Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography
Abstract
:1. Introduction
2. Materials and Methods
2.1. Gene Construct Design, Protein Expression, and Purification
2.2. Crystallization of Protein with Drug by Using Soaking Method
2.3. Transport of Microcrystals
2.4. Injection of Microcrystals
2.5. Data Collection at LCLS
2.6. Data Processing
2.7. Structure Determination
3. Results
3.1. Serial Crystallography Based Faster High-Throughput Drug Screening at an XFEL
3.2. Determining Mpro Structures with Mitigated Radiation Damage at Near Physiological Temperature
3.3. Interpretation of Experimental Findings
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sample ID | PDB ID | Total Run Time | Effective Run Time | Runs | Run Number | Total Number Frames Collected | Total Number Frames with More Than 30 Bragg Reflections and a Signal to Noise Ratio > 4.5 | Hit Rate (%) | Total Number of Crystal Lattices Indexed to the Appropriate Unit Cell | Total Number of Crystal Lattices Merged into the Final SFX Dataset |
---|---|---|---|---|---|---|---|---|---|---|
Mpro3 | 7VJY | 5 h 20 min | 4 h 20 min | 20–80 | 60 | 1,465,292 | 279,428 | 19.07 | 73,605 | 68,314 |
Mpro4 | 7VK3 | 3 h 28 min | 2 h 59 min | 82–115 | 33 | 1,121,297 | 182,774 | 16.3 | 33,384 | 67,918 |
Mpro5 | 7CWB | 2 h 59 min | 2 h 52 min | 118–144 | 26 | 1,163,413 | 208,839 | 17.95 | 168,655 | 160,510 |
Mpro6 | 7VK4 | 2 h 18 min | 2 h 18 min | 235–257 | 22 | 790,492 | 229,422 | 29.02 | 315,158 | 313,250 |
Mpro7 | 7VK8 | 1 h 25 min | 45 min | 176–188 | 12 | 327,156 | 2078 | 0.63 | 1177 | 947 |
Mpro8 | N/A | N/A | N/A | N/A | N/A | N/A | 13 | N/A | N/A | N/A |
Mpro9 | 7VK1 | 3 h 04 min | 3 h 04 min | 145–174 | 29 | 1,148,580 | 51,112 | 4.45 | 33,384 | 30,732 |
Mpro10 | 7VK0 | 2 h 11 min | 2 h 11 min | 264–291 | 27 | 858,742 | 932,35 | 10.86 | 116,315 | 115,577 |
Mpro11 | 7VJZ | 2 h 07 min | 1 h 57 min | 189–213 | 24 | 669,237 | 132,447 | 19.79 | 70,272 | 65,579 |
Mpro12 | 7VK5 | 1 h 30 min | 1 h 30 min | 292–310 | 18 | 609,036 | 80,383 | 13.19 | 120,147 | 119,430 |
Mpro13 | 7VK2 | 2 h 47 min | 2 h 47 min | 216–234 | 18 | 873,323 | 180,625 | 20.68 | 88,551 | 81,563 |
Mpro14 | 7VJW | 1 h 12 min | 1 h 12 min | 312–234 | 7 | 192,352 | 15,899 | 8.26 | 22,470 | 21,278 |
Mpro15 | N/A | N/A | N/A | N/A | N/A | N/A | 0 | N/A | N/A | N/A |
Mpro16 | 7CWC | 1 h 53 min | 1 h 53 min | 320–340 | 20 | 686,808 | 214,355 | 31.21 | 15,7976 | 156,512 |
Mpro17 | 7VK7 | 1 h 28 min | 1 h 28 min | 341–357 | 16 | 439,830 | 86,490 | 19.66 | 174,120 | 173,796 |
Mpro18 | 7VJX | 1 h 22 min | 1 h 22 min | 358–372 | 15 | 466,014 | 111,815 | 23.99 | 170,214 | 169,695 |
Mpro19 | 7VK6 | 36 min | 36 min | 373–375 | 3 | 326,781 | 78,455 | 24.01 | 111,145 | 107,895 |
Dataset | Mpro3 | Mpro4 | Mpro5 | Mpro6 | Mpro7 |
