READDI protocol: Crystallisation of CHIKV nsP3 macrodomain

Jasmin Aschenbrenner, Peter Marples, Daren Fearon, michael fairhead, Andre Schutzer de Godoy

Published: 2024-07-02 DOI: 10.17504/protocols.io.x54v92jzzl3e/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 https://doi.org/10.1107/S2059798320014114

Abstract

Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months or even years. Millions of people have been infected with CHIKV, mostly in low- and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. The crystallization protocol and buffer conditions used to obtain reproducible Chikungunya Virus nsP3 macrodomain 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:

21.6µL``11mg/mL Sample

2.88mL

10.08µL Sample Seeds, dilution 1:100

Crystallisation screen composition:

0.1Molarity (M) Tris-NaOH 7.8 0.1Molarity (M) Potassium thiocyanate0.1Molarity (M) Sodium bromide25 % v/v PEG Smear Broad Stock solutions used:

1Molarity (M) Tris adjusted to 7.8 with NaOH

1Molarity (M) Potassium thiocyanate

1Molarity (M) Sodium bromide

50% v/v PEG Smear Broad

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 75``11mg/mL Sample to each lens using the SPT mosquito.

Dispense 40 to each lens using the SPT mosquito.

Dispense 35 to each lens using the SPT mosquito.

Drop ratio: 15:8:7 ratio (75 nl Sample : 40 nl Crystallisation solution: 35 nl Seeds)

Final drop volume: 150 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.

Citation

The crystals reach their maximum size after 24-48 h.Crystals typically form as single crystals at the bottom of the drop or on the drop-air interface.Morphology: typically thin rectangles with pointed ends.Size: ~100 μm in length and ~50 μm in width, depth of the crystals is ~10 μmAppearance : glass shard.Average resolution: 1.5 ÅSpace group: P3₁Unit cell: 87 Å, 87 Å, 85 Å 90.00°, 90.00°, 120.00°

An example of a drop containing CHIKV 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): 1.64

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