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Yorodumi- PDB-7jpe: Room Temperature Structure of SARS-CoV-2 Nsp10/Nsp16 Methyltransf... -
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-Basic information
Entry | Database: PDB / ID: 7jpe | |||||||||
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Title | Room Temperature Structure of SARS-CoV-2 Nsp10/Nsp16 Methyltransferase in a Complex with m7GpppA Cap-0 and SAM Determined by Fixed-Target Serial Crystallography | |||||||||
Components |
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Keywords | VIRAL PROTEIN / SARS CoV-2 / Serial Crystallography / Methyltransferase / m7GpppA / Cap-0 / S-adenosylhomomethionine / Structural Genomics / Center for Structural Genomics of Infectious Diseases / CSGID | |||||||||
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 / Translation of Replicase and Assembly of the Replication Transcription Complex / TRAF3-dependent IRF activation pathway / 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 / 5'-3' DNA helicase activity / SARS coronavirus main proteinase / host cell endosome / host cell endoplasmic reticulum-Golgi intermediate compartment / 3'-5'-RNA exonuclease activity / 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 / SARS-CoV-2 modulates host translation machinery / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / mRNA (guanine-N7)-methyltransferase / host cell Golgi apparatus / methyltransferase cap1 / symbiont-mediated perturbation of host ubiquitin-like protein modification / mRNA (nucleoside-2'-O-)-methyltransferase activity / DNA helicase / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / single-stranded RNA binding / host cell endoplasmic reticulum membrane / host cell perinuclear region of cytoplasm / viral protein processing / lyase activity / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / RNA helicase / induction by virus of host autophagy / copper ion binding / RNA-directed RNA polymerase / 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 | |||||||||
Biological species | Severe acute respiratory syndrome coronavirus 2 | |||||||||
Method | X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.18 Å | |||||||||
Authors | Wilamowski, M. / Sherrell, D.A. / Minasov, G. / Kim, Y. / Shuvalova, L. / Lavens, A. / Chard, R. / Rosas-Lemus, M. / Maltseva, N. / Jedrzejczak, R. ...Wilamowski, M. / Sherrell, D.A. / Minasov, G. / Kim, Y. / Shuvalova, L. / Lavens, A. / Chard, R. / Rosas-Lemus, M. / Maltseva, N. / Jedrzejczak, R. / Michalska, K. / Satchell, K.J.F. / Joachimiak, A. / Center for Structural Genomics of Infectious Diseases (CSGID) | |||||||||
Citation | Journal: Proc.Natl.Acad.Sci.USA / Year: 2021 Title: 2'-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography. Authors: Wilamowski, M. / Sherrell, D.A. / Minasov, G. / Kim, Y. / Shuvalova, L. / Lavens, A. / Chard, R. / Maltseva, N. / Jedrzejczak, R. / Rosas-Lemus, M. / Saint, N. / Foster, I.T. / Michalska, K. ...Authors: Wilamowski, M. / Sherrell, D.A. / Minasov, G. / Kim, Y. / Shuvalova, L. / Lavens, A. / Chard, R. / Maltseva, N. / Jedrzejczak, R. / Rosas-Lemus, M. / Saint, N. / Foster, I.T. / Michalska, K. / Satchell, K.J.F. / Joachimiak, A. | |||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7jpe.cif.gz | 186.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7jpe.ent.gz | 146.2 KB | Display | PDB format |
PDBx/mmJSON format | 7jpe.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7jpe_validation.pdf.gz | 2 MB | Display | wwPDB validaton report |
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Full document | 7jpe_full_validation.pdf.gz | 2 MB | Display | |
Data in XML | 7jpe_validation.xml.gz | 17.6 KB | Display | |
Data in CIF | 7jpe_validation.cif.gz | 24.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/jp/7jpe ftp://data.pdbj.org/pub/pdb/validation_reports/jp/7jpe | HTTPS FTP |
-Related structure data
Related structure data | 6xkmC 7jheC 7jibC 6wq3S C: citing same article (ref.) S: Starting model for refinement |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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Unit cell |
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-Components
-Protein , 2 types, 2 molecules AB
#1: Protein | Mass: 33627.504 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Severe acute respiratory syndrome coronavirus 2 Gene: rep, 1a-1b / Plasmid: pMCSG53 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Magic References: UniProt: P0DTD1, Transferases; Transferring one-carbon groups; Methyltransferases |
