key: cord-1053465-klsv11ct
authors: Ertem, Fatma Betul; Guven, Omur; Buyukdag, Cengizhan; Gocenler, Oktay; Ayan, Esra; Yuksel, Busra; Gul, Mehmet; Usta, Gozde; Cakılkaya, Barıs; Johnson, J. Austin; Dao, E. Han; Su, Zhen; Poitevin, Frederic; Yoon, Chun Hong; Kupitz, Christopher; Hayes, Brandon; Liang, Mengning; Hunter, Mark S.; Batyuk, Alexander; Sierra, Raymond G.; Ketawala, Gihan; Botha, Sabine; Dağ, Çağdaş; DeMirci, Hasan
title: Protocol for Structure Determination of SARS-CoV-2 Main Protease at Near-physiological-temperature by Serial Femtosecond Crystallography
date: 2022-01-24
journal: STAR Protoc
DOI: 10.1016/j.xpro.2022.101158
sha: 9fd8f61daa091a44b2b0e78074f8f49dc455485b
doc_id: 1053465
cord_uid: klsv11ct

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps of structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography. For complete details on the use and execution of this protocol, please refer to Durdagi et al. (2021).

Timing: 15 days 1.

Obtain the gene sequence from UniProt database (UniProt ID: P0DTD1).

Add a C-terminal stop codon.

Escherichia coli (E. coli) is selected as the host expression organism. 4. pET28a(+) (Addgene: Cat#69864-3) should be selected as the target vector. The plasmid encodes kanamycin resistance and 6x Histidine tag at N and C terminus which are cleavable with thrombin enzyme (Figure 1 ).

Select NdeI and BamHI restriction cleavage sites at 5' and 3' ends.

Timing: 20 minutes 6. Prepare kanamycin, chloramphenicol, and isopropyl-beta-d-thiogalactopyranoside (IPTG) stock solutions for E. coli BL21 (DE3) Rosetta™ 2 culture. Rosetta™ 2 contains a chloramphenicol-resistant plasmid to enhance protein expression by providing tRNAs for rare codons.

7.

Weigh 2.25 g of kanamycin and dissolve it in 45 ml EDI ultrapure water Stock concentration is 50 mg/ml. Store at -20°C. 8.

Weigh 1.575 g of chloramphenicol and dissolve it in 45 ml 100% ethanol . Stock concentration is 35 mg/ml. Store at -20°C. 9.

Weigh 4.20 g of IPTGand dissolve it in 45 ml of EDI ultrapure water. Stock concentration is 0.4 M. Store at -20°C.

Timing: 6 hours 10. Prepare Luria-Bertani (LB) Agar plates for bacterial cell culture. a. Weigh 10 g of LB Agar and dissolve it in 150 ml of EDI ultrapure water in a 500 ml volume bottle. b. Add 100 ml more EDI ultrapure water to bring the final volume to 250 ml. c. Close the lid loosely and autoclave it at 121°C for 20 minutes.

CRITICAL: Never fill bottles more than ⅔ volume. Loosely closing the lid allows gas exchange and prevents pressure build-up that may lead to over-pressurization.

d. Take the bottle out of the autoclave and wait until it cools down to ~60°C e. Add 250 µl (1:1000) of chloramphanicol and kanamycin from stock solutions.

CRITICAL: Antibiotics must be added after cooling down as they degrade at high temperatures.

f. Pour the agar mixture into the Petri dishes until 1:4 volume is filled by rotating the dish to spread agar equally under aseptic conditions inside the fume hood g. Wait until the agar solidifies and cover the edges of the plate with parafilm. h. Store it at 4°C upside down.

CRITICAL: Keeping the agar plates upside down prevents humidity on the surface of the LB-Agar.

