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- EMDB-13020: CryoEM structure of DNA Polymerase alpha - primase bound to SARS ... -

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Basic information

Entry
Database: EMDB / ID: EMD-13020
TitleCryoEM structure of DNA Polymerase alpha - primase bound to SARS CoV nsp1
Map data3D refinement map
Sample
  • Complex: Complex of DNA polymerase alpha - primase bound to SARS COV-2 nsp1
    • Protein or peptide: DNA polymerase alpha catalytic subunit
    • Protein or peptide: DNA polymerase alpha subunit BDNA polymerase
    • Protein or peptide: DNA primase small subunitPrimase
    • Protein or peptide: DNA primase large subunitPrimase
    • Protein or peptide: Non-structural protein 1
  • Ligand: ZINC ION
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster
Function / homology
Function and homology information


DNA primase AEP / positive regulation of DNA primase activity / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / Polymerase switching / Processive synthesis on the lagging strand / regulation of type I interferon production ...DNA primase AEP / positive regulation of DNA primase activity / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / Polymerase switching / Processive synthesis on the lagging strand / regulation of type I interferon production / alpha DNA polymerase:primase complex / Removal of the Flap Intermediate / Assembly of the SARS-CoV-1 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / Transcription of SARS-CoV-1 sgRNAs / DNA primase activity / Polymerase switching on the C-strand of the telomere / lagging strand elongation / DNA replication, synthesis of primer / mitotic DNA replication initiation / Translation of Replicase and Assembly of the Replication Transcription Complex / Replication of the SARS-CoV-1 genome / K48-linked deubiquitinase activity / DNA strand elongation involved in DNA replication / host cell endoplasmic reticulum / leading strand elongation / DNA synthesis involved in DNA repair / K63-linked deubiquitinase activity / G1/S-Specific Transcription / SARS-CoV-1 modulates host translation machinery / DNA replication origin binding / DNA replication initiation / Activation of the pre-replicative complex / viral genome replication / methyltransferase activity / Defective pyroptosis / nuclear matrix / double-strand break repair via nonhomologous end joining / protein import into nucleus / SARS-CoV-1 activates/modulates innate immune responses / single-stranded DNA binding / nuclear envelope / methylation / 4 iron, 4 sulfur cluster binding / double membrane vesicle viral factory outer membrane / SARS coronavirus main proteinase / host cell endosome / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / endonuclease activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / DNA replication / single-stranded RNA binding / DNA-directed DNA polymerase / host cell perinuclear region of cytoplasm / DNA-directed DNA polymerase activity / viral protein processing / lyase activity / induction by virus of host autophagy / symbiont-mediated suppression of host gene expression / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / nucleotide binding / DNA repair / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / chromatin binding / chromatin / nucleolus / protein kinase binding / magnesium ion binding / proteolysis / DNA binding / zinc ion binding / nucleoplasm / membrane / identical protein binding / metal ion binding / nucleus / cytosol
Similarity search - Function
DNA polymerase alpha, subunit B, N-terminal domain superfamily / 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, subunit B, N-terminal domain superfamily / 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 / Non-structural protein 3, SUD-N macrodomain, SARS-CoV / DNA polymerase family B, thumb 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-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP3, N-terminal, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal / NSP1 globular domain superfamily, betacoronavirus / Non-structural protein 2, SARS-CoV-like / Coronavirus 3Ecto domain profile. / : / Betacoronavirus Nsp3e group 2-specific marker (G2M) domain profile. / NSP1, C-terminal domain, betacoronavirus / Betacoronavirus Nsp3c-M domain profile. / NSP1, globular domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus replicase NSP1 / Betacoronavirus single-stranded poly(A) binding domain / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / Betacoronavirus Nsp3c-C domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein NSP3, nucleic acid-binding domain superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Papain-like protease, N-terminal domain superfamily, coronavirus / Papain-like viral protease, palm and finger domains, coronavirus / : / Coronavirus (CoV) Nsp2 middle domain profile. