Co-crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P1 21 1 with ligands
Jasmin Aschenbrenner, Peter Marples, Daren Fearon
crystallisation
XChem
ASAP
AViDD
CMD
Diamond Light Source
i04-1
SARS-CoV-2
nsp3
macrodomain
co-crystallisation
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 with seeding, we describe specific buffer conditions and procedures to produce high-quality crystals of ligand-bound nsp3-mac.
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 used for crystallisation was expressed and purified using the following protocol.
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: 2 dilution Sample seeds
Protein, compounds and buffer requirements:
115.2µL``21.6mg/mL Sample
2.88mL
28.8µL Sample seeds, dilution 1:2
12µL``100millimolar (mM) Sample ligand in DMSO
Crystallisation screen composition:
0.1Molarity (M) MES 6.5
30% w/v PEG 3000
Stock solutions used:
1Molarity (M) MES adjusted to 6.5 with HCl
50% w/v PEG 3000
Dispense 30µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 40nL Compound (~ 5% of final drop volume) to each lens using the Echo liquid handler.
Dispense 400nL``21.6mg/mL Sample to each lens using the SPT mosquito.
Dispense 300nL to each lens using the SPT mosquito.
Dispense 100nL to each lens using the SPT mosquito.
Drop ratio: 400 nL Sample : 300 nL reservoir solution: 100 nL seeds: 40 nL compounds
Final drop volume: 840 nL
Incubate at 20°C for 24h 0m 0s to maximum of 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 ~12 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