Timing: 4 hours 11. For small-scale protein production, weigh 10 g tryptone, 5 g yeast extract, and 10 g sodium chloride (NaCl) per liter and mix the powders well. 12. Measure 500 ml EDI ultrapure water using a graduated cylinder. 13. Add 25 g of LB medium to the graduated cylinder containing 500 ml EDI ultrapure water. 14. Then, add EDI ultrapure water until the total volume reaches 1 L. 15. Place the graduated cylinder on a magnetic stirrer and add a magnetic fish to mix the solution. 16. Aliquot 100 ml of LB medium into 250 ml glass Erlenmeyer flasks. 17. Cover the lids of the flasks with aluminum foil. Place autoclave tape on top of the foil. 18. Sterilize LB media in an autoclave at liquid mode (121°C, 20 minutes). J o u r n a l P r e -p r o o f 19. Store them in a 4°C fridge once the flasks cool down. 20. For large-scale protein production, weigh 120 g tryptone, 60 g yeast extract, and 120 g NaCl and mix the powders well. 21. Measure 12 L EDI ultrapure water by using a graduated cylinder. 22. Add 12 L EDI ultrapure water to a demijohn. 23. Add the mixed powder to 12 L EDI ultrapure water in the demijohn. 24. Place the demijohn on a stirrer and add a magnetic fish to mix the solution. 25. Aliquot 2 L LB medium into 2.8 L glass Erlenmeyer flasks. 26. Cover the lids of the flask with aluminum foilPlace autoclave tape on top of the foil. 27. Sterilize the LB media in an autoclave at liquid mode (121°C 20 minutes). 28. Store them in a 4°C fridge once the flasks cool down.

CRITICAL Proper autoclave temperature (121°C) can be checked by autoclave tape color changes at this temperature. However, sterilization time is also crucial, and the color change of tape does not precisely represent sterilization. Sastry et al., 2013) https://www.schrod inger.com/products/ maestro PROPKA (Bas et al., 2008) https://pypi.org/pro ject/propka/ R (Bio3D package) (Grant et al., 2006; Yao and Grant, 2013) https://www.rproject.org/ J o u r n a l P r e -p r o o f Step-by-step method details J o u r n a l P r e -p r o o f 

Take Rosetta™ 2 competent cells from -80°C and place them on ice.

Take 50 µl of Rosetta™ 2 competent cells into a sterile 1.5 ml Eppendorf tube once the competent cells are fully dissolved.

Thaw both the component cells and the plasmid on ice for 20 minutes.

Add 2 µl of the plasmid into the competent cells. 6.

Mix the plasmid gently through the bacterial solution by pipetting. 7.

Incubate the cells for 20 minutes more on ice. 8.

After incubation, give them a heat shock at 42°C for 45 seconds.

CRITICAL: The heating timing and temperature are crucial for transformation accuracy. Do not wait less or more than 45 seconds and exceed 42°C.

Put the cells back on ice for 2 minutes. 10. Add 500 µl of room temperature (22°C) LB medium into the Eppendorf tube which contains transformed competent cells. 11. Incubate the culture tube in a incubator at 37°C for 90 minutes. 12. After incubation, centrifuge the tube for 2 minutes at 1500 rpm (238 g) at 22 °C. 13. Discard most of the supernatant by inverting the tube once and dissolve the pellet with the leftover supernatant. 14. Light the Bunsen burner. 15. Sterilize the spreader with alcohol (70% ethanol) and burn the tip of the spreader. Or use a commercial sterile spreader. 16. Spread 50 µl of cell suspension by the sterilized spreader into kanamycin (50 μg/ml) and chloramphenicol (35 μg/mL)-containing agar plate that is preheated to 37°C. 17. Put the plate upside down into a 37°C incubator. 18. Incubate it for 18h. J o u r n a l P r e -p r o o f 19. Take out 150 mL LB from 4°C after for 18h incubation. 20. Warm it at 22 °C. 21. Add kanamycin and chloramphenicol antibiotics to 150 mL LB medium in a 1:1000 ratio. 22. Inoculate the cells into LB medium by making several streaks in different directions with the help of a pipette tip. 23. Incubate the starter culture in the incubator shaker at 37°C 110 rpm for 18h ( Figure 2 ). 24. Add 2 ml (1:1000) kanamycin and 2 ml (1:1000) chloramphenicol antibiotics to 6 X 2 L LB media prepared beforehand. 25. Take out the starter culture from the incubator shaker. 26. Split the starter culture into 2 L glass Erlenmeyer flasks containing LB equally. 27. Incubate the 2 L flasks in the incubator shaker at 37°C 110 rpm until the growth OD reaches 0.8-1.2 at 600 nm.

