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Yorodumi- PDB-8vd7: MicroED structure of SARS-CoV-2 main protease (MPro/3CLPro) with ... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8vd7 | ||||||||||||
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Title | MicroED structure of SARS-CoV-2 main protease (MPro/3CLPro) with missing cone eliminated by suspended drop | ||||||||||||
Components | 3C-like proteinase nsp5 | ||||||||||||
Keywords | VIRAL PROTEIN / Cysteine Protease / Viral Protease / Viral Assembly | ||||||||||||
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 / SARS coronavirus main proteinase / host cell endoplasmic reticulum-Golgi intermediate compartment / 3'-5'-RNA exonuclease activity / symbiont-mediated suppression of host NF-kappaB cascade / 5'-3' DNA helicase activity / 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 / mRNA (guanine-N7)-methyltransferase / omega peptidase activity / methyltransferase cap1 / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / SARS-CoV-2 modulates host translation machinery / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / DNA helicase / mRNA (nucleoside-2'-O-)-methyltransferase activity / mRNA 5'-cap (guanine-N7-)-methyltransferase activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / host cell perinuclear region of cytoplasm / single-stranded RNA binding / host cell endoplasmic reticulum membrane / viral protein processing / lyase activity / RNA helicase / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / induction by virus of host autophagy / viral translational frameshifting / RNA-directed RNA polymerase / copper ion binding / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / lipid binding / DNA-templated transcription / 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 | ELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.15 Å | ||||||||||||
Authors | Bu, G. / Gillman, C. / Danelius, E. / Hattne, J. / Nannenga, B.L. / Gonen, T. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: J Struct Biol X / Year: 2024 Title: Eliminating the missing cone challenge through innovative approaches. Authors: Cody Gillman / Guanhong Bu / Emma Danelius / Johan Hattne / Brent L Nannenga / Tamir Gonen / Abstract: Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle ...Microcrystal electron diffraction (MicroED) has emerged as a powerful technique for unraveling molecular structures from microcrystals too small for X-ray diffraction. However, a significant hurdle arises with plate-like crystals that consistently orient themselves flat on the electron microscopy grid. If the normal of the plate correlates with the axes of the crystal lattice, the crystal orientations accessible for measurement are restricted because the crystal cannot be arbitrarily rotated. This limits the information that can be acquired, resulting in a missing cone of information. We recently introduced a novel crystallization strategy called suspended drop crystallization and proposed that crystals in a suspended drop could effectively address the challenge of preferred crystal orientation. Here we demonstrate the success of the suspended drop approach in eliminating the missing cone in two samples that crystallize as thin plates: bovine liver catalase and the SARS‑CoV‑2 main protease (Mpro). This innovative solution proves indispensable for crystals exhibiting systematic preferred orientations, unlocking new possibilities for structure determination by MicroED. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8vd7.cif.gz | 76.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8vd7.ent.gz | 52.7 KB | Display | PDB format |
PDBx/mmJSON format | 8vd7.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8vd7_validation.pdf.gz | 955.6 KB | Display | wwPDB validaton report |
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Full document | 8vd7_full_validation.pdf.gz | 958 KB | Display | |
Data in XML | 8vd7_validation.xml.gz | 13.9 KB | Display | |
Data in CIF | 8vd7_validation.cif.gz | 19.7 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/vd/8vd7 ftp://data.pdbj.org/pub/pdb/validation_reports/vd/8vd7 | HTTPS FTP |
-Related structure data
Related structure data | 43144MC 9bdjC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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Unit cell |
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Components on special symmetry positions |
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-Components
#1: Protein | Mass: 33825.547 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 (E. coli) References: UniProt: P0DTD1, SARS coronavirus main proteinase |
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#2: Chemical | ChemComp-CL / |
#3: Water | ChemComp-HOH / |
Has ligand of interest | N |
-Experimental details
-Experiment
Experiment | Method: ELECTRON CRYSTALLOGRAPHY |
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EM experiment | Aggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography |
-Sample preparation
Component | Name: SARS-CoV-2 main protease (Mpro) / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Source (natural) | Organism: Severe acute respiratory syndrome coronavirus 2 |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 6.5 / Details: 0.1 M MES pH 6.5, 20% PEG 3350, 5% DMSO. |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293 K |
-Data collection
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company | ||||||||||||||||||||||||||||||||||||||||||
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Microscopy | Model: FEI TITAN KRIOS | ||||||||||||||||||||||||||||||||||||||||||
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM | ||||||||||||||||||||||||||||||||||||||||||
Electron lens | Mode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm | ||||||||||||||||||||||||||||||||||||||||||
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 85 K / Temperature (min): 75 K | ||||||||||||||||||||||||||||||||||||||||||
Image recording | Average exposure time: 1 sec. / Electron dose: 0.0025 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 420 | ||||||||||||||||||||||||||||||||||||||||||
EM diffraction | Camera length: 2480 mm / Tilt angle list: -40,70 | ||||||||||||||||||||||||||||||||||||||||||
EM diffraction shell |
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EM diffraction stats | Fourier space coverage: 95.5 % / High resolution: 2.15 Å / Num. of intensities measured: 123734 / Num. of structure factors: 14825 / Phase error rejection criteria: none / Rmerge: 25.6 | ||||||||||||||||||||||||||||||||||||||||||
Reflection | Biso Wilson estimate: 32.44 Å2 |
-Processing
Software | Name: REFMAC / Version: 5.8.0425 / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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EM 3D crystal entity | ∠α: 90 ° / ∠β: 101.074 ° / ∠γ: 90 ° / A: 115.54 Å / B: 55.53 Å / C: 45.37 Å / Space group name: C2 / Space group num: 5 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.15 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Target criteria: Rwork/Rfree gap of less than 5% | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Method to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.15→44.525 Å / Cor.coef. Fo:Fc: 0.957 / Cor.coef. Fo:Fc free: 0.938 / SU B: 12.217 / SU ML: 0.271 / Cross valid method: FREE R-VALUE / ESU R: 0.338 / ESU R Free: 0.226
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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 BULK SOLVENT | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 44.804 Å2
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