Apr 25, 2025
Version 2
Crystallisation of Enterovirus coxsackievirus A16 2A protease in space group C2 used for apo, fragment screen and follow compounds (PDB 8POA) V.2
- Ryan Lithgo1,2,3,
- Peter Marples1,2,3,
- Lizbé Koekemoer4,5,3,
- Daren Fearon1,2,3
- 1Diamond Light Source;
- 2Research Complex at Harwell;
- 3ASAP Discovery Consortium;
- 4Centre of Medicines Discovery;
- 5University of Oxford
- Ryan Lithgo: The principle crystallographer on the Coxsackievirus A16 project.;
- ASAP Discovery
Protocol Citation: Ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon 2025. Crystallisation of Enterovirus coxsackievirus A16 2A protease in space group C2 used for apo, fragment screen and follow compounds (PDB 8POA). protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49kdzgo5/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: April 25, 2025
Last Modified: April 25, 2025
Protocol Integer ID: 162007
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, Coxsackievirus, A16, crystallisation of enterovirus coxsackievirus a16, coxsackievirus a16 crystal, 2a protease of the virus, enterovirus coxsackievirus a16, picornaviridae coxsackievirus a16, assembly of capsid protein, final stages of viral replication, cleavage from the poly protein, capsid protein, antiviral activity, virus, crystallographic fragment screening, poly protein, throughput crystallographic fragment screening, viral replication, protein production protocol, pdb 8poa, 2a protease in space group c2, 2a protease, formation of mature virion, protein production, protease, crystallisation, target for pandemic preparedness, solvent
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
Picornaviridae coxsackievirus A16 is the causative agent of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of the virus is responsible for self-cleavage from the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. Inhibition deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. This protocol was used to grow coxsackievirus A16 crystals (PDB 8POA) that were used in high-throughput crystallographic fragment screening, and follow up compounds on the target. In this new version we added: the group deposition code; details about the fragment screen and solvent tolerance; also the protein production protocol.
Materials
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP
1 Molarity (M) MES 6.7 , Molecular Dimensions, Catalog # MD2-013-PH 6.7
50% w/v PEG 20000, Molecular Dimensions, Catalog # MD2-250-16
Purified SARS CoV-2 Coxsackievirus A16 protein (20 mg/mL ) in 10 millimolar (mM) HEPES, 7.5 , 0.5 Molarity (M) NaCl, 5% glycerol, 0.5 millimolar (mM) TCEP
Protein construct https://www.addgene.org/204809/
Protocol materials
Human Coxsackievirus A16 strain G10 2A proteaseaddgeneCatalog #228632
Troubleshooting
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
Protein expression and purification
We used the following protein expression and purification protocol for the plasmid Human Coxsackievirus A16 strain G10 2A proteaseaddgeneCatalog #228632
Protocol
CREATED BY
Korvus Wang
Equipment needed
Crystallization experiment
1d
1: 1000 dilution Sample seeds
Protein and buffer requirements:
43.2 µL 20 mg/mL Sample
3.36 mL Crystallization screen
14.4 µL Sample seeds, dilution 1:1000
Crystallisation screen composition:
13.5 % PEG 20000
0.1 Molarity (M) MES 6.7
Stock solutions used:
1 Molarity (M) MES 6.7
50% w/v PEG 20000
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 35 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 µL 20 mg/mL Sample to each lens using the SPT mosquito.
Dispense 150 µL Crystallisation screen to each lens using the SPT mosquito.
Dispense 50 µL Seeds to each lens using the SPT mosquito.
Drop ratio: 3:3:1 ratio (150 nl Sample : 150 nl reservoir solution: 50 nl seeds)
Final drop volume: 350 nl
Incubate at 20 °C for 24:00:00 h in Formulatrix Rock Imager.
Imaging Schedule: The first images are taken after 12hrs and the imaging schedule follows a Fibonacci sequence of days for further collections.
1d
Crystal typically form after ~24hrs
Expected result
Crystals typically reach their maximum size after ~24 h.
Morphology: typically rectangles.
Size: ~75 μm in length and ~10 μm in width, depth of the crystals is ~10 μm, giving a
rectangular appearance
Average resolution: 1.6 Å
Space group: C2
Unit cell: 86 Å, 57 Å, 32 Å
90°, 95°, 90°
An example of a drop containing Coxsackievirus A16 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): 1.21
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
Solvent tolerance
Fragment and compound soaking
Conditions used 5-10% DMSO, for 1h
Results
The results of this fragment screen was deposited under the PDB code G_1002288 https://www.rcsb.org/groups/summary/entry/G_1002288