Oct 02, 2024

Public workspaceCrystallisation of MERS-CoV Mpro

 Forked from Crystallization of MERS-CoV Mpro
DOI
dx.doi.org/10.17504/protocols.io.8epv5rn84g1b/v1
  • 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
Peter Marples
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DOI: dx.doi.org/10.17504/protocols.io.8epv5rn84g1b/v1
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-COV-MPRO
Protocol Citation: blake.h.balcomb, Peter Marples, Lizbé Koekemoer, Daren Fearon, Charlie Tomlinson 2024. Crystallisation of MERS-CoV Mpro. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rn84g1b/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: June 26, 2024
Last Modified: October 02, 2024
Protocol Integer ID: 102453
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, Research complex at Harwell, 8R5J, MERS, MERS Mpro, Mpro
Funders Acknowledgement:
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 (Concentration17 mg/mL ) in Concentration10 millimolar (mM) HEPES, Ph7.5 , Concentration0.5 Molarity (M) NaCl, 5% glycerol, Concentration0.5 millimolar (mM) TCEP
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
MERS-CoV Mpro expression and purification
MERS-CoV Mpro expression and purification
The protein used for crystallisation was expressed and purified using the following protocol.
Protocol
MERS-CoV Mpro large scale purification protocol
NAME
MERS-CoV Mpro large scale purification protocol
CREATED BY
Korvus Wang

Equipment needed
Equipment needed
Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
Equipment
Mosquito HV
NAME
High Volume 16-Channel Robotic Liquid Handler
TYPE
SPT LabTech
BRAND
3097-01057
SKU
https://www.sptlabtech.com/products/liquid-handling/mosquito-hv/
LINK
P100 8 multi-channel pipette
SwissCI 3 lens plate

Crystallisation experiment
Crystallisation experiment
1d
1d
Prepare seed stock:
Protocol
Diamond XChem Seeding Protocol
NAME
Diamond XChem Seeding Protocol
CREATED BY
Peter Marples
1: 1000 dilution SampleSample seeds
17m 40s
Protein and buffer requirements:
Amount32 µL Concentration17 mg/mL SampleSample
Amount3.36 mL Crystallisation screen
Amount14.4 µL SampleSample seeds, dilution 1:1000

Crystallisation screen composition:
Concentration0.2 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 Amount35 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount150 nL Concentration17 mg/mL SampleSample to each lens using the SPT mosquito.
Dispense Amount20 nL Seeds to each lens using the SPT mosquito.
Dispense Amount130 nL Crystallisation screen to each lens using the SPT mosquito.

Drop ratio: 15:13:2 ratio (150 nl SampleSample : 150 nl reservoir solution: 20 nl seeds)
Final drop volume: 300 nl
10m
Incubate at Temperature20 °C for Duration24:00:00 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
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