May 13, 2025

Public workspaceCrystallisation of SARS-CoV-2 MPro in P212121 for compound soaking V.2

Version 1 is forked from Crystallization of SARS-CoV-2 Mpro
DOI
https://dx.doi.org/10.17504/protocols.io.261ge5emyg47/v2
Crystallisation of SARS-CoV-2 MPro in P212121 for compound soaking
  • 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 SARS-CoV-2 Mpro project.;
  • ASAP Discovery
Peter Marples
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DOI: https://dx.doi.org/10.17504/protocols.io.261ge5emyg47/v2
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-COV-MPRO
Protocol Citation: Blake Balcomb, Peter Marples, Lizbé Koekemoer, Charlie Tomlinson, Daren Fearon 2025. Crystallisation of SARS-CoV-2 MPro in P212121 for compound soaking. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5emyg47/v2Version created by Mary-Ann Xavier
Manuscript citation:
Melissa L. Boby et al., Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors.Science382,eabo7201(2023).DOI:10.1126/science.abo7201
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 24, 2025
Last Modified: May 13, 2025
Protocol Integer ID: 123294
Keywords: crystallisation, XChem, ASAP, AViDD, Diamond Light Source, i04-1, Mpro, SARS-CoV-2, crystallisation of sar, p212121 for compound, reproducible sar, crystallization protocol, xchem fragment screening, severe acute respiratory syndrome coronavirus, crystallisation, solvent test, strategies for pandemic preparedness, pandemic preparedness, sar, cov mpro
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. In this new version we added the solvent test, soaking conditions, seeding reference paper and addgene ids used in this experiment.
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

Protocol materials
Reagent SARS-CoV-2 isolate: Wuhan-Hu-1 Mpro (3CL protease)addgeneCatalog #228666
ReagentSARS-CoV-2 Mpro protease (C145S mutant - catalytically inactive); aliases: 3CLpro, 3C-likeaddgeneCatalog #204769
Troubleshooting
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
SARS-CoV-2 Mpro expression and purification
The protein used for crystallisation was expressed and purified using the following protocol and plasmid Reagent SARS-CoV-2 isolate: Wuhan-Hu-1 Mpro (3CL protease)addgeneCatalog #228666
Protocol
NAME
SARS-CoV-2 Mpro small scale expression and purification protocol
CREATED BY
Korvus Wang



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
1d
Preparation of seed stock using imatureReagentSARS-CoV-2 Mpro protease (C145S mutant - catalytically inactive); aliases: 3CLpro, 3C-likeaddgeneCatalog #204769 Mpro and mature protein: Protocol for obtaining SARS-CoV-2 Mpro P212121 crystal form can be found here: DOI: https://doi.org/10.1016/j.jmb.2021.167118.


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 Crystallisation screen
Amount14.4 µL SampleSample P212121 crystal form seeds (see step 3), dilution 1:250



Crystallisation screen composition:
Concentration0.1 Molarity (M) MES Ph6.7
11% w/v 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 nL SampleSample to each lens using the SPT mosquito.
Dispense Amount150 nL Crystallisation screen to each lens using the SPT mosquito.
Dispense Amount50 nL Seeds P212121 stock 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
Soaking Tolerance
Crystals can tolerate up to 20% DMSO, 1-2h
Protocol
XChem solvent test
NAME

XChem solvent test

CREATED BY
Peter Marples

Compound Soaking
Crystals can tolerate up to 20% DMSO, 1-2h

Protocol
XChem crystallographic fragment screening
NAME
XChem crystallographic fragment screening
CREATED BY
Peter Marples

Protocol references
For SARS-CoV-2 Main protease in orthorhombic space group
https://www.rcsb.org/structure/7MBG
https://www.sciencedirect.com/science/article/pii/S0022283621003429#s0045


Group deposition SARS-CoV-2 main protease in complex with inhibitors from the COVID Moonshot
https://www.rcsb.org/structure/7GMB
https://www.science.org/doi/10.1126/science.abo7201