Oct 02, 2024
Crystallisation of Enterovirus coxsackievirus A16 2A protease
- ryan Lithgo1,2,
- Peter Marples1,2,
- Lizbé Koekemoer3,
- Daren Fearon1,2
- 1Diamond Light Source;
- 2Research Complex at Harwell;
- 3Centre of Medicines Discovery, University of Oxford
- ryan Lithgo: The principle crystallographer on the Coxsackievirus A16 project.;
- ASAP Discovery
Protocol Citation: ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon 2024. Crystallisation of Enterovirus coxsackievirus A16 2A protease. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g715eqgwz/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: 102455
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, Coxsackievirus, A16
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
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 that were applied high-throughput crystallographic fragment screening on the target.
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/
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
Enterovirus coxsackievirus A16 2A protease expression and purification
Enterovirus coxsackievirus A16 2A protease expression and purification
The protein used for crystallisation was expressed and purified using the following protocol.
Protocol
NAME
Enterovirus coxsackievirus A16 2A protease small scale expression and purification protocolCREATED BY
Korvus Wang
Equipment needed
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
Crystallisation experiment
Crystallisation experiment
1d
1d
Protocol
NAME
Diamond XChem Seeding ProtocolCREATED BY
Peter Marples
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 % w/v 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 nL 20 mg/mL Sample to each lens using the SPT mosquito.
Dispense 150 nL Crystallisation screen to each lens using the SPT mosquito.
Dispense 50 nL 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 ~36 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
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
Protocol references
Crystallographic Fragment Screen of Coxsackievirus A16 2A Protease identifies new opportunities for the development of broad-spectrum anti-enterovirals, https://doi.org/10.1101/2024.04.29.591684