The growth should take almost 3 hours. Make sure to check the optical density value every 30 minutes.

28. The temperature of the incubator shaker is then lowered to 16°C to cool the cultures. 29. Add 2 ml (1:1000) 0.4 M isopropyl-beta-d-thiogalactopyranoside (IPTG) to each flask once the cultures cool down. 30. Induce the cultures for 72 hours at 16°C and 110 rpm. 31. After three days of induction, transfer the cultures into 750 ml Thermo Bottles. 32. Harvest the cells by centrifugation in an Allegra X-15R Centrifuge at 4°C (precooled) 3500 rpm (2850 g) for 45 min. 33. After centrifugation, discard the supernatant. 34. Place four of the centrifuge tubes on crushed ice. 35. Collect pellets with the help of a sterilized spoon. 36. Use the back of the spoon for gently transferring the pellets into 4 X 50 mL centrifuge tubes 37. Add 25 ml of supernatant to each flask (when the pellet becomes fluid and cannot be collected by spoon) with a serological pipette to dissolve the remaining pellets. 38. Transfer dissolved pellets to the same falcon tubes which contain pellets. 39. Collect the remaining dissolved pellets and supernatants until the centrifuge tubes are completely empty. 40. Spin down 50 mL centrifuge tubes containing bacterial pellets. 41. Discard the remaining supernatant. 42. Store the bacterial pellets in the -80°C freezer.

Pause point: The cell pellets can be stored at -80°C until the next step is planned and started. 66. Take the centrifuge tubes from the rotor and transfer supernatants to a sterilized glass bottle that is placed in an ice box. 67.

Power off the centrifuge after cleaning with a paper towel. 68.

Prepare AKTA GO FPLC system for Nickel affinity purification. a. Turn on the AKTA GO FPLC protein purification system. b. Clean all the tubes with EDI ultrapure water to discard the remains of the previous run. c. Place the Nickel affinity column to the column port of AKTA GO FPLC.

CRITICAL: The column should be packed with 2 ml Ni-agarose beads for each liter of bacterial culture. We expressed 12 L culture and 24 ml resin used for the Nickel affinity column.

d. Wash the column with 3x column volume water. e. Wash the column with 2x column volume buffer B.

J o u r n a l P r e -p r o o f f. Equilibrate the column with 3x column volume buffer A (running buffer).

69. Load the sample to the column with 2.5 mL per minute speed. 70. Take a 40 µl sample from the load and flowthrough and mix with 10 µl loading dye for SDS-PAGE gel electrophoresis. 71. Wash the column with 5x column volume of buffer A after loading of the sample is completed. 72. Elute the sample with 2x column volume of buffer B. 73. Take a 40 µl sample from the elution and and mix with 10 µl loading dye. Then, check all samples with SDS-PAGE gel electrophoresis.

Pause point: Elution can be kept at 4°C for 1-2 days until the next step is planned. Longer storage at 4°C can allow the proteolytic activity of other proteases present as impurities within the eluent.

74. Cut an appropriate length of dialysis membrane and leave it in 22°C water for 10 minutes to soften. 75. Close one end of the dialysis membrane with a clip and put the elution sample from the previous step inside. 76. Take a 40 µl elution sample and mix with 10 µl loading dye for SDS-PAGE gel electrophoresis. 77. Add 1 µl (2 units 

Timing: 3 days Crystallize Mpro for structural data collection.