---|---|---|---|---|---|
PDB ID | 7VJY | 7VK3 | 7CWB | 7VK4 | 7VK8 |
Data collection | |||||
Beamline | LCLS (MFX) | LCLS (MFX) | LCLS (MFX) | LCLS (MFX) | LCLS (MFX) |
Space group | C121 | P212121 | C121 | P212121 | C121 |
Cell dimensions | |||||
a, b, c (Å) | 114.0, 53.5, 45.0 | 69.3, 104.4, 105.7 | 114.0, 53.5, 45.0 | 68.9, 103.9, 105.2 | 115.3, 55.2, 45.7 |
α, β, γ (°) | 90.0, 102.0, 90.0 | 90.0, 90.0, 90.0 | 90.0, 102.0, 90.0 | 90.0, 90.0, 90.0 | 90.0, 101.3, 90.0 |
Resolution (Å) 1 | 48.24–1.73 (1.80–1.73) 1 | 46.8–2.14 (2.22–2.14) 1 | 55.0–1.9 (1.98–1.90) 1 | 46.9–2.14 (2.22–2.14) 1 | 31.92–2.34 (2.43–2.34) 1 |
Rsplit | 1.09 (36.1) | 1.12 (54.4) | 0.63 (0.94) | 0.49 (45.2) | 5.94 (13.5) |
CC1/2 | 0.998 (0.179) | 0.982 (0.089) | 0.996 (0.598) | 0.998 (0.331) | 0.477 (0.194) |
I/σI | 0.25 | 0.22 | 0.57 | 0.23 | 0.97 |
CC* | 0.997 (0.552) | 0.998 (0.405) | 0.999 (0.865) | 1.000 (0.705) | 0.804 (0.410) |
Completeness (%) | 100.0 (100.0) | 100.0 (26.0) | 100.0 (100.0) | 100.0 (100.0) | 99.2 (98.0) |
Redundancy | 387 | 1779 | 825 | 5066 | 9.2 |
Refinement | |||||
Resolution (Å) | 48.2–1.9 (1.95–1.90) 1 | 46.8–2.22 (2.29–2.22) 1 | 34.0–1.9 (1.95–1.90) 1 | 46.9–2.1 (2.15–2.10) 1 | 31.9–2.4 (2.51–2.40) 1 |
No. reflections | 21,030 (1359) | 42,380 (3277) | 21,029 (1359) | 44,742 (3028) | 11,028 (1197) |
Rwork/Rfree | 0.23/0.28 (0.49/0.52) | 0.24/0.28 (0.41/0.43) | 0.22/0.26 (0.43/0.49) | 0.21/0.23 (0.30/0.34) | 0.22/0.25 (0.34/0.41) |
No. atoms | |||||
Protein | 2427 | 4710 | 2447 | 4675 | 2435 |
Ligand/Ion/Water | 60 | 114 | 113 | 7 | 67 |
B-factors | |||||
Protein | 38.59 | 53.56 | 42.86 | 64.78 | 31.50 |
Ligand/Ion/Water | 38.02 | 52.13 | 51.0 | 32.72 | 24.57 |
Coordinate errors | 0.39 | 0.39 | 0.34 | 0.29 | 0.49 |
R.m.s deviations | |||||
Bond lengths (Å) | 0.010 | 0.004 | 0.007 | 0.013 | 0.004 |
Bond angles (°) | 1.183 | 0.873 | 0.869 | 1.567 | 0.746 |
Ramachandran plot | |||||
Favored (%) | 293 (96.4) | 573 (96.3) | 294 (96.7) | 573 (97.3) | 290 (96.4) |
Allowed (%) | 6 (2.0) | 17 (2.9) | 9 (3.0) | 12 (2.0) | 9 (3.0) |
Disallowed (%) | 5 (1.6) | 5 (0.8) | 1 (0.3) | 4 (0.7) | 2 (0.7) |
Dataset | Mpro9 | Mpro10 | Mpro11 | Mpro12 | Mpro13 |
PDB ID | 7VK1 | 7VK0 | 7VJZ | 7VK5 | 7VK2 |
Data collection 1 | |||||
Beamline | LCLS (MFX) | LCLS (MFX) | LCLS (MFX) | LCLS (MFX) | LCLS (MFX) |
Space group | C121 | P212121 | C121 | P212121 | C121 |
Cell dimensions | |||||
a, b, c (Å) | 114.0, 53.5, 45.0 | 69.2, 104.3, 105.7 | 115.7, 55.2, 45.6 | 69.2, 104.3, 105.6 | 115.0, 54.8, 45.4 |
α, β, γ (°) | 90.0, 102.0, 90.0 | 90.0, 90.0, 90.0 | 90.0, 101.2, 90.0 | 90.0, 90.0, 90.0 | 90.0, 101.4, 90.0 |
Resolution (Å) 1 | 34.076–1.83 (1.90–1.83) 1 | 46.77–2.14 (2.22–2.14) 1 | 34.74–1.93 (2.11–1.93) 1 | 47.11–2.14 (2.22–2.14) 1 | 34.6–2.04 (2.11–2.04) 1 |
