Crystallization of MERS-CoV Mpro

Peter Marples, Lizbé Koekemoer, Charlie Tomlinson, Daren Fearon, blake.h.balcomb

Published: 2024-04-26 DOI: 10.17504/protocols.io.ewov194x7lr2/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 http://doi.org/10.1107/S2059798320014114

Abstract

The COVID-19 pandemic has highlighted the need to identify novel therapeutic interventions and strategies for pandemic preparedness. Other than Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there are several human coronaviruses that are of pandemic concern, these include SARS-CoV and Middle Eastern Respiratory Syndrome (MERS-CoV). MERS-CoV is a zoonotic virus that was first discovered in 2012. The disease has spread rapidly with large outbreaks as recent as 2015 and 2018. Currently there is no therapeutic intervention for MERS-CoV with 35% of reported cases resulting in human death. Like-wise to SARS-CoV-2, MERS-CoV produces a main protease (Mpro) which is essential for viral replication and therefore an attractive target to inhibit the virus.

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:

32µL``17mg/mL Sample

3.36mL

14.4µL Sample seeds, dilution 1:1000

Crystallisation screen composition:

0.2Molarity (M) Sodium malonate dibasic monohydrate

20% w/v PEG 3350

Stock solutions used:

JCSG+ condition 2-30

Note

For long term storage keep the Crystallisation screen in the fridge at 4°C.

Dispense 35µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.

Dispense 150``17mg/mL Sample to each lens using the SPT mosquito.

Dispense 20 to each lens using the SPT mosquito.

Dispense 130 to each lens using the SPT mosquito.

Drop ratio: 15:13:2 ratio (150 nl Sample : 150 nl reservoir solution: 20 nl seeds)

Final drop volume: 300 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.

Crystal form after ~24 h.

Citation

The crystals reach their maximum size after 24 h.Morphology: typically thin needles or rectangles with pointed ends.Size: ~100 μm in length and ~2 μm in width, depth of the crystals is ~2 μmAppearance : glass shard.Average resolution: 2.2 ÅSpace group: C222₁Unit cell: 87 Å, 94 Å, 155 Å 90.00°, 90.00°, 90.00°

An example of a drop containing MERS-CoV Mpro 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): 1.78

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