Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P1 21 1
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 http://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 consistently produce high-quality crystals suitable for XChem fragment screening. The method yields crystals that diffract to an average resolution of 1.5 Å, enabling high-resolution structural studies and can also be used for compound development through co-crystallization experiments.
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.
SARS-CoV-2 nsp3 macrodomain His-SUMO tagged expression and purification protocol for crystallization (c004)
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: 2 dilution Sample seeds
Protein and buffer requirements:
115.2µL``21.6mg/mL Sample
2.88mL
28.8µL Sample seeds, dilution 1:2
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 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: 4:3:1 ratio (400 nl Sample : 300 nl reservoir solution: 100 nl seeds)
Final drop volume: 800 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 ~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
