EMDB-13021: CryoEM structure of DNA polymerase alpha - primase bound to SARS ...

Basic information

• Entry: Database: EMDB / ID: EMD-13021
• Title: CryoEM structure of DNA polymerase alpha - primase bound to SARS CoV nsp1 protein
• Map data: 3D refinement map

Sample

• Complex: CryoEM structure of DNA polymerase alpha - primase bound to SARS CoV nsp1 protein

Function / homology

Function:

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 / alpha DNA polymerase:primase complex / regulation of type I interferon production / Processive synthesis on the lagging strand / Assembly of the SARS-CoV-1 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / Removal of the Flap Intermediate / Transcription of SARS-CoV-1 sgRNAs / DNA primase activity / Polymerase switching on the C-strand of the telomere / lagging strand elongation / mitotic DNA replication initiation / DNA replication, synthesis of primer / 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 / K63-linked deubiquitinase activity / leading strand elongation / DNA synthesis involved in DNA repair / G1/S-Specific Transcription / DNA replication origin binding / SARS-CoV-1 modulates host translation machinery / DNA replication initiation / Activation of the pre-replicative complex / viral genome replication / Defective pyroptosis / methyltransferase activity / 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 / 4 iron, 4 sulfur cluster binding / methylation / 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 / DNA-directed DNA polymerase / single-stranded RNA binding / 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

Domain/homology:

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

Component:

DNA polymerase alpha catalytic subunit / Replicase polyprotein 1a / DNA primase small subunit / DNA primase large subunit / DNA polymerase alpha subunit B

• Biological species: Homo sapiens (human)
• Method: single particle reconstruction / cryo EM / Resolution: 4.4 Å
• Authors: Kilkenny ML / Pellegrini L

Funding support

United Kingdom, 1 items

Organization	Grant number	Country
Wellcome Trust	104641/Z/14/Z	United Kingdom

• Citation: Journal: 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 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

Deposition	Jun 1, 2021	-
Header (metadata) release	Nov 10, 2021	-
Map release	Nov 10, 2021	-
Update	Feb 23, 2022	-
Current status	Feb 23, 2022	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_13021.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: 3D refinement map
• Voxel size: X=Y=Z: 1.304 Å

Density

Contour Level	By AUTHOR: 0.0075 / Movie #1: 0.0075
Minimum - Maximum	-0.015939752 - 0.08067985
Average (Standard dev.)	0.0005205729 (±0.003353054)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 160 160 160 Spacing 160 160 160
Cell	A=B=C: 208.64 Å α=β=γ: 90.0 °

CCP4 map header:

mode	Image stored as Reals
Å/pix. X/Y/Z	1.304	1.304	1.304
M x/y/z	160	160	160
origin x/y/z	0.000	0.000	0.000
length x/y/z	208.640	208.640	208.640
α/β/γ	90.000	90.000	90.000
MAP C/R/S	1	2	3
start NC/NR/NS	0	0	0
NC/NR/NS	160	160	160
D min/max/mean	-0.016	0.081	0.001

Supplemental data

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

• File: emd_13021_additional_1.map
• Annotation: Sharpened map, calculated from the the two half maps using local, anisotropic sharpening in Phenix.

Half map: Half map #1

• File: emd_13021_half_map_1.map
• Annotation: Half map #1

Half map: half map #2

• File: emd_13021_half_map_2.map
• Annotation: half map #2

Sample components

Entire : CryoEM structure of DNA polymerase alpha - primase bound to SARS ...

• Entire: Name: CryoEM structure of DNA polymerase alpha - primase bound to SARS CoV nsp1 protein

Components

• Complex: CryoEM structure of DNA polymerase alpha - primase bound to SARS CoV nsp1 protein

Supramolecule #1: CryoEM structure of DNA polymerase alpha - primase bound to SARS ...

• Supramolecule: Name: CryoEM structure of DNA polymerase alpha - primase bound to SARS CoV nsp1 protein; type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
• Source (natural): Organism: Homo sapiens (human)
• Recombinant expression: Organism: Spodoptera frugiperda (fall armyworm)
• Molecular weight: Theoretical: 310 KDa

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.3 mg/mL

Buffer

pH: 7.2
Component:

Concentration	Name
25.0 mM	Hepes
150.0 mM	KCl
1.0 mM	DTT

• Grid: Model: 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
• Vitrification: Cryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
• Details: The nsp1 protein was added in 10-fold stoichiometric excess

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -2.5 µm / Nominal defocus min: -0.7000000000000001 µm / Nominal magnification: 130000
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Image recording: Film 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/Ų
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 709068
• CTF correction: Software - Name: CTFFIND (ver. 4.1)
• Startup model: Type of model: OTHER; Details: The initial 3D model was generated using a Stochastic Gradient Descent algorithm as implemented in Relion 3.1.
• Initial angle assignment: Type: OTHER / Software - Name: RELION (ver. 3.1) / Details: As implemented in Relion 3.1.
• Final 3D classification: Software - Name: RELION (ver. 3.1)
• Final angle assignment: Type: OTHER / Software - Name: RELION (ver. 3.1) / Details: As implemented in Relion 3.1.
• Final reconstruction: Applied symmetry - Point group: C₁ (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 233476

Atomic model buiding 1

Initial model

(PDB ID:

5exr

,

7k3n

,

2hsx

)

• Refinement: Space: REAL / Protocol: RIGID BODY FIT