Apr 26, 2024
Crystallization of MERS-CoV Mpro
- blake.h.balcomb 1,2,
- Peter Marples1,2,
- Lizbé Koekemoer3,
- Daren Fearon1,2,
- Charlie Tomlinson1,2
- 1Diamond Light Source;
- 2Research Complex at Harwell;
- 3Centre of Medicines Discovery, University of Oxford
- blake.h.balcomb : The principle crystallographer on the MERS Mpro project.;
- ASAP Discovery
Protocol Citation: blake.h.balcomb , Peter Marples, Lizbé Koekemoer, Daren Fearon, Charlie Tomlinson 2024. Crystallization of MERS-CoV Mpro. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov194x7lr2/v1
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: April 26, 2024
Last Modified: April 26, 2024
Protocol Integer ID: 98845
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, Research complex at Harwell, 8R5J, MERS, MERS Mpro, Mpro, acute respiratory syndrome coronavirus, several human coronavirus, severe acute respiratory syndrome coronavirus, other than severe acute respiratory syndrome coronavirus, zoonotic virus, essential for viral replication, therapeutic intervention for mer, cov mpro, viral replication, cov, pandemic concern, virus, strategies for pandemic preparedness, crystallization of mer, large outbreak, mer, pandemic preparedness, middle eastern respiratory syndrome, wise to sar, sar, main protease, protease
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.
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
Troubleshooting
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
Equipment needed
Formulatrix Rock Imager (or incubator of choice)
Equipment
Mosquito HV
NAME
High Volume 16-Channel Robotic Liquid Handler
TYPE
SPT LabTech
BRAND
3097-01057
SKU
LINK
P100 8 multi-channel pipette
Crystallization experiment
1d
1: 1000 dilution Sample seeds
17m 40s
Protein and buffer requirements:
32 µL 17 mg/mL Sample
3.36 mL Crystallization 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 µL 17 mg/mL Sample to each lens using the SPT mosquito.
Dispense 20 µL Seeds to each lens using the SPT mosquito.
Dispense 130 µL 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 ~24 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
Protocol references
N/A