Rsplit | 1.45 (7.1) | 0.83 (36.6) | 0.94 (12.6) | 0.75 (56.4) | 0.81 (18.1) |
CC1/2 | 0.980 (0.255) | 0.995 (0.130) | 0.990 (0.387) | 0.996 (0.238) | 0.993 (0.474) |
I/σI | 1.06 | 0.27 | 0.52 | 0.20 | 0.32 |
CC* | 0.995 (0.949) | 0.999 (0.480) | 0.997 (0.747) | 0.999 (0.620) | 0.998 (0.802) |
Completeness (%) | 87.6 (0.56) | 100.0 (100.0) | 100.0 (100.0) | 100.0 (99.0) | 100.0 (100.0) |
Redundancy | 166 | 2378 | 400 | 310 | 497 |
Refinement | |||||
Resolution (Å) | 34.1–1.93 (1.99–1.93) 1 | 46.8–2.1 (2.15–2.10) 1 | 34.7–1.9 (1.95–1.90) 1 | 47.1–2.17 (2.23–2.17) 1 | 32.0–2.0 (2.05–2.00) 1 |
No. reflections | 17,608 (949) | 45,326 (3045) | 22,355 (1441) | 41,092 (2971) | 18,846 (1314) |
Rwork/Rfree | 0.21/0.26 (0.47/0.57) | 0.23/0.24 (0.49/0.46) | 0.21/0.25 (0.32/0.36) | 0.23/0.27 (0.44/0.45) | 0.23/0.27 (0.46/0.47) |
No. atoms | |||||
Protein | 2439 | 4662 | 2447 | 4710 | 2447 |
Ligand/Ion/Water | 112 | 104 | 55 | 79 | 40 |
B-factors | |||||
Protein | 45.68 | 58.42 | 44.43 | 57.56 | 49.02 |
Ligand/Ion/Water | 51.48 | 57.40 | 43.69 | 52.70 | 46.00 |
Coordinate errors | 0.37 | 0.35 | 0.29 | 0.38 | 0.37 |
R.m.s deviations | |||||
Bond lengths (Å) | 0.005 | 0.011 | 0.005 | 0.004 | 0.003 |
Bond angles (°) | 0.746 | 1.535 | 0.744 | 0.907 | 0.635 |
Ramachandran plot | |||||
Favored (%) | 293 (96.4) | 574 (62.5) | 299 (98.4) | 571 (96.5) | 296 (97.4) |
Allowed (%) | 7 (2.3) | 15 (2.5) | 3 (1.0) | 15 (2.6) | 5 (1.6) |
Disallowed (%) | 4 (1.3) | 6 (1.0) | 2 (0.7) | 6 (1.0) | 3 (1.0) |
Dataset | Mpro14 | Mpro16 | Mpro17 | Mpro18 | Mpro19 |
PDB ID | 7VJW | 7CWC | 7VK7 | 7VJX | 7VK6 |
Data collection 1 | |||||
Beamline | LCLS (MFX) | LCLS (MFX) | LCLS (MFX) | LCLS (MFX) | LCLS (MFX) |
Space group | P212121 | P212121 | P212121 | P212121 | P212121 |
Cell dimensions | |||||
a, b, c (Å) | 69.1, 104.1, 105.5 | 69.2, 104.3, 105.6 | 69.1, 104.3, 105.5 | 69.2, 104.3, 105.7 | 104.3, 105.5, 69.1 |
α, β, γ (°) | 90.0, 90.0, 90.0 | 90.0, 90.0, 90.0 | 90.0, 90.0, 90.0 | 90.0, 90.0, 90.0 | 90.0, 90.0, 90.0 |
Resolution (Å) 1 | 34.55–2.34 (2.43–2.34) 1 | 75.0–2.1 (2.14–2.10) 1 | 41.93–2.24 (2.32–2.24) 1 | 46.77–2.14 (2.22–2.14) 1 | 16.82–2.14 (2.22–2.14) 1 |
Rsplit | 1.54 (8.3) | 0.59 (3.33) | 0.84 (2.3) | 0.61 (−498.4) | 1.03 (165.7) |
CC1/2 | 0.979 (0.217) | 0.997 (0.634) | 0.995 (0.793) | 0.997 (0.384) | 0.993 (0.121) |
I/σI | 1.29 | 0.33 | 5.25 | −0.02 | 0.08 |
CC* | 0.995 (0.649) | 0.999 (0.678) | 0.999 (0.941) | 0.999 (0.509) | 0.998 (0.465) |
Completeness (%) | 100.0 (100.0) | 100.0 (100.0) | 100.0 (100.0) | 100.0 (96.0) | 100.0 (100.0) |
Redundancy | 449 | 3105 (2025) | 4683 | 4516 | 2370 |
Refinement | |||||
Resolution (Å) | 34.6–2.2 (2.25–2.20) 1 | 42.0–2.1 (2.15–2.10) 1 | 41.9–2.4 (2.48–2.40) 1 | 46.8–2.2 (2.26–2.20) 1 | 16.8–2.25 (2.39–2.25) 1 |
