Crystallization of SARS-CoV-2 N Protein

Peter Marples, Lizbé Koekemoer, Daren Fearon

Published: 2024-04-26 DOI: 10.17504/protocols.io.4r3l2q4b3l1y/v1

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 crystallization protocol and buffer conditions used to obtain reproducible SARS C0V-2 Nucelocapsid crystals suitable for XChem fragment screening.

Steps

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: 100 dilution Sample seeds

Protein and buffer requirements:

57.6µL``20mg/mL Sample

2.88mL

5.76µL Sample seeds, dilution 1:100

Crystallisation screen composition:

0.1Molarity (M) HEPES 7.8

10 % isopropanol

23% w/v PEG 4000

Stock solutions used:

1Molarity (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 30µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.

Dispense 200``20mg/mL Sample to each lens using the SPT mosquito.

Dispense 180 to each lens using the SPT mosquito.

Dispense 20 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 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.

Citation

The crystals reach their maximum size after 24 h.Crystals typically form as single crystals as six sided shardsMorphology: six sided shardSize: ~100 μm in length and ~30 μm in width, depth of the crystals is ~30 μmAppearance : glass shard.Average resolution: 2.0 ÅSpace group: I4₁Unit 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