Aug 21, 2025
Version 2
Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P43 V.2
- Jasmin Aschenbrenner1,
- Peter Marples1,
- Charlie Tomlinson1,
- Lizbé Koekemoer2,
- Daren Fearon1
- 1Diamond Light Source, Research Complex at Harwell, ASAP Discovery Consortium;
- 2Centre of Medicines Discovery, University of Oxford, ASAP Discovery Consortium
- Jasmin Aschenbrenner: The principle crystallographer on the SARS-CoV-2 nsp3 macrodomain project;
- ASAP Discovery
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-SARS-COV-2-NSP3-MAC1
Protocol Citation: Jasmin Aschenbrenner, Peter Marples, Charlie Tomlinson, Lizbé Koekemoer, Daren Fearon 2025. Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P43. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw1qb2lmk/v2Version created by Mary-Ann Xavier
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 05, 2025
Last Modified: August 21, 2025
Protocol Integer ID: 219643
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, SARS-CoV-2, nsp3, macrodomain, crystallisation protocol for sar, crystallization method for the sar, quality crystals suitable for xchem fragment screening, crystallisation protocol, optimized crystallization method, xchem fragment screening, quality crystal, crystal, severe acute respiratory syndrome coronavirus, improved pandemic preparedness strategy, sar
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: Grant ID: U19AI171399
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). In this version we added the Addgene id and changed the results section.
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP
1 Molarity (M) CHES, 9.5 , Molecular Dimensions, Catalog # MD2-008-PH 9.5
50% w/v PEG 3000, Molecular Dimensions, Catalog # MD2-100-8
Purified SARS-CoV-2 nsp3 macrodomain protein (47 mg/mL ) in 10 millimolar (mM) HEPES, 7.5 , 0.5 Molarity (M) NaCl, 5% glycerol, 0.5 millimolar (mM) TCEP
Protocol materials
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate:Wuhan-Hu-1 nsp3 macrodomainaddgeneCatalog #228643
Troubleshooting
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
SARS-CoV-2 nsp3 macrodomain expression and purification
The protein used for crystallisation was expressed and purified using the following protocol.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate:Wuhan-Hu-1 nsp3 macrodomainaddgeneCatalog #228643
Protocol
CREATED BY
Korvus Wang
Equipment needed
Crystallization experiment
1d
Protein and buffer requirements:
45 µL 47 mg/mL Sample
2.0 mL Crystallization screen
Crystallisation screen composition:
100 millimolar (mM) CHES, 9.5
30% w/v PEG 3000
Stock solutions used:
1 Molarity (M) CHES adjusted to 9.5 with NaOH
50% w/v PEG 3000
Note
The crystallisation screen can be stored in a Duran bottle or aliquoted into 96 deep well block for easy dispensing into SwissCI 3 lens plates.
For long-term storage keep the Crystallisation screen in the fridge at 4°C.
Dispense 20 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 µL 47 mg/mL Sample to each lens using the SPT mosquito.
Dispense 150 µL Crystallisation screen 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 48:00:00 h 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.
2d
Crystal form after ~24 h.
Expected result
The crystals reach their maximum size after 48 h.
Crystals typically form as single crystals.
Morphology: typically large cubes or rectangles.
Size: ~300 μm in length and ~150 μm in width, depth of the crystals is ~100 μm
Appearance: large crystal chunks.
Average resolution: 1.2 Å
Space group: P43
Unit cell: 89 Å, 89 Å, 39 Å
90.00°, 90.00°, 90.00°
An example of a drop containing SARS-CoV-2 nsp3 macrodomain crystals
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
Protocol references
Protocol based on
Marion Schuller et al., Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking. Sci. Adv. 7, eabf8711 (2021).