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- PDB-8e4r: Room-temperature X-ray structure of SARS-CoV-2 main protease H41A... -

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Basic information

Entry
Database: PDB / ID: 8e4r
TitleRoom-temperature X-ray structure of SARS-CoV-2 main protease H41A miniprecursor mutant in complex with GC373
ComponentsReplicase polyprotein 1ab
KeywordsHYDROLASE / homodimer / cysteine protease / precursor enzyme / H41A mutation
Function / homology
Function and homology information


protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs ...protein guanylyltransferase activity / RNA endonuclease activity, producing 3'-phosphomonoesters / mRNA guanylyltransferase activity / 5'-3' RNA helicase activity / Lyases; Phosphorus-oxygen lyases / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of TBK1 activity / Assembly of the SARS-CoV-2 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / ISG15-specific peptidase activity / Transcription of SARS-CoV-2 sgRNAs / TRAF3-dependent IRF activation pathway / Translation of Replicase and Assembly of the Replication Transcription Complex / Replication of the SARS-CoV-2 genome / snRNP Assembly / double membrane vesicle viral factory outer membrane / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / SARS coronavirus main proteinase / host cell endoplasmic reticulum-Golgi intermediate compartment / 3'-5'-RNA exonuclease activity / 5'-3' DNA helicase activity / symbiont-mediated suppression of host NF-kappaB cascade / host cell endosome / symbiont-mediated suppression of host toll-like receptor signaling pathway / 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 / SARS-CoV-2 modulates host translation machinery / mRNA (guanine-N7)-methyltransferase / methyltransferase cap1 / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA helicase / mRNA (nucleoside-2'-O-)-methyltransferase activity / ubiquitinyl hydrolase 1 / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell perinuclear region of cytoplasm / viral protein processing / host cell endoplasmic reticulum membrane / lyase activity / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / induction by virus of host autophagy / copper ion binding / RNA-directed RNA polymerase / viral translational frameshifting / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / DNA-templated transcription / lipid binding / host cell nucleus / SARS-CoV-2 activates/modulates innate and adaptive immune responses / ATP hydrolysis activity / proteolysis / RNA binding / zinc ion binding / ATP binding / membrane
Similarity search - Function
RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : ...RNA-dependent RNA polymerase, SARS-CoV-like / Nonstructural protein 14, betacoronavirus / NSP15, NendoU domain, coronavirus / Nonstructural protein 15, middle domain, alpha/betacoronavirus / Nonstructural protein 15, N-terminal domain, alpha/beta-coronavirus / NSP14, guanine-N7-methyltransferase domain, coronavirus / Coronavirus (CoV) guanine-N7-methyltransferase (N7-MTase) domain profile. / Coronavirus (CoV) Nsp15 N-terminal oligomerization domain profile. / NSP12 RNA-dependent RNA polymerase, coronavirus / : / : / : / Coronavirus Nonstructural protein 13, 1B domain / Coronavirus Non-structural protein 13, zinc-binding domain / Coronavirus Nonstructural protein 13, stalk domain / Coronavirus Nsp12 RNA-dependent RNA polymerase (RdRp) domain profile. / Coronavirus Nsp12 Interface domain profile. / Non-structural protein NSP15, N-terminal domain superfamily, coronavirus / Non-structural protein NSP15, middle domain superfamily / Coronavirus replicase NSP15, N-terminal oligomerization / Nonstructural protein 15, middle domain, coronavirus / Nonstructural protein 13, 1B domain, coronavirus / Nidovirus 2-O-methyltransferase / Coronavirus replicase NSP15, middle domain / Coronavirus replicase NSP15, N-terminal oligomerisation / Nidovirus 2'-O-methyltransferase (2'-O-MTase) domain profile. / Non-structural protein NSP16, coronavirus-like / Non-structural protein 14, coronavirus / RNA polymerase, N-terminal, coronavirus / Nidovirus 3'-5' exoribonuclease domain / Coronavirus 2'-O-methyltransferase / Coronavirus proofreading exoribonuclease / Coronavirus RNA-dependent RNA polymerase, N-terminal / Nidovirus 3'-5' exoribonuclease (ExoN) domain profile. / Nonstructural protein 13, zinc-binding domain, coronavirus-like / Coronaviridae zinc-binding (CV ZBD) domain profile. / Arterivirus Nsp11 N-terminal/Coronavirus NSP15 middle domain / Arterivirus Nsp11 N-terminal/coronavirus NSP15 middle (AV-Nsp11N/CoV-Nsp15M) domain profile. / Nidoviral uridylate-specific endoribonuclease (NendoU) domain profile. / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain / Nidovirus RdRp-associated nucleotidyl transferase (NiRAN) domain profile. / Endoribonuclease EndoU-like / NendoU domain, nidovirus / Coronavirus replicase NSP15, uridylate-specific endoribonuclease / : / Lipocalin signature. / DNA2/NAM7 helicase-like, C-terminal / AAA domain / (+) RNA virus helicase core domain / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / (+)RNA virus helicase core domain profile. / 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 / Carbamoyl-phosphate synthase subdomain signature 2. / 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 / NSP1, globular domain, alpha/betacoronavirus / Coronavirus (CoV) Nsp2 middle domain profile. / 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 / : / : / Coronavirus replicase NSP7 / Peptidase family C16 domain profile. / Coronavirus 3Ecto domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile.
Similarity search - Domain/homology
Chem-K36 / Replicase polyprotein 1ab
Similarity search - Component
Biological speciesSevere acute respiratory syndrome coronavirus 2
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsCoates, L. / Kneller, D.W. / Kovalevsky, A.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: J.Mol.Biol. / Year: 2022
Title: Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor.
Authors: Kovalevsky, A. / Coates, L. / Kneller, D.W. / Ghirlando, R. / Aniana, A. / Nashed, N.T. / Louis, J.M.
History
DepositionAug 18, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 23, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 30, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.title
Revision 1.2Oct 25, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.3Oct 23, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Replicase polyprotein 1ab
B: Replicase polyprotein 1ab
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,9784
Polymers69,0072
Non-polymers9712
Water3,693205
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2290 Å2
ΔGint-9 kcal/mol
Surface area25740 Å2
MethodPISA
Unit cell
Length a, b, c (Å)55.843, 100.126, 59.957
Angle α, β, γ (deg.)90.00, 108.34, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Replicase polyprotein 1ab / pp1ab / ORF1ab polyprotein