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / NSP1, globular domain, alpha/betacoronavirus / : / Coronavirus (CoV) Nsp3 Y domain profile. / Coronavirus (CoV) Nsp1 globular domain profile. / Coronavirus replicase NSP2, N-terminal / Nonstructural protein 2, N-terminal domain, coronavirus / Coronavirus replicase NSP2, C-terminal / Non-structural protein 2, C-terminal domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp8 cofactor domain profile. / Coronavirus Nsp9 single-stranded RNA (ssRNA)-binding domain profile. / Coronavirus (CoV) ExoN/MTase coactivator domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / Papain-like protease, thumb domain superfamily, coronavirus / Coronavirus replicase NSP7 / Peptidase family C16 domain profile. / Non-structural protein NSP7, coronavirus / Peptidase C30, coronavirus / Peptidase C16, coronavirus / Non-structural protein NSP9, coronavirus / Non-structural protein NSP8, coronavirus / RNA synthesis protein NSP10, coronavirus / Non-structural protein NSP4, C-terminal, coronavirus / RNA synthesis protein NSP10 superfamily, coronavirus / Non-structural protein NSP9 superfamily, coronavirus / Non-structural protein NSP7 superfamily, coronavirus / Non-structural protein NSP8 superfamily, coronavirus / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Peptidase C30, domain 3, coronavirus / Non-structural protein 6, coronavirus / Coronavirus replicase NSP3, C-terminal / Non-structural protein NSP4, N-terminal, coronavirus / Coronavirus endopeptidase C30
Similarity search - Domain/homology
DNA polymerase alpha catalytic subunit / Replicase polyprotein 1a / DNA primase small subunit / DNA primase large subunit / DNA polymerase alpha subunit B
Similarity search - Component
Biological speciesHomo sapiens (human) / Severe acute respiratory syndrome coronavirus
Methodsingle particle reconstruction / cryo EM / Resolution: 4.12 Å
AuthorsKilkenny ML / Pellegrini L
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust104641/Z/14/Z United Kingdom
CitationJournal: Protein Sci / Year: 2022
Title: Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with DNA polymerase α-primase.
Authors: Mairi L Kilkenny / Charlotte E Veale / Amir Guppy / Steven W Hardwick / Dimitri Y Chirgadze / Neil J Rzechorzek / Joseph D Maman / Luca Pellegrini /
Abstract: The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-are under intense ...The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-are under intense current scrutiny to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent proteomic screens of the interactions between viral and host proteins have identified the human proteins targeted by SARS-CoV-2. The DNA polymerase α (Pol α)-primase complex or primosome-responsible for initiating DNA synthesis during genomic duplication-was identified as a target of nonstructural protein 1 (nsp1), a major virulence factor in the SARS-CoV-2 infection. Here, we validate the published reports of the interaction of nsp1 with the primosome by demonstrating direct binding with purified recombinant components and providing a biochemical characterization of their interaction. Furthermore, we provide a structural basis for the interaction by elucidating the cryo-electron microscopy structure of nsp1 bound to the primosome. Our findings provide biochemical evidence for the reported targeting of Pol α by the virulence factor nsp1 and suggest that SARS-CoV-2 interferes with Pol α's putative role in the immune response during the viral infection.
History
DepositionJun 1, 2021-
Header (metadata) releaseNov 10, 2021-
Map releaseNov 10, 2021-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.008
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  • Surface view with fitted model
  • Atomic models: PDB-7opl
  • Surface level: 0.008
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13020.png

Downloads & links

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Map

FileDownload / File: emd_13020.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D refinement map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 160 pix.
= 210.24 Å		1.31 Å/pix.
x 160 pix.
= 210.24 Å		1.31 Å/pix.
x 160 pix.
= 210.24 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.314 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.007 / Movie #1: 0.008
Minimum - Maximum-0.015819203 - 0.075377904
Average (Standard dev.)0.0004861129 (±0.0034148486)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 210.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3141.3141.314
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z210.240210.240210.240
α/β/γ90.00090.00090.000
start NX/NY/NZ7810892
NX/NY/NZ127111124
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.0160.0750.000

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Supplemental data

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Additional map: Sharpened map, calculated from the two half maps...