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#2: Protein | Mass: 15075.190 Da / Num. of mol.: 1 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 BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): Magic / References: UniProt: P0DTD1 |
-Non-polymers , 5 types, 132 molecules
#3: Chemical | ChemComp-GTA / | ||
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#4: Chemical | ChemComp-SAM / | ||
#5: Chemical | ChemComp-M7G / | ||
#6: Chemical | #7: Water | ChemComp-HOH / | |
-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: X-RAY DIFFRACTION / Number of used crystals: 1 |
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-Sample preparation
Crystal | Density Matthews: 4.49 Å3/Da / Density % sol: 72.6 % |
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Crystal grow | Temperature: 297 K / Method: batch mode / pH: 6.5 Details: Protein: 4.0 mg/ml (NSP10/NSP16 1:1), 0.15 M CaCl, 0.01 M Tris-HCl, 2 mM SAM, 1 mM TCEP, 5% Glycerol, pH 7.5. Precipitation buffer: 0.1M MES pH 6.5, 0.9 M NaF. Batch crystallization: 100 ul ...Details: Protein: 4.0 mg/ml (NSP10/NSP16 1:1), 0.15 M CaCl, 0.01 M Tris-HCl, 2 mM SAM, 1 mM TCEP, 5% Glycerol, pH 7.5. Precipitation buffer: 0.1M MES pH 6.5, 0.9 M NaF. Batch crystallization: 100 ul of protein mixed with 100 ul of precipitation buffer in 500 ul polypropylane tube. Two days before data collection 1 mM EDTA was added to batch crystallization. Crystal were soaked with m7GpppA (0.5 mM) for 10 minutes before data collection. |
-Data collection
Diffraction | Mean temperature: 293 K / Serial crystal experiment: Y |
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Diffraction source | Source: SYNCHROTRON / Site: APS / Beamline: 19-ID / Wavelength: 0.97918 Å |
Detector | Type: DECTRIS PILATUS3 X 6M / Detector: PIXEL / Date: Aug 2, 2020 Details: Sagittally focusing mono and vertically focusing mirror |
Radiation | Monochromator: double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray |
Radiation wavelength | Wavelength: 0.97918 Å / Relative weight: 1 |
Reflection | Resolution: 2.18→49.72 Å / Num. obs: 46273 / % possible obs: 100 % / Redundancy: 50.51 % / Biso Wilson estimate: 22.8 Å2 / CC1/2: 0.95 / Net I/σ(I): 2.55 |
Reflection shell | Resolution: 2.18→2.22 Å / Redundancy: 11.42 % / Mean I/σ(I) obs: 0.53 / Num. unique obs: 2290 / CC1/2: 0.398 / % possible all: 99.7 |
Serial crystallography sample delivery | Description: Nylon Mesh / Method: fixed target |
Serial crystallography sample delivery fixed target | Crystals per unit: 1 / Description: SBC Mesh Holder / Details: start/stop raster over area / Motion control: SmarAct Motors viaPMAC / Sample dehydration prevention: 6 um Mylar sandwich / Sample holding: mesh Sample solvent: 50 mM MES pH 6.5, 0.45 M NaF, 75 mM NaCl, 5 mM Tris-HCl, 1 mM SAM, 1 mM EDTA, 0.5 mM TCEP, 2.5% Glycerol, 0.5 mM m7GpppA Sample unit size: 90 µm / Support base: xyz stage / Velocity horizontal: 10 / Velocity vertical: 10 |
Serial crystallography data reduction | Crystal hits: 5734 / Frame hits: 74120 / Frames indexed: 5734 / Frames total: 74120 / Lattices indexed: 4704 |
-Processing
Software |
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Refinement | Method to determine structure: MOLECULAR REPLACEMENT Starting model: 6WQ3 Resolution: 2.18→49.72 Å / Cor.coef. Fo:Fc: 0.937 / Cor.coef. Fo:Fc free: 0.923 / SU B: 21.975 / SU ML: 0.237 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.163 / ESU R Free: 0.147 / Stereochemistry target values: MAXIMUM LIKELIHOOD Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
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Solvent computation | Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso max: 140.52 Å2 / Biso mean: 37.222 Å2 / Biso min: 4.16 Å2
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Refinement step | Cycle: final / Resolution: 2.18→49.72 Å
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Refine LS restraints |
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LS refinement shell | Resolution: 2.18→2.237 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
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Refinement TLS params. | Method: refined / Refine-ID: X-RAY DIFFRACTION
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Refinement TLS group |
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