Apr 26, 2024

Public workspaceCrystallization of Enterovirus coxsackievirus A16 2A protease

DOI
dx.doi.org/10.17504/protocols.io.3byl49kdzgo5/v1
Crystallization of Enterovirus coxsackievirus A16 2A protease
  • 1Diamond Light Source;
  • 2Research Complex at Harwell;
  • 3Centre of Medicines Discovery, University of Oxford
  • ryan Lithgo: The principle crystallographer on the Coxsackievirus A16 project.;
Lizbé Koekemoer
Open access
DOI: dx.doi.org/10.17504/protocols.io.3byl49kdzgo5/v1
Protocol Citation: ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon 2024. Crystallization of Enterovirus coxsackievirus A16 2A protease. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl49kdzgo5/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: 98841
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
https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP

Concentration1 Molarity (M) MES Ph6.7 , 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 (Concentration20 mg/mL ) in Concentration10 millimolar (mM) HEPES, Ph7.5 , Concentration0.5 Molarity (M) NaCl, 5% glycerol, Concentration0.5 millimolar (mM) TCEP

Protein construct https://www.addgene.org/204809/
Safety warnings
Attention
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
Equipment needed
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
Crystallization experiment
1d
Prepare seed stock:
Protocol
Diamond XChem Seeding Protocol
NAME
Diamond XChem Seeding Protocol
CREATED BY
Peter Marples
1: 1000 dilution SampleSample seeds
Protein and buffer requirements:
Amount43.2 µL Concentration20 mg/mL SampleSample
Amount3.36 mL Crystallization screen
Amount14.4 µL SampleSample seeds, dilution 1:1000
Crystallisation screen composition:
13.5 % PEG 20000
Concentration0.1 Molarity (M) MES Ph6.7


Stock solutions used:
Concentration1 Molarity (M) MES Ph6.7
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 Amount35 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount150 undetermined Concentration20 mg/mL SampleSample to each lens using the SPT mosquito.
Dispense Amount150 undetermined Crystallisation screen to each lens using the SPT mosquito.
Dispense Amount50 undetermined 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
Incubate at Temperature20 °C for Duration24:00:00 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.
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
N/A