97. The crystallization screening is performed by using commercially available crystallization screen kits.

The protocol for each screen can be reached at https://kuybim.ku.edu.tr/wp-content/uploads/2021/12/Crystallization-screens.pdf 98. Sitting drop, microbatch screening (under oil), performed by mixing the protein and crystallization screens at a 1:1 ratio, is used for crystallization.

99. Thaw the protein samples, which are stored at -80°C, on ice. 100. Prepare all required materials and tools while the protein samples are thawing ( 

ii.

Set the other one to 16.6 µl (12 repeats) for paraffin oil loading ( Figure  4 .2). iii.

Set a 0.1-2.5 µl micropipette to 0.83 µl for crystallization screen loading. b) Label 72-well Terasaki plate (15 µl volume) with protein name, date, crystallization screen name, and initials of the researcher's name performing the experiment (Figure 4) . c) Fill a 50 ml falcon tube with paraffin oil and put it into a proper rack. d) Take the crystallization screen sets (3 boxes, each including 48 different screening conditions). 101. Open the lid of the Terasaki plate. 102. Use the electronic micropipette set to 0.83 µl, start loading the protein samples of 25 mg/ml concentration into the first two columns (12 wells) in the Terasaki plate (Figure 4.1) . 103. Load and mix 0.83 µl of each crystallization condition by pipetting gently in a well containing 0.83 µl protein sample (Figure 4 .2). Repeat this step until each of the 12 protein samples in the wells has been mixed with one condition. 104. Cover these 12 wells with paraffin oil using the electronic micropipette set to 16.6 µl to minimize evaporation ( c. Crystal seeds, obtained from step a by vortexing, were added to the 100 µl protein reservoir mixture solution. d. After seeding and scaling crystals to 100 µl volume, this seeded solution is used to further inoculate 1:1 1 ml protein reservoir mixture solution. e. Wait 2-3 days for crystal formation. f. Mix 10 ml protein sample with 10 ml crystallization condition in a falcon tube. g. Similarly, the 1 ml seeded crystal solution is used to inoculate the 10 ml protein solution. 111. The crystals are grown on a large-scale within four days.

CRITICAL: The crystallization screens must be kept at 4℃ to prevent evaporation. Moreover, screening conditions are aliquoted in 0.5 ml Eppendorf tubes, and the Eppendorf tubes containing the screens must be spun down after a few uses (e.g., four times) for the same purpose.

112. Cover the 50 ml centrifuge tube containing the crystals with three layers of organic cotton 

Collect and process the SFX data from Mpro crystals for structure .determination 118. Load the 1.6 ml sample reservoir with Mpro crystals in the unaltered mother liquor. 119. Use Microfluidic Electrokinetic Sample Holder (MESH) (Sierra et al., 2012; Sierra et al., 2016) to inject the samples. The measure of the long-fused silica sample capillary should be 200 μm ID × 360 μm OD × 1.0 m. Troubleshooting 5 120. The voltage should be 2500-3000 V and the counter electrode should be grounded. 121. Adjust sample flow rate between 2.5 and 8 µl per minute. 122. SFX experiments are conducted at the ambient temperature at the SLAC National Accelerator Laboratory (Menlo Park, CA) 123. Focus the LCLS X-ray beam with a vertically polarized pulse 30 femtosecond (fs) duration with refractive beryllium compound lenses. 124. The beam size should be ~6 × 6 μm full width half maximum (FWHM). 125. The pulse energy and photon energy should be 0.8 mJ and 9.8 keV (1.25 Å) respectively. 126. The repetition rate should be 120 Hz. 127. Before data collection, Psocake (www.github.com/lcls-psana/psocake) was used to determine the initial diffraction geometry of the ePix10k 2-megapixel detector (ePix10k2M). For data processing, Cheetah was used. Crystal hit rates were monitored using OM. J o u r n a l P r e -p r o o f i. To perform detector readout calibration into the physically meaningful layout by assembling multi-panel detector tiles (asics). ii. Subtract the photon background through radial averaging/median kernel. iii. Obtain the interactive histogram including Analog to Digital Units (ADUs) in region-of-interest (ROI). iv. Do detector translation through lab coordinates (x, y, z). b. Data processing:

i. Use the peakfinder8 algorithm in Cheetah to determine the hit finding parameters. Consider a minimum pixel count above 2 and adc-threshold of 500 with a minimum signal to noise ratio of 7 as a peak. Classify an image with a minimum of 20 peaks as a crystal hit. i.

