Crystallization of Enterovirus coxsackievirus A16 2A protease
Peter Marples, Lizbé Koekemoer, Daren Fearon, ryan Lithgo
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 http://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.
Steps
Equipment needed
Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
Equipment
| Value | Label |
|---|---|
| Mosquito HV | NAME |
| High Volume 16-Channel Robotic Liquid Handler | TYPE |
| SPT LabTech | BRAND |
| 3097-01057 | SKU |
P100 8 multi-channel pipette
SwissCI 3 lens plate
Crystallization experiment
Prepare seed stock: seed stock:
Diamond XChem Seeding Protocol1: 1000 dilution Sample seeds
Protein and buffer requirements:
43.2µL``20mg/mL``Sample
3.36mL
14.4µL Sample seeds, dilution 1:1000
Crystallisation screen composition:
13.5 % PEG 20000
0.1Molarity (M) MES 6.7
Stock solutions used:
1Molarity (M) MES 6.7
50% w/v PEG 20000
Dispense 35µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150``20mg/mL``Sample to each lens using the SPT mosquito.
Dispense 150 to each lens using the SPT mosquito.
Dispense 50 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 24h 0m 0s 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.
Crystal typically form after ~24hrs
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
