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 / 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 / Assembly of the SARS-CoV-1 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / Transcription of SARS-CoV-1 sgRNAs / Polymerase switching / : / Processive synthesis on the lagging strand / DNA replication, synthesis of primer / lagging strand elongation / Removal of the Flap Intermediate / Polymerase switching on the C-strand of the telomere / 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 / host cell endoplasmic reticulum / K63-linked deubiquitinase activity / DNA synthesis involved in DNA repair / DNA strand elongation involved in DNA replication / leading strand elongation / G1/S-Specific Transcription / SARS-CoV-1 modulates host translation machinery / DNA replication origin binding / Activation of the pre-replicative complex / DNA replication initiation / viral genome replication / Defective pyroptosis / methyltransferase activity / double-strand break repair via nonhomologous end joining / nuclear matrix / protein import into nucleus / SARS-CoV-1 activates/modulates innate immune responses / nuclear envelope / single-stranded DNA binding / 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 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / omega peptidase activity / endonuclease activity / host cell Golgi apparatus / methylation / symbiont-mediated perturbation of host ubiquitin-like protein modification / ubiquitinyl hydrolase 1 / DNA-directed DNA polymerase / cysteine-type deubiquitinase activity / DNA-directed DNA polymerase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / DNA replication / single-stranded RNA binding / regulation of autophagy / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / ciliary basal body / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / symbiont-mediated suppression of host gene expression / viral translational frameshifting / symbiont-mediated activation of host autophagy / cysteine-type endopeptidase activity / DNA repair / RNA-directed RNA polymerase activity / nucleotide binding / intracellular membrane-bounded organelle / chromatin binding / protein kinase binding / chromatin / nucleolus / magnesium ion binding / proteolysis / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / identical protein binding / nucleus / membrane / cytosol

Domain/homology:

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 / 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, multifunctional domain / 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 / 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 / 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 / NSP1, C-terminal domain, betacoronavirus / : / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / Betacoronavirus Nsp3e group 2-specific marker (G2M) 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, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Non-structural protein NSP3, nucleic acid-binding domain superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Papain-like viral protease, palm and finger domains, coronavirus / Papain-like protease, N-terminal domain superfamily, coronavirus / Coronavirus replicase NSP2, N-terminal / : / Coronavirus replicase NSP2, C-terminal / Coronavirus (CoV) Nsp2 middle domain profile. / NSP1, globular domain, alpha/betacoronavirus / Coronavirus (CoV) Nsp1 globular domain profile. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / Nonstructural protein 2, N-terminal domain, coronavirus / Non-structural protein 2, C-terminal domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / Peptidase family C16 domain profile. / : / : / Coronavirus replicase NSP7 / Coronavirus 3Ecto 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. / Coronavirus (CoV) Nsp3 Y domain profile. / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / Coronavirus main protease (M-pro) domain profile. / Peptidase C30, coronavirus / Peptidase C16, coronavirus / Non-structural protein NSP9, coronavirus / Non-structural protein NSP7, 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 / Papain-like protease, thumb domain superfamily, coronavirus / Peptidase C30, domain 3, coronavirus

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
• 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
• 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 FIELD / 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
• 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.
• 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
• Initial angle assignment: Type: OTHER / Software - Name: RELION (ver. 3.1) / Details: As implemented in Relion 3.1.
• Final 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)

Atomic model buiding 1

Initial model

(PDB ID:

5exr

,

7k3n

,

2hsx

)

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