Generate the small data providing each event including hits, resolution. ii.

The hits were calibrated (detector gain threshold applied) and converted to HDF5 file format using Cheetah, including entries for the image data, instrument data and event codes. iii.

Observable hot pixels and bad detector areas (such as the panel edges) were masked out during peakfinding by applying a mask in HDF5 format (consisting of zeros and ones, which gets multiplied with the image data).

iv. Optimize the detector geometry by updating the mask as well as optimizing the mapping of the detector panels to the lab frame. The latter can be done using geoptimiser, which is included in the CrystFEL package up to version 0. 9.1.

Do CrystFEL indexing of the diffraction patterns applying the correct unit cell and Bravais lattice type once these have been successfully determined.

CRITICAL: Once a correct indexing solution has been found, the predicted Bragg reflection positions can be displayed overlaid onto the image. For an indexing solution to be correct, it is important for any observed peaks to align with these predictions.

vi.

Obtain CrystFEL stream file that contains a list of intensity values for every reflection predicted by the indexing solution in every indexable pattern. vii.

Reiterate through the detector geometry optimization, adjusting the panel positions relative to the beam ensuring the most accurate indexing results.

128. Index the hits by using CrystFEL version 0.9.0. CrystFEL (Github Repository: https://github.com/taw10/crystfel) is a free and open-source software package to process the data from serial crystallography (SX) experiments characterized by XFEL and with other X-ray sources. It processes the data in which "snapshot" diffraction patterns are provided in a "serial" manner. Likewise, consider a minimum pixel count above 2 and adc-threshold of 500 with a minimum signal to noise ratio of 7 as a peak.

b. Determine the unit cell parameters and generate unit cell file using XGANDALF, DirAx, MOSFLM, and XDS in this order via indexamajig.

CRITICAL: Use cell axis tolerances of 5 Å and angle tolerances of 5 degrees (set this from the tolerance option in CrystFEL) to index the data after finding an approximate cell.

c. After finding the approximate cell and optimizing the detector geometry, HDF5 data is re-indexed through the indexamajig algorithm. d. Set the integration radii to 2,3,5 and switch the multi-option to index multiple crystal lattices in a single image. e. If an "indexing ambiguity" imposed by the serial nature of data collection is present for the particular pointgroup (reference) it can be resolved using the ambigator algorithm, and the stream file is produced by re-indexing. f. Integrate and merge the indexed reflections by using partialator (or process_hkl) a core program of CrystFEL and get .hkl, .hkl1, .hkl2 files.. i. "Merged data (.hkl)" provides the calculation of I/σ, completeness etc. via check_hkl algorithm. ii.

"Merged half data set A (.hkl1)" and "Merged half data set B (.hkl2)" provide the calculation of Rsplit, CC* etc. via compare_hkl algorithm.

iii.

The previous two steps provide data collection statistics that are used to determine the resolutions cut-offs for the data, so as to maximize signal without incorporating an abundance of noise. g. Apply the unity model over 3 iterations and set the max-ADU to 7500. h. Export .mtz file from "Merged data (.hkl)" file via create_mtz algorithm using CCP4's F2MTZ algorithm. i. Scale and cut the reflection intensity list from CrystFEL by using the TRUNCATE software. Refer to the Wilson plot as well as the aforementioned data collection statistics to determine the ideal high resolution cut-off.