Fileemd_13020_additional_1.map
AnnotationSharpened map, calculated from the two half maps using local, anisotropic sharpening in Phenix.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map #1

Fileemd_13020_half_map_1.map
AnnotationHalf map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map #2

Fileemd_13020_half_map_2.map
AnnotationHalf map #2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of DNA polymerase alpha - primase bound to SARS COV-2 nsp1

EntireName: Complex of DNA polymerase alpha - primase bound to SARS COV-2 nsp1
Components
  • Complex: Complex of DNA polymerase alpha - primase bound to SARS COV-2 nsp1
    • Protein or peptide: DNA polymerase alpha catalytic subunit
    • Protein or peptide: DNA polymerase alpha subunit BDNA polymerase
    • Protein or peptide: DNA primase small subunitPrimase
    • Protein or peptide: DNA primase large subunitPrimase
    • Protein or peptide: Non-structural protein 1
  • Ligand: ZINC ION
  • Ligand: IRON/SULFUR CLUSTERIron–sulfur cluster

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Supramolecule #1: Complex of DNA polymerase alpha - primase bound to SARS COV-2 nsp1

SupramoleculeName: Complex of DNA polymerase alpha - primase bound to SARS COV-2 nsp1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 310 KDa

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Macromolecule #1: DNA polymerase alpha catalytic subunit

MacromoleculeName: DNA polymerase alpha catalytic subunit / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 133.702562 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSAWSHPQFE KGGGSGGGSG GGSWSHPQFE KLEVLFQGPE FGADEEQVFH FYWLDAYEDQ YNQPGVVFLF GKVWIESAET HVSCCVMVK NIERTLYFLP REMKIDLNTG KETGTPISMK DVYEEFDEKI ATKYKIMKFK SKPVEKNYAF EIPDVPEKSE Y LEVKYSAE ...String:
MSAWSHPQFE KGGGSGGGSG GGSWSHPQFE KLEVLFQGPE FGADEEQVFH FYWLDAYEDQ YNQPGVVFLF GKVWIESAET HVSCCVMVK NIERTLYFLP REMKIDLNTG KETGTPISMK DVYEEFDEKI ATKYKIMKFK SKPVEKNYAF EIPDVPEKSE Y LEVKYSAE MPQLPQDLKG ETFSHVFGTN TSSLELFLMN RKIKGPCWLE VKSPQLLNQP VSWCKVEAMA LKPDLVNVIK DV SPPPLVV MAFSMKTMQN AKNHQNEIIA MAALVHHSFA LDKAAPKPPF QSHFCVVSKP KDCIFPYAFK EVIEKKNVKV EVA ATERTL LGFFLAKVHK IDPDIIVGHN IYGFELEVLL QRINVCKAPH WSKIGRLKRS NMPKLGGRSG FGERNATCGR MICD VEISA KELIRCKSYH LSELVQQILK TERVVIPMEN IQNMYSESSQ LLYLLEHTWK DAKFILQIMC ELNVLPLALQ ITNIA GNIM SRTLMGGRSE RNEFLLLHAF YENNYIVPDK QIFRKPQQKL GDEDEEIDGD TNKYKKGRKK AAYAGGLVLD PKVGFY DKF ILLLDFNSLY PSIIQEFNIC FTTVQRVASE AQKVTEDGEQ EQIPELPDPS LEMGILPREI RKLVERRKQV KQLMKQQ DL NPDLILQYDI RQKALKLTAN SMYGCLGFSY SRFYAKPLAA LVTYKGREIL MHTKEMVQKM NLEVIYGDTD SIMINTNS T NLEEVFKLGN KVKSEVNKLY KLLEIDIDGV FKSLLLLKKK KYAALVVEPT SDGNYVTKQE LKGLDIVRRD WCDLAKDTG NFVIGQILSD QSRDTIVENI QKRLIEIGEN VLNGSVPVSQ FEINKALTKD PQDYPDKKSL PHVHVALWIN SQGGRKVKAG DTVSYVICQ DGSNLTASQR AYAPEQLQKQ DNLTIDTQYY LAQQIHPVVA RICEPIDGID AVLIATWLGL DPTQFRVHHY H KDEENDAL LGGPAQLTDE EKYRDCERFK CPCPTCGTEN IYDNVFDGSG TDMEPSLYRC SNIDCKASPL TFTVQLSNKL IM DIRRFIK KYYDGWLICE EPTCRNRTRH LPLQFSRTGP LCPACMKATL QPEYSDKSLY TQLCFYRYIF DAECALEKLT TDH EKDKLK KQFFTPKVLQ DYRKLKNTAE QFLSRSGYSE VNLSKLFAGC AVKS

