Apr 26, 2024

Public workspaceCrystallization of SARS-CoV-2 Mpro

DOI
https://dx.doi.org/10.17504/protocols.io.kqdg326y7v25/v1
Crystallization of SARS-CoV-2 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 SARS Mpro project.;
  • ASAP Discovery
Lizbé Koekemoer
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DOI: https://dx.doi.org/10.17504/protocols.io.kqdg326y7v25/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. Crystallization of SARS-CoV-2 Mpro. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg326y7v25/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: 98844
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, SARS CoV, SARS Cov-2 Mpro, Mpro, crystallization of sar, crystals suitable for xchem fragment screening, reproducible sar, acute respiratory syndrome coronavirus, severe acute respiratory syndrome coronavirus, crystallization protocol, cov mpro, xchem fragment screening, crystallization, sar, strategies for pandemic preparedness, pandemic preparedness, crystal
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 against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This protocol outlines the crystallization protocol and buffer conditions used to obtain reproducible SARS-CoV-2 Mpro crystals suitable for XChem fragment screening.
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP

Concentration1 Molarity (M) MES adjusted to Ph6.7 with HCl, Molecular Dimensions, Catalog # MD2-013-PH 6.7
50% w/v PEG 4000, Molecular Dimensions, Catalog # MD2-250-11
99.9% DMSO, Molecular Dimensions, Catalog # MD2-250-159

Purified SARS-CoV-2 Mpro protein (Concentration5 mg/mL ) in Concentration10 millimolar (mM) HEPES, Ph7.5 , Concentration0.5 Molarity (M) NaCl, 5% glycerol, Concentration0.5 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)
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

Crystallization experiment
1d
Prepare seed stock:
Protocol
Diamond XChem Seeding Protocol
NAME
Diamond XChem Seeding Protocol
CREATED BY
Peter Marples
1: 250 dilution SampleSample seeds
17m 40s
Protein and buffer requirements:
Amount43.2 µL Concentration5 mg/mL SampleSample
Amount1.92 mL Crystallization screen
Amount14.4 µL SampleSample seeds, dilution 1:250

Crystallisation screen composition:
Concentration0.1 Molarity (M) MES Ph6.7
11% PEG 4000
5% DMSO

Stock solutions used:
Concentration1 Molarity (M) MES adjusted to Ph6.7 with HCl
50% w/v PEG 4000
99.9% DMSO

Note
The crystallisation screen can be stored in a duran bottle or aliquoted into 96 deep well block for easy dispensing into SwissCI 3 lens plates.

For long term storage keep the Crystallisation screen in the fridge at 4°C.

Dispense Amount20 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount150 µL Concentration5 mg/mL SampleSample to each lens using the SPT mosquito.
Dispense Amount150 µL Crystallisation screen to each lens using the SPT mosquito.
Dispense Amount50 µL Seeds to each lens using the SPT mosquito.

Drop ratio: 3:3:1 ratio (150 nl SampleSample : 150 nl reservoir solution: 50 nl seeds)
Final drop volume: 350 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 ~24 h.
Expected result
The crystals reach their maximum size after 48 h.

Crystals typically form either as stacked playes or in small clusters containng 4-6 crystals.

Morphology: plates.
Size: ~100 μm in length and ~20 μm in width, depth of the crystals is ~10 μm
Appearance: glass shard.
Average resolution: 1.8 Å
Space group: P212121
Unit cell: 67 Å, 99 Å, 102 Å
90.00°, 90.00°, 90.00°
An example of a drop containing SARS-CoV-2 Mpro protease 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