Oct 02, 2024

Public workspaceCrystallisation of SARS-CoV-2 N Protein

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.8epv5r8ydg1b/v1
  • 1Diamond Light Source;
  • 2Research Complex at Harwell;
  • 3Centre of Medicines Discovery, University of Oxford
  • ASAP Discovery
Peter Marples
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DOI: dx.doi.org/10.17504/protocols.io.8epv5r8ydg1b/v1
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-SARS-COV-2-NPROTEIN
Protocol Citation: Peter Marples, Lizbé Koekemoer, Daren Fearon 2024. Crystallisation of SARS-CoV-2 N Protein. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5r8ydg1b/v1
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: October 02, 2024
Last Modified: October 02, 2024
Protocol Integer ID: 108829
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, SARS CoV-2 Nucleocapsid, Nucleocapsid, N-protein, Research complex at Harwell
Funders Acknowledgement:
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 C0V-2 Nucelocapsid crystals suitable for XChem fragment screening.
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP

Concentration1 Molarity (M) HEPES adjusted to Ph7.8 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 (Concentration20 mg/mL ) in Concentration10 millimolar (mM) HEPES, Ph7.5 , Concentration0.5 Molarity (M) NaCl, 5% glycerol, Concentration0.5 millimolar (mM) TCEP

Construct used protein residues 250-364
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
Equipment needed
Equipment needed
Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
Equipment
Mosquito HV
NAME
High Volume 16-Channel Robotic Liquid Handler
TYPE
SPT LabTech
BRAND
3097-01057
SKU
https://www.sptlabtech.com/products/liquid-handling/mosquito-hv/
LINK
P100 8 multi-channel pipette
SwissCI 3 lens plate

Crystallisation experiment
Crystallisation experiment
1d
1d
Prepare seed stock:
Protocol
Diamond XChem Seeding Protocol
NAME
Diamond XChem Seeding Protocol
CREATED BY
Peter Marples
1: 100 dilution SampleSample seeds
Protein and buffer requirements:
Amount57.6 µL Concentration20 mg/mL SampleSample
Amount2.88 mL Crystallization screen
Amount5.76 µL SampleSample seeds, dilution 1:100

Crystallisation screen composition:
Concentration0.1 Molarity (M) HEPES Ph7.8
10 % isopropanol
23% w/v PEG 4000

Stock solutions used:
Concentration1 Molarity (M) HEPES adjusted to Ph7.8 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 Amount30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount200 nL Concentration20 mg/mL SampleSample to each lens using the SPT mosquito.
Dispense Amount180 nL Crystallisation screen to each lens using the SPT mosquito.
Dispense Amount20 nL Seeds to each lens using the SPT mosquito.

Drop ratio: 5:9:1 ratio (100 nL SampleSample : 180 nL reservoir solution: 20 nL seeds)
Final drop volume: 300 nl
Incubate at Temperature20 °C for Duration24:00:00 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
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
Protocol references
N/A