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Macromolecule #2: DNA polymerase alpha subunit B

MacromoleculeName: DNA polymerase alpha subunit B / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 49.855434 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSSFSPSAT PSQKYNSRSN RGEVVTSFGL AQGVSWSGRG GAGNISLKVL GCPEALTGSY KSMFQKLPDI REVLTCKIEE LGSELKEHY KIEAFTPLLA PAQEPVTLLG QIGCDSNGKL NNKSVILEGD REHSSGAQIP VDLSELKEYS LFPGQVVIME G INTTGRKL ...String:
MGSSFSPSAT PSQKYNSRSN RGEVVTSFGL AQGVSWSGRG GAGNISLKVL GCPEALTGSY KSMFQKLPDI REVLTCKIEE LGSELKEHY KIEAFTPLLA PAQEPVTLLG QIGCDSNGKL NNKSVILEGD REHSSGAQIP VDLSELKEYS LFPGQVVIME G INTTGRKL VATKLYEGVP LPFYQPTEED ADFEQSMVLV ACGPYTTSDS ITYDPLLDLI AVINHDRPDV CILFGPFLDA KH EQVENCL LTSPFEDIFK QCLRTIIEGT RSSGSHLVFV PSLRDVHHEP VYPQPPFSYS DLSREDKKQV QFVSEPCSLS ING VIFGLT STDLLFHLGA EEISSSSGTS DRFSRILKHI LTQRSYYPLY PPQEDMAIDY ESFYVYAQLP VTPDVLIIPS ELRY FVKDV LGCVCVNPGR LTKGQVGGTF ARLYLRRPAA DGAERQSPCI AVQVVRI

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Macromolecule #3: DNA primase small subunit

MacromoleculeName: DNA primase small subunit / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 52.590801 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MHHHHHHHHH HGENLYFQGT SMETFDPTEL PELLKLYYRR LFPYSQYYRW LNYGGVIKNY FQHREFSFTL KDDIYIRYQS FNNQSDLEK EMQKMNPYKI DIGAVYSHRP NQHNTVKLGA FQAQEKELVF DIDMTDYDDV RRCCSSADIC PKCWTLMTMA I RIIDRALK ...String:
MHHHHHHHHH HGENLYFQGT SMETFDPTEL PELLKLYYRR LFPYSQYYRW LNYGGVIKNY FQHREFSFTL KDDIYIRYQS FNNQSDLEK EMQKMNPYKI DIGAVYSHRP NQHNTVKLGA FQAQEKELVF DIDMTDYDDV RRCCSSADIC PKCWTLMTMA I RIIDRALK EDFGFKHRLW VYSGRRGVHC WVCDESVRKL SSAVRSGIVE YLSLVKGGQD VKKKVHLSEK IHPFIRKSIN II KKYFEEY ALVNQDILEN KESWDKILAL VPETIHDELQ QSFQKSHNSL QRWEHLKKVA SRYQNNIKND KYGPWLEWEI MLQ YCFPRL DINVSKGINH LLKSPFSVHP KTGRISVPID LQKVDQFDPF TVPTISFICR ELDAISTNEE EKEENEAESD VKHR TRDYK KTSLAPYVKV FEHFLENLDK SRKGELLKKS DLQKDF

