Crystallization of Enterovirus D68 3C protease

Peter Marples, Lizbé Koekemoer, Daren Fearon, ryan Lithgo

Published: 2024-04-26 DOI: 10.17504/protocols.io.5qpvoky29l4o/v1

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.

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: 1 000 000 dilution Sample seeds

Protein and buffer requirements:

14.4µL``35mg/mL``Sample

2.88mL

7.2µL seeds, dilution 1:1 000 000

Crystallisation screen composition:

0.1Molarity (M) Tris 7.8

0.2Molarity (M) Ammonium acetate

26% w/v PEG 3350

Stock solutions used:

1Molarity (M) Tris adjusted to 7.8 with NaOH

1Molarity (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 30µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.

Dispense 50``35mg/mL``Sample to each lens using the SPT mosquito.

Dispense 100 to each lens using the SPT mosquito.

Dispense 25 to each lens using the SPT mosquito.

Drop ratio: 2:4:1

Final drop volume: 175 nl

Incubate at 20°C for 24h 0m 0s 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.

Citation

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 aglass shard appearanceAverage resolution: 1.5 ÅSpace group: P2₁Unit 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