Timing: 3 days

Calculate the crystal structure of Mpro protein.

j. Once a list of scaled and merged structure factor amplitudes has been obtained using CrystFEL and associated software as described above, export the SFX mtz file (White et al., 2012 ) (Movie S1).

129. Scan the existing structures in the database (Protein Data Bank) using the SWISS-MODEL (Waterhouse et al., 2018) 

Repeat steps 133-136 to iteratively improve your structure. Ideally, an appropriate structure has a minimum number of ramachandran outliers with r-free and r-work values less than 2, if not then less than 3. Troubleshooting 10 134.

When you are satisfied with your structure, open the structure and map in coot again, Check the individual water molecules in the Coot program and retain positions with strong differential density. Those which do not, can be deleted by selecting 'Delete Item…' in the right action bar, selecting 'Water' in the pop-up, and clicking on the desired water molecule. Troubleshooting 11 135.

Strong empty differential densities can be filled with water by selecting 'Place Atom At Pointer…' in the right action bar, selecting 'Water' in the pop-up, and selecting 'OK'.

CRITICAL: Standardl, adding water molecules should be the last step of structure refinement, only after the protein model has been fully refined.

136. Upload the final PDBx/mmCIF format to the PDB database.

a. Before submission, convert the coordinate file to mmCIF format using this link: https://pdb-extract.wwpdb.org/ b. After receiving the mmCIF file, start the PDB submission from the submission page:

https://deposit-pdbj.wwpdb.org/deposition/new c. After getting an email about the ID and password, enter the ID and password, and click to start deposition. d. Make sure having all the required files: a) .pdb file b) .mtz file c) .log file d) .map file e) .lp file e. Upload the .mmCIF file to the system. f. Complete the required elements specified in the requirement charts list. CRITICAL: If you get any errors at this step, try to fix the errors by opening the corresponding PDB in a text file format.

We expect to obtain 200 mg/ml of pure protein from 12 L bacterial culture. Regular LB medium and 3 to 4 days of induction time improves growth. After conducting reverse Nickel affinity purification, dialysis and concentration, we obtain 80 mg/ml protein. Using the commercial crystallization conditions with crystal seeding would yield more reliable results.After SARS-CoV-2 Mpro data is processed and visualized, the final aim will be achieved. Accordingly, detailed information about the interaction between Mpro and inhibitors can be obtained by examining the alternative ligand-binding pocket conformations in detail via Molecular Dynamics (MD) simulations, docking and drug repurposing studies.

J o u r n a l P r e -p r o o f Limitations After obtaining the crystals, sending them to another country for data collection poses the possibility of damaging the crystals.

Problem 1

High pressure when loading the protein into the column.

This problem may be due to the sample not centrifuging for enough time or because it contains too much target protein. To prevent this, the sample can be passed through a 0.22 um filter before it is loaded on the column, or the sample can be divided into 2 fractions, reducing the total load of the column, and the purification can be conducted in 2 sections.

Majority of the protein is not cut by PreScission Protease.

The concentration of the restriction enzyme and the incubation time can be increased. Since commercial PreScission Protease is very expensive, it can be produced with a cleavable His-tag to overcome enzyme availability problems and eliminate contamination after cleavage as the histidine tags will bind to the column with the cleaved tag during reverse Ni-NTA column chromatography.

Protein impurity is not overcome with single-step Ni-NTA column chromatography.

Reverse-nickel affinity chromatography after PreScission Protease cleavage is performed to get rid of non-specific bindings. Size exclusion chromatography can also be used.

Problem 4

Proteins do not form crystals.

Crystallization of proteins can be performed at different temperatures and humidity, or the waiting time required for crystallization can be extended.

The injector is clogged.

Changing the injector and using a micro-capillary can solve the problem.

The hatch door gets stuck and does not close.

Call the responsible department to fix the door.

Bragg spots are not found during peak finding/hit finding on Psocake.

The intensity and position of the pixel can be displayed and examined by hovering the mouse over the corresponding pixel.

When the interaction point or the detector motor position has changed, detector geometry optimization is necessary.