No. reflections | 39,287 (2628) | 45,238 (3044) | 30,465 (2599) | 39,396 (2558) | 36,768 (5902) |
Rwork/Rfree | 0.22/0.28 (0.37/0.39) | 0.22/0.26 (0.37/0.44) | 0.21/0.24 (0.34/0.36) | 0.25/0.28 (0.37/0.37) | 0.23/0.27 (0.37/0.38) |
No. atoms | |||||
Protein | 4691 | 4710 | 4711 | 4710 | 4695 |
Ligand/Ion/Water | 63 | 166 | 61 | 97 | 103 |
B-factors | |||||
Protein | 56.07 | 65.66 | 77.72 | 62.20 | 60.48 |
Ligand/Ion/Water | 56.04 | 73.9 | 72.10 | 55.62 | 56.41 |
Coordinate errors | 0.40 | 0.34 | 0.37 | 0.39 | 0.42 |
R.m.s deviations | |||||
Bond lengths (Å) | 0.008 | 0.003 | 0.002 | 0.001 | 0.002 |
Bond angles (°) | 0.935 | 0.619 | 0.466 | 0.401 | 0.494 |
Ramachandran plot | |||||
Favored (%) | 568 (95.5) | 580 (97.5) | 571 (96.0) | 571 (96.0) | 576 (96.8) |
Allowed (%) | 23 (3.9) | 14 (2.4) | 19 (3.2) | 22 (3.7) | 16 (2.7) |
Disallowed (%) | 4 (0.7) | 1 (0.1) | 5 (0.8) | 2 (0.3) | 3 (0.5) |
Mpro03 | Mpro07 | Mpro09 | Mpro11 | |
---|---|---|---|---|
Mpro07 | 0.543 | - | - | - |
Mpro09 | 0.130 | 0.517 | - | - |
Mpro11 | 0.484 | 0.266 | 0.456 | - |
Mpro13 | 0.347 | 0.267 | 0.336 | 0.176 |
Mpro04 | Mpro06 | Mpro10 | Mpro12 | Mpro14 | Mpro17 | Mpro18 | |
---|---|---|---|---|---|---|---|
Mpro06 | 0.184 | - | - | - | - | - | - |
Mpro10 | 0.132 | 0.183 | - | - | - | - | - |
Mpro12 | 0.134 | 0.165 | 0.142 | - | - | - | - |
Mpro14 | 0.154 | 0.138 | 0.153 | 0.154 | - | - | - |
Mpro17 | 0.201 | 0.192 | 0.182 | 0.184 | 0.175 | - | - |
Mpro18 | 0.160 | 0.182 | 0.144 | 0.156 | 0.145 | 0.161 | - |
Mpro19 | 0.1180 | 0.187 | 0.172 | 0.168 | 0.158 | 0.157 | 0.137 |
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Guven, O.; Gul, M.; Ayan, E.; Johnson, J.A.; Cakilkaya, B.; Usta, G.; Ertem, F.B.; Tokay, N.; Yuksel, B.; Gocenler, O.; et al. Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography. Crystals 2021, 11, 1579. https://doi.org/10.3390/cryst11121579
Guven O, Gul M, Ayan E, Johnson JA, Cakilkaya B, Usta G, Ertem FB, Tokay N, Yuksel B, Gocenler O, et al. Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography. Crystals. 2021; 11(12):1579. https://doi.org/10.3390/cryst11121579
Chicago/Turabian StyleGuven, Omur, Mehmet Gul, Esra Ayan, J Austin Johnson, Baris Cakilkaya, Gozde Usta, Fatma Betul Ertem, Nurettin Tokay, Busra Yuksel, Oktay Gocenler, and et al. 2021. "Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography" Crystals 11, no. 12: 1579. https://doi.org/10.3390/cryst11121579
APA StyleGuven, O., Gul, M., Ayan, E., Johnson, J. A., Cakilkaya, B., Usta, G., Ertem, F. B., Tokay, N., Yuksel, B., Gocenler, O., Buyukdag, C., Botha, S., Ketawala, G., Su, Z., Hayes, B., Poitevin, F., Batyuk, A., Yoon, C. H., Kupitz, C., ... DeMirci, H. (2021). Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography. Crystals, 11(12), 1579. https://doi.org/10.3390/cryst11121579