Apr 26, 2024

Public workspaceCrystallization of Enterovirus D68 3C protease V.1

DOI
dx.doi.org/10.17504/protocols.io.5qpvoky29l4o/v1
  • 1Diamond Light Source;
  • 2Research Complex at Harwell;
  • 3Centre of Medicines Discovery, University of Oxford
  • Ryan Lithgo: The principle crystallographer on the Enterovirus 3C protease ProB project.;
  • ASAP Discovery
Lizbé Koekemoer
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DOI: dx.doi.org/10.17504/protocols.io.5qpvoky29l4o/v1
Protocol Citation: Ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon 2024. Crystallization of Enterovirus D68 3C protease. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoky29l4o/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: 98840
Keywords: crystallisation, 3C protease, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, D68 3C 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.
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP

Concentration1 Molarity (M) Tris adjusted to Ph7.8 with NaOH, Molecular Dimensions, Catalog # MD2-027-PH 7.8
Concentration1 Molarity (M) Ammonium acetate, Molecular Dimensions, Catalog # MD2-002-PH
50% w/v PEG 3350, Molecular Dimensions, Catalog # MD2-250-9

Purified D683C protein (Concentration35 mg/mL ) in Concentration10 millimolar (mM) HEPES, Ph7.5 , Concentration0.5 Molarity (M) NaCl, 5% glycerol, Concentration0.5 millimolar (mM) TCEP
Safety warnings
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
1d
Prepare seed stock:
Protocol
Diamond XChem Seeding Protocol
NAME
Diamond XChem Seeding Protocol
CREATED BY
Peter Marples
1: 1 000 000 dilution SampleSample seeds
Protein and buffer requirements:
Amount14.4 µL Concentration35 mg/mL SampleSample
Amount2.88 mL Crystallization screen
Amount7.2 µL seeds, dilution 1:1 000 000

Crystallisation screen composition:
Concentration0.1 Molarity (M) Tris Ph7.8
Concentration0.2 Molarity (M) Ammonium acetate
26% w/v PEG 3350

Stock solutions used:
Concentration1 Molarity (M) Tris adjusted to Ph7.8 with NaOH
Concentration1 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 Amount30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount50 µL Concentration35 mg/mL SampleSample to each lens using the SPT mosquito.
Dispense Amount100 µL Crystallisation screen to each lens using the SPT mosquito.
Dispense Amount25 µL Seeds to each lens using the SPT mosquito.

Drop ratio: 2:4:1
Final drop volume: 175 nl
Incubate at Temperature20 °C for Duration24:00:00 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
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
N/A