May 13, 2025
Version 2
Crystallisation of MERS-CoV Mpro in C2221 used for compound soaking V.2
Version 1 is forked from Crystallization of MERS-CoV Mpro
- Blake Balcomb1,2,3,
- Peter Marples1,2,3,
- Lizbé Koekemoer4,5,3,
- Charlie Tomlinson1,2,3,
- Daren Fearon1,2,3
- 1Diamond Light Source;
- 2Research Complex at Harwell;
- 3ASAP Discovery Consortium;
- 4Centre of Medicines Discovery;
- 5University of Oxford
- Blake Balcomb: The principle crystallographer on the MERS-CoV Mpro project.;
- ASAP Discovery
Protocol Citation: Blake Balcomb, Peter Marples, Lizbé Koekemoer, Charlie Tomlinson, Daren Fearon 2025. Crystallisation of MERS-CoV Mpro in C2221 used for compound soaking. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rn84g1b/v2Version created by Mary-Ann Xavier
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 21, 2025
Last Modified: May 13, 2025
Protocol Integer ID: 123150
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, Research complex at Harwell, 8R5J, MERS, MERS Mpro, Mpro, severe acute respiratory syndrome coronavirus, several human coronavirus, other than severe acute respiratory syndrome coronavirus, cov mpro in c2221, therapeutic intervention for mer, zoonotic virus, essential for viral replication, cov mpro, crystallisation of mer, cov, protein purification protocol, covid, virus, strategies for pandemic preparedness, viral replication, mer, pandemic concern, pandemic preparedness, main protease, pandemic, middle eastern respiratory syndrome, protein, wise to sar, sar
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Acknowledgements:
Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot OX11 0QX, UK
Research Complex at Harwell, Harwell Science and Innovation Campus, Didcot OX11 0FA, UK
Oxford Lab Technologies crystal shifter https://doi.org/10.1107/S2059798320014114
Abstract
The COVID-19 pandemic has highlighted the need to identify novel therapeutic interventions and strategies for pandemic preparedness. Other than Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several human coronaviruses that are of pandemic concern, these include SARS-CoV and Middle Eastern Respiratory Syndrome (MERS-CoV). MERS-CoV is a zoonotic virus that was first discovered in 2012. The disease has spread rapidly with large outbreaks as recent as 2015 and 2018. Currently there is no therapeutic intervention for MERS-CoV with 35% of reported cases resulting in human death. Like-wise to SARS-CoV-2, MERS-CoV produces a main protease (Mpro) which is essential for viral replication and therefore an attractive target to inhibit the virus. In this new version we added the protein purification protocol and Addgene id, together with the solvent test and compound soaking conditions.
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP
JCSG+ condition 2-30, Molecular Dimensions, Catalog # MDSR-37-250-2-30
Purified MERS-CoV Mpro protein (17 mg/mL ) in 10 millimolar (mM) HEPES, 7.5 , 0.5 Molarity (M) NaCl, 5% glycerol, 0.5 millimolar (mM) TCEP
Protocol materials
MERS-CoV Mpro strain HCoV-EMC 3CL protease domainaddgeneCatalog #228646
Troubleshooting
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
MERS-CoV Mpro expression and purification
The protein used for crystallisation was expressed and purified using the following protocol. MERS-CoV Mpro strain HCoV-EMC 3CL protease domainaddgeneCatalog #228646
Equipment needed
Crystallisation experiment
1d
1: 1000 dilution Sample seeds
17m 40s
Protein and buffer requirements:
32 µL 17 mg/mL Sample
3.36 mL Crystallisation screen
14.4 µL Sample seeds, dilution 1:1000
Crystallisation screen composition:
0.2 Molarity (M) Sodium malonate dibasic monohydrate
20% w/v PEG 3350
Stock solutions used:
JCSG+ condition 2-30
Note
For long term storage keep the crystallisation screen in the fridge at 4°C.
Dispense 35 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 nL 17 mg/mL Sample to each lens using the SPT mosquito.
Dispense 25 nL Seeds to each lens using the SPT mosquito.
Dispense 130 nL Crystallisation screen to each lens using the SPT mosquito.
Drop ratio: 15:13:2 ratio (150 nl Sample : 150 nl reservoir solution: 20 nl seeds)
Final drop volume: 300 nl
10m
Incubate at 20 °C for 24:00:00 h in Formulatrix Rock Imager.
Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections.
1d
Crystal form after ~12 h.
Expected result
The crystals reach their maximum size after 24 h.
Morphology: typically thin needles or rectangles with pointed ends.
Size: ~100 μm in length and ~2 μm in width, depth of the crystals is ~2 μm
Appearance: glass shard.
Average resolution: 2.2 Å
Space group: C2221
Unit cell: 87 Å, 94 Å, 155 Å
90.00°, 90.00°, 90.00°
An example of a drop containing MERS-CoV Mpro crystals.
Data collection at Synchrotron
Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength: 0.9212 Å
Resolution (Å): 1.78
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 9.50e+11
Soaking Tolerance
Compound Soaking
Crystals can tolerate up to 20% DMSO, 1-2h
Protocol references
N/A