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Macromolecule #4: DNA primase large subunit

MacromoleculeName: DNA primase large subunit / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 58.890918 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEFSGRKWRK LRLAGDQRNA SYPHCLQFYL QPPSENISLI EFENLAIDRV KLLKSVENLG VSYVKGTEQY QSKLESELRK LKFSYRENL EDEYEPRRRD HISHFILRLA YCQSEELRRW FIQQEMDLLR FRFSILPKDK IQDFLKDSQL QFEAISDEEK T LREQEIVA ...String:
MEFSGRKWRK LRLAGDQRNA SYPHCLQFYL QPPSENISLI EFENLAIDRV KLLKSVENLG VSYVKGTEQY QSKLESELRK LKFSYRENL EDEYEPRRRD HISHFILRLA YCQSEELRRW FIQQEMDLLR FRFSILPKDK IQDFLKDSQL QFEAISDEEK T LREQEIVA SSPSLSGLKL GFESIYKIPF ADALDLFRGR KVYLEDGFAY VPLKDIVAII LNEFRAKLSK ALALTARSLP AV QSDERLQ PLLNHLSHSY TGQDYSTQGN VGKISLDQID LLSTKSFPPC MRQLHKALRE NHHLRHGGRM QYGLFLKGIG LTL EQALQF WKQEFIKGKM DPDKFDKGYS YNIRHSFGKE GKRTDYTPFS CLKIILSNPP SQGDYHGCPF RHSDPELLKQ KLQS YKISP GGISQILDLV KGTHYQVACQ KYFEMIHNVD DCGFSLNHPN QFFCESQRIL NGGKDIKKEP IQPETPQPKP SVQKT KDAS SALASLNSSL EMDMEGLEDY FSEDS

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Macromolecule #5: Non-structural protein 1

MacromoleculeName: Non-structural protein 1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Severe acute respiratory syndrome coronavirus
Molecular weightTheoretical: 12.955852 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
GSMGHVQLSL PVLQVRDVLV RGFGDSVEEA LSEAREHLKN GTCGLVELEK GVLPQLEQPY VFIKRSDALS TNHGHKVVEL VAEMDGIQY GRSGITLGVL VPHVGETPIA YRNVLLRKNG

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Macromolecule #6: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #7: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 7 / Number of copies: 1 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER / Iron–sulfur cluster

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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

Concentration0.3 mg/mL
BufferpH: 7.2
Component:
ConcentrationName
25.0 mMHepes
150.0 mMKCl
1.0 1mMDTT
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsThe nsp1 protein was added in 10-fold stoichiometric excess

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -0.0007 µm / Nominal defocus min: -0.0025 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 2919 / Average exposure time: 1.31 sec. / Average electron dose: 46.91 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 709068
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: OTHER
Details: The initial 3D model was generated using a Stochastic Gradient Descent algorithm as implemented in Relion 3.1.
Initial angle assignmentType: OTHER / Software - Name: RELION (ver. 3.1) / Details: As implemented in Relion 3.1.
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: OTHER / Software - Name: RELION (ver. 3.1) / Details: As implemented in Relion 3.1.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.12 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 233476

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Atomic model buiding 1

Initial model
PDB IDChain

5exr

chain_id: A

5exr

chain_id: B

5exr

chain_id: C

5exr

chain_id: D

7k3n

chain_id: E

2hsx

chain_id: E
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7opl:
CryoEM structure of DNA Polymerase alpha - primase bound to SARS CoV nsp1

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