RFRFSILPKD KIQDFLKDSQ LQFEAISDEE KTLREQEIVA ...String:
MEFSGRKWRK LRLAGDQRNA SYPHCLQFYL QPPSENISLI EFENLAIDRV KLLKSVENLG VSYVKGTEQ YQSKLESELR KLKFSYRENL EDEYEPRRRD HISHFILRLA YCQSEELRRW F IQQEMDLL RFRFSILPKD KIQDFLKDSQ LQFEAISDEE KTLREQEIVA SSPSLSGLKL GF ESIYKIP FADALDLFRG RKVYLEDGFA YVPLKDIVAI ILNEFRAKLS KALALTARSL PAV QSDERL QPLLNHLSHS YTGQDYSTQG NVGKISLDQI DLLSTKSFPP CMRQLHKALR ENHH LRHGG RMQYGLFLKG IGLTLEQALQ FWKQEFIKGK MDPDKFDKGY SYNIRHSFGK EGKRT DYTP FSCLKIILSN PPSQGDYHGC PFRHSDPELL KQKLQSYKIS PGGISQILDL VKGTHY QVA CQKYFEMIHN VDDCGFSLNH PNQFFCESQR ILNGGKDIKK EPIQPETPQP KPSVQKT KD ASSALASLNS SLEMDMEGLE DYFSEDS

UniProtKB: DNA primase large subunit

-
Macromolecule #4: DNA primase small subunit

MacromoleculeName: DNA primase small subunit / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: GSMETFDPTE LPELLKLYYR RLFPYSQYYR WLNYGGVIKN YFQHREFSFT LKDDIYIRYQ SF NNQSDLE KEMQKMNPYK IDIGAVYSHR PNQHNTVKLG AFQAQEKELV FDIDMTDYDD VRR CCSSAD ICPKCWTLMT MAIRIIDRAL KEDFGFKHRL WVYSGRRGVH ...String:
GSMETFDPTE LPELLKLYYR RLFPYSQYYR WLNYGGVIKN YFQHREFSFT LKDDIYIRYQ SF NNQSDLE KEMQKMNPYK IDIGAVYSHR PNQHNTVKLG AFQAQEKELV FDIDMTDYDD VRR CCSSAD ICPKCWTLMT MAIRIIDRAL KEDFGFKHRL WVYSGRRGVH CWVCDESVRK LSSA VRSGI VEYLSLVKGG QDVKKKVHLS EKIHPFIRKS INIIKKYFEE YALVNQDILE NKESW DKIL ALVPETIHDE LQQSFQKSHN SLQRWEHLKK VASRYQNNIK NDKYGPWLEW EIMLQY CFP RLDINVSKGI NHLLKSPFSV HPKTGRISVP IDLQKVDQFD PFTVPTISFI CRELDAI ST NEEEKEENEA ESDVKHRTRD YKKTSLAPYV KVFEHFLENL DKSRKGELLK KSDLQKDF

UniProtKB: DNA primase small subunit

-
Experimental details

-
Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration0.009 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
20.0 mMC8H18N2O4SHEPES
5.0 mMMgCl2Magnesium chloride
1.0 mMC9H15O6PTCEP
StainingType: NEGATIVE / Material: Uranyl Formate
Details: 2.5 uL sample was applied to grid for 1 minute, then blotted with filter paper, washed for 5 seconds with DI water, blotted, washed for 5 seconds in stain, blotted, then stained for 90 seconds.

-
Electron microscopy

MicroscopeFEI TECNAI 20
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1083 / Average electron dose: 20.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 1.2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.5 µm

+
Image processing

Particle selectionNumber selected: 1628779
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 10.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 1.4) / Number images used: 21150
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 3 / Avg.num./class: 16112 / Software - Name: cryoSPARC (ver. 4.1)
FSC plot (resolution estimation)

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more