Potential solution J o u r n a l P r e -p r o o f

If you have silver behenate data, find the region-of-interest widget that is shown a green ring which is the new detector center. With a mouse, this widget can be moved or resized around x, y. Adjust the detector distance (z) in the Diffraction Geometry panel until resolution rings overlap silver behenate ring positions. Thus, the newly determined detector position is ready to be deployed in the calibration folder. In CrystFEL, indexed Bragg peaks can be used to optimize the beam centre using the detectorshift script.

Problem 9

There can be assorted reasons for indexing failure including detector center offset, wrong detector distance or numerous false peaks found.

A convenient way is to be able to get feedback and tweak each parameter if indexing has succeeded or not.

Problem 10 PDB structure does not have appropriate r-free/r-work values or is otherwise unsatisfactory after repeated refinement.

Many tools exist for refinement.Further information can be sought in the COOT, PHENIX and other websites through google searches or at their respective websites. COOT: https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ PHENİX: https://phenix-online.org/ R-free/r-work values greater than 6 suggests total randomness. High R values indicate that MPRO models do not fit well into your map. You could therefore consider why radical changes would occur in your map -either poor data or protein denaturation.

The PDB structure has too many Ramachandran outliers.

A few Ramachandran outliers may be expected as part of enzymatic function. If you do not believe this to be the case, further rounds of refinement may be necessary to reduce the number of outliers.

Resource availability

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, [Hasan DeMirci] (hdemirci@ku.edu.tr).

Any unique reagents/materials used in this study are available from the lead contact with a completed Materials Transfer Agreement.

The 3D electron density map of SARS-CoV-2 Main protease has been deposited in the Protein DataBank under accession numbers 7CWB. Any additional information required to reanalyze the data reported is available from the lead contact upon request. 

PHENIX: A comprehensive Python-based system for macromolecular structure solution

Software for high-throughput reduction and analysis of serial femtosecond X-ray diffraction data

Very fast prediction and rationalization of pKa values for protein-ligand complexes

Linac Coherent Light Source data analysis using psana

Nearphysiological-temperature serial femtosecond X-ray crystallography reveals novel conformations of SARS-CoV-2 main protease active site for improved drug repurposing

Coot : model-building tools for molecular graphics

Bio3d: an R package for the comparative analysis of protein structures

Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

Nanoflow electrospinning serial femtosecond crystallography

SWISS-MODEL: Homology modelling of protein structures and complexes

CrystFEL: A software suite for snapshot serial crystallography

Overview of the CCP4 suite and current developments

Domain-Opening and Dynamic Coupling in the α-Subunit of Heterotrimeric G Proteins

Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved αketoamide inhibitors

Plasmid map of Native Mpro construct and multiple cloning site of pET28a(+) vector. SnapGene Viewer (GSL

The authors gratefully acknowledge use of the services and facilities of the Koç University IsBank Infectious Disease Center (KUIS-CID). HD acknowledges support from National Science Foundation (NSF) Science and Technology Centers grant NSF-1231306 (Biology with X-ray Lasers, BioXFEL). This publication has been produced benefiting from the 2232 International 

[Please disclose competing interests for all submitted content by completing and submitting the "declaration of interests" form. Include a "declaration of interests" section in the text of all articles even if there are no interests declared.]

131. Review all residues in the relevant structure one by one in Coot (Emsley and Cowtan, 2004) software. Plot. An indicator next to these values will display green for acceptable, yellow and orange for moderately acceptable, and red for inacceptable. g. Select Validate -> Open Ramachandran Plot -> name.pdb to view a ramachandran plot.Red dots are outliers and may be selected for further inspection. These can be minimized through iterative refinement of the residue and surrounding residues.h. After the coot refinement is finished, click File -> Save Coordinates to save the resultant structure.132. Use the model structure's coordinates for initial rigid-body refinement executing the phenix.refine command with the parameters given below using this new PDB file and the .mtz file in "Terminal" of macOS or Linux: