Jun 05, 2025
Version 2
Crystallisation of Enterovirus D68 3C protease V.2
Forked from a private protocol
- 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 Enterovirus 3C protease;
- ASAP Discovery
Protocol Citation: Ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon 2025. Crystallisation of Enterovirus D68 3C protease. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzjp45lx1/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: June 05, 2025
Last Modified: June 05, 2025
Protocol Integer ID: 219632
Keywords: crystallisation, 3C protease, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, D68 3C protease, crystallisation of enterovirus d68 3c protease, enterovirus d68 3c protease, pathogen enterovirus d68, enterovirus d68, emerging pathogen enterovirus d68, d68 3c prob crystal, spectrum antiviral, 3c protease of ev, antiviral drug, essential role in the viral life cycle, compound screening on d68, viral life cycle, 3c protease, crystallization, protein, crystallisation, protein expression, throughput crystallographic, d68, future pandemic, purification protocol, purification, protease
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 development of effective broad-spectrum antivirals forms an important part of preparing for future pandemics. A cause for concern is the currently emerging pathogen Enterovirus D68 (EV-D68) which primarily spreads through respiratory routes causing mostly mild to severe respiratory illness but, in severe cases, acute flaccid myelitis. The 3C protease of EV-D68 is a potential target for the development of antiviral drugs due to its essential role in the viral life cycle and high sequence conservation. This protocol was used to grow D68 3C ProB crystals that were applied high-throughput crystallographic follow up compound screening on D68 3C. In this version we correct the protein expression and purification protocol, and also added the Addgene id. Also, this protocol should be deprecated and we will add modifications to the following protocol.
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP
1 Molarity (M) Tris adjusted to 7.8 with NaOH, Molecular Dimensions, Catalog # MD2-027-PH 7.8
1 Molarity (M) Ammonium acetate, Molecular Dimensions, Catalog # MD2-002-PH
50% w/v PEG 3350, Molecular Dimensions, Catalog # MD2-250-9
Purified D683C protein (35 mg/mL ) in 10 millimolar (mM) HEPES, 7.5 , 0.5 Molarity (M) NaCl, 5% glycerol, 0.5 millimolar (mM) TCEP
Protocol materials
Enterovirus D68 (EV-D68) strain EV-D68 STL 2014 12 3C protease.addgeneCatalog #228644
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
Enterovirus D68 3C protease expression and purification
The protein used for crystallisation was expressed and purified using the following protocol.Enterovirus D68 (EV-D68) strain EV-D68 STL 2014 12 3C protease.addgeneCatalog #228644
Protocol
CREATED BY
korvus.wang
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
1d
1: 1 000 000 dilution Sample seeds
Protein and buffer requirements:
14.4 µL 35 mg/mL Sample
2.88 mL Crystallisation screen
7.2 µL seeds, dilution 1:1 000 000
ccCrystallisation screen composition:
0.1 Molarity (M) Tris 7.8
0.2 Molarity (M) Ammonium acetate
26% w/v PEG 3350
Stock solutions used:
1 Molarity (M) Tris adjusted to 7.8 with NaOH
1 Molarity (M) Ammonium acetate
50% w/v PEG 3350
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 30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 50 µL 35 mg/mL Sample to each lens using the SPT mosquito.
Dispense 100 µL Crystallisation screen to each lens using the SPT mosquito.
Dispense 25 µL Seeds to each lens using the SPT mosquito.
Drop ratio: 2:4:1
Final drop volume: 175 nl
Incubate at 20 °C for 24:00:00 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
Expected result
Crystals typically appear after 24 hours and reach their maximum size after ~24 h with some precipitation often remaining.
Morphology: small shards.
Size: ~40 μm in length and ~40 μm in width, depth of the crystals is ~20 μm, giving a
glass shard appearance
Average resolution: 1.5 Å
Space group: P21
Unit cell: 39.7 Å, 105 Å, 43.5 Å
90.00°, 110.00°, 90.00°
An example of a drop containing D68 3C 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): 1.62
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 Enterovirus D68 3C protease and iterative design of lead-like compounds using structure-guided expansions, https://doi.org/10.1101/2024.04.29.591650