Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P43
Jasmin Aschenbrenner, Peter Marples, Lizbé Koekemoer, Charlie Tomlinson, Daren Fearon
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 demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protocol details an optimized crystallization method for the SARS-CoV-2 nsp3 macrodomain, a potential drug target. Using sitting drop vapor diffusion, we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening. The method yields crystals that diffract to an average resolution of 1.2 Å, enabling high-resolution structural studies.
All structures solved during the development of tool compounds for the SARS-CoV-2 nsp3 macrodomain are deposited on the PDB (Group deposition: G_1002283).
Steps
SARS-CoV-2 nsp3 macrodomain expression and purification
The protein usedfor crystallisation was expressed and purified using the following protocol.
SARS-CoV-2 nsp3 macrodomain expression and purification protocol for crystallization
Equipment needed
Formulatrix Rock Imager (or incubator of choice)
P100 multi-channel pipette
Crystallization experiment
Protein and buffer requirements:
45µL``47mg/mL Sample
2.0mL
Crystallisation screen composition:
100millimolar (mM) CHES, 9.5
30% w/v PEG 3000
Stock solutions used:
1Molarity (M) CHES adjusted to 9.5 with NaOH
50% w/v PEG 3000
Dispense 20µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150nL``47mg/mL Sample to each lens using the SPT mosquito.
Dispense 150nL to each lens using the SPT mosquito.
Drop ratio: 1:1 ratio (150 nL Sample : 150 nL reservoir solution)
Final drop volume: 300 nl
Incubate at 20°C for 48h 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): 1.62
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 3.80e+12
