Helicase of Type 2 Porcine Reproductive and Respiratory Syndrome Virus Strain HV Reveals a Unique Structure
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cloning, Expression, and Purification of PRRSV nsp10
2.2. Reductive Methylation
2.3. DNA Preparations
2.4. Crystallization and Data Collection
2.5. Structure Determination
2.6. NTPase Assay
2.7. Unwinding Assay
2.8. Electrophoretic Mobility Shift Assay
2.9. Model Generation
2.10. Small-Angle X-ray Scattering
3. Results
3.1. Overall Domain Organization of HP-PRRSV nsp10
3.2. Biochemical Characterization of PRRSV nsp10
3.3. Structure of the ZBD of PRRSV nsp10
3.4. The Novel CTD Essential for the Overall Structure and Enzymatic Activities
3.5. Regions Critical for Enzymatic Activities
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Parameters | Methylated PRRSV nsp10 (6JDR) | PRRSV nsp10 (6JDU) | Truncated PRRSV nsp10 (6JDS) | |
---|---|---|---|---|
Data collection | Wavelength | 0.979 | 0.979 | 0.979 |
Space group | P43212 | P43212 | P43212 | |
Cell dimensions | a, b, c (Å) | 93.16, 93.16, 148.11 | 92.91, 92.91, 147.70 | 99.98, 99.98, 83.37 |
α, β, γ (°) | 90, 90, 90 | 90, 90, 90 | 90, 90, 90 | |
Resolution (Å) 1 | 50.00–2.50 (2.54–2.50) | 50.00–2.80 (2.85–2.80) | 50.00–2.50 (2.54–2.50) | |
Rmerge (%) | 9.9 (85.4) | 11.9 (70.3) | 8.0 (82.7) | |
I/σ | 31.1 (2.5) | 19.9 (2.7) | 36.4 (4.8) | |
Completeness (%) | 99.9 (99.9) | 97.7 (81.9) | 99.6 (93.6) | |
Total No. of reflections | 1492609 | 1424962 | 518654 | |
Unique reflections | 23333 | 16581 | 15236 | |
Redundancy | 64.0 | 85.9 | 34.0 | |
Refinement | Resolution (Å) | 50.00–2.50 (2.56–2.50) | 50.00–2.81 (2.88–2.81) | 50.00–2.50 (2.57–2.50) |
No. of reflections | 21997 (1513) | 15074 (877) | 14161 (853) | |
Rwork/Rfree (%) | 23.5/27.2 | 22.5/23.8 | 21.1/23.1 | |
No. of atoms | ||||
Protein | 3254 | 3162 | 1908 | |
Ligand/ions | 10 | 6 | 4 | |
Water | 193 | 168 | 174 | |
B-factors (Å2) | Protein | 65.9 | 51.0 | 38.7 |
Ligand/ion | 65.5 | 39.0 | 30.9 | |
Water | 55.0 | 35.0 | 38.1 | |
rms deviations | Bond lengths (Å) | 0.004 | 0.007 | 0.004 |
Bond angles (°) | 0.897 | 1.147 | 0.846 | |
Ramachandran Plot (%) 2 | 96.7/2.8/0.5 | 96.2/3.3/0.5 | 97.6/2.4/0 |
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Tang, C.; Deng, Z.; Li, X.; Yang, M.; Tian, Z.; Chen, Z.; Wang, G.; Wu, W.; Feng, W.-h.; Zhang, G.; et al. Helicase of Type 2 Porcine Reproductive and Respiratory Syndrome Virus Strain HV Reveals a Unique Structure. Viruses 2020, 12, 215. https://doi.org/10.3390/v12020215
Tang C, Deng Z, Li X, Yang M, Tian Z, Chen Z, Wang G, Wu W, Feng W-h, Zhang G, et al. Helicase of Type 2 Porcine Reproductive and Respiratory Syndrome Virus Strain HV Reveals a Unique Structure. Viruses. 2020; 12(2):215. https://doi.org/10.3390/v12020215
Chicago/Turabian StyleTang, Chenjun, Zengqin Deng, Xiaorong Li, Meiting Yang, Zizi Tian, Zhenhang Chen, Guoguo Wang, Wei Wu, Wen-hai Feng, Gongyi Zhang, and et al. 2020. "Helicase of Type 2 Porcine Reproductive and Respiratory Syndrome Virus Strain HV Reveals a Unique Structure" Viruses 12, no. 2: 215. https://doi.org/10.3390/v12020215