Crystallization of SARS-CoV-2 Mpro
Peter Marples, Lizbé Koekemoer, Charlie Tomlinson, Daren Fearon, blake.h.balcomb
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 against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This protocol outlines the crystallization protocol and buffer conditions used to obtain reproducible SARS-CoV-2 Mpro crystals suitable for XChem fragment screening.
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: 250 dilution Sample seeds
Protein and buffer requirements:
43.2µL``5mg/mL Sample
1.92mL
14.4µL Sample seeds, dilution 1:250
Crystallisation screen composition:
0.1Molarity (M) MES 6.7
11% PEG 4000
5% DMSO
Stock solutions used:
1Molarity (M) MES adjusted to 6.7 with HCl
50% w/v PEG 4000
99.9% DMSO
Dispense 20µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150``5mg/mL Sample to each lens using the SPT mosquito.
Dispense 150 to each lens using the SPT mosquito.
Dispense 50 to each lens using the SPT mosquito.
Drop ratio: 3:3:1 ratio (150 nl Sample : 150 nl reservoir solution: 50 nl seeds)
Final drop volume: 350 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