Mass: 34503.262 Da / Num. of mol.: 2 / Mutation: Q0E, H41A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2
Gene: rep, 1a-1b / Production host: Escherichia coli (E. coli) / References: UniProt: P0DTD1
#2: Chemical ChemComp-K36 / (1S,2S)-2-({N-[(benzyloxy)carbonyl]-L-leucyl}amino)-1-hydroxy-3-[(3S)-2-oxopyrrolidin-3-yl]propane-1-sulfonic acid / GC376


Type: peptide-like / Mass: 485.551 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H31N3O8S / Feature type: SUBJECT OF INVESTIGATION / Comment: medication, antivirus*YM
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 205 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.31 Å3/Da / Density % sol: 46.65 %
Crystal growTemperature: 287 K / Method: vapor diffusion, sitting drop / Details: 18-21% PEG3350, 0.1 M Bis-Tris pH 6.5 or pH 7.0

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Data collection

DiffractionMean temperature: 293 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 HF / Wavelength: 1.5418 Å
DetectorType: DECTRIS EIGER R 4M / Detector: PIXEL / Date: Oct 28, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.8→100.13 Å / Num. obs: 57453 / % possible obs: 99.2 % / Redundancy: 4.6 % / CC1/2: 0.992 / Rmerge(I) obs: 0.066 / Net I/σ(I): 21.18
Reflection shellResolution: 1.8→1.86 Å / Redundancy: 3.1 % / Rmerge(I) obs: 0.446 / Mean I/σ(I) obs: 1.83 / Num. unique obs: 5646 / CC1/2: 0.812 / % possible all: 97.4

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
CrysalisProdata reduction
CrysalisProdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6WQF

6wqf


Resolution: 1.8→28.24 Å / SU ML: 0.18 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 20.06 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.195 2780 4.85 %random
Rwork0.1648 ---
obs0.1663 57371 99.12 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.8→28.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4657 0 58 205 4920
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0074884
X-RAY DIFFRACTIONf_angle_d0.9296648
X-RAY DIFFRACTIONf_dihedral_angle_d7.82674
X-RAY DIFFRACTIONf_chiral_restr0.057751
X-RAY DIFFRACTIONf_plane_restr0.007862
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.830.30541110.25462681X-RAY DIFFRACTION97
1.83-1.860.30071250.2422682X-RAY DIFFRACTION98
1.86-1.90.24341300.21782681X-RAY DIFFRACTION98
1.9-1.940.25171470.20462736X-RAY DIFFRACTION98
1.94-1.980.23731470.19522681X-RAY DIFFRACTION99
1.98-2.030.24471550.1952663X-RAY DIFFRACTION99
2.03-2.080.22871500.19422728X-RAY DIFFRACTION99
2.08-2.130.19371480.17722712X-RAY DIFFRACTION99
2.13-2.20.22451510.17672671X-RAY DIFFRACTION99
2.2-2.270.211510.17152734X-RAY DIFFRACTION100
2.27-2.350.19231330.17142765X-RAY DIFFRACTION100
2.35-2.440.22741320.17952747X-RAY DIFFRACTION100
2.44-2.550.22871280.1782761X-RAY DIFFRACTION100
2.55-2.690.19971560.17792750X-RAY DIFFRACTION100
2.69-2.860.19331470.18092755X-RAY DIFFRACTION100
2.86-3.080.22581360.17092746X-RAY DIFFRACTION100
3.08-3.390.1791270.16692770X-RAY DIFFRACTION100
3.39-3.880.1541340.14252772X-RAY DIFFRACTION100
3.88-4.880.16671330.11762789X-RAY DIFFRACTION100
4.88-28.240.13891390.13622767X-RAY DIFFRACTION98

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