Crystallization of MERS-CoV Mpro
Peter Marples, Lizbé Koekemoer, Charlie Tomlinson, Daren Fearon, blake.h.balcomb
crystallisation
XChem
ASAP
AViDD
CMD
Diamond Light Source
i04-1
Research complex at Harwell
8R5J
MERS
MERS Mpro
Mpro
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 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)
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
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
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.
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
