Oct 16, 2025
Version 2
Crystallisation of SARS-CoV-2 C terminal nucleocapsid protein for fragment screening and compound soaking V.2
Forked from a private protocol
- Peter Marples1,2,3,
- Lizbé Koekemoer4,3,
- Daren Fearon1,2,3
- 1Diamond Light Source;
- 2Research Complex at Harwel;
- 3ASAP Discovery Consortium;
- 4Centre of Medicines Discovery, University of Oxford
- ASAP Discovery
Protocol Citation: Peter Marples, Lizbé Koekemoer, Daren Fearon 2025. Crystallisation of SARS-CoV-2 C terminal nucleocapsid protein for fragment screening and compound soaking. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r8ydg1b/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: August 20, 2025
Last Modified: October 16, 2025
Protocol Integer ID: 225096
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, SARS CoV-2 Nucleocapsid, Nucleocapsid, N-protein, Research complex at Harwell, crystallisation of sar, crystals suitable for xchem fragment screening, reproducible sar, xchem fragment screening, crystallization protocol, crystallisation, sar, crystal, fragment screening, fragment soaking condition
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 crystallization protocol and buffer conditions used to obtain reproducible SARS CoV-2 Nucleocapsid crystals suitable for XChem fragment screening. In this new version we added the Addgene id, together with the expression and purification protocol. We also added the solvent tolerance test and the compound/fragment soaking conditions.
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) HEPES adjusted to 7.8 with NaOH, Molecular Dimensions, Catalog # MD2-011-PH 7.8
70 % isopropanol, Sigma Aldrich, Catalog # 563935
50% w/v PEG 4000, Molecular Dimensions, Catalog # MD2-250-11
Purified SARS CoV-2 Nucleocapsid protein (20 mg/mL ) in 10 millimolar (mM) HEPES, 7.5 , 0.5 Molarity (M) NaCl, 5% glycerol, 0.5 millimolar (mM) TCEP
Construct used protein residues 250-364
Protocol materials
SARS-CoV-2 isolate : Wuhan-Hu-1 Nucleocapsid Protein C terminaladdgeneCatalog #228641
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
C-terminal protein expression and purification
For protein heterolog expression we used the plasmid SARS-CoV-2 isolate : Wuhan-Hu-1 Nucleocapsid Protein C terminaladdgeneCatalog #228641 and the following protocol.
Equipment needed
Formulatrix Rock Imager (or incubator of choice)
P100 8 multi-channel pipette
Crystallisation experiment
1d
1: 100 dilution Sample seeds
Protein and buffer requirements:
57.6 µL 20 mg/mL Sample
2.88 mL Crystallization screen
5.76 µL Sample seeds, dilution 1:100
Crystallisation screen composition:
0.1 Molarity (M) HEPES 7.8
10 % isopropanol
23% w/v PEG 4000
Stock solutions used:
1 Molarity (M) HEPES adjusted to 7.8 with NaOH
70 % isopropanol
50% w/v PEG 4000
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 30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 200 µL 20 mg/mL Sample to each lens using the SPT mosquito.
Dispense 180 µL Crystallisation screen to each lens using the SPT mosquito.
Dispense 20 µL Seeds to each lens using the SPT mosquito.
Drop ratio: 5:9:1 ratio (100 nL Sample : 180 nL reservoir solution: 20 nL seeds)
Final drop volume: 300 nl
Incubate at 20 °C for 24:00:00 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.
1d
Crystal form after ~12 h.
Expected result
The crystals reach their maximum size after 24 h.
Crystals typically form as single crystals as six sided shards
Morphology: six sided shard
Size: ~100 μm in length and ~30 μm in width, depth of the crystals is ~30 μm
Appearance: glass shard.
Average resolution: 2.0 Å
Space group: I41
Unit cell: 89, 89, 40
90.00°, 90.00°, 90.00°
An example of a drop containing SARS N-protein 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): 1.68
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
Soaking tolerance test
Crystals can tolerate up to 20% DMSO, for up to 03:00:00
Compound soaking conditions
Crystals can tolerate up to 20% DMSO, for up to 02:00:00
Protocol references
N/A