Jul 02, 2024

Public workspaceREADDI protocol: Crystallisation of CHIKV nsP3 macrodomain

DOI
dx.doi.org/10.17504/protocols.io.x54v92jzzl3e/v1
  • 1Diamond Light Source;
  • 2Research Complex at Harwell;
  • 3university of oxford;
  • 4University of Sao Paulo
  • ASAP Discovery
Lizbé Koekemoer
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DOI: dx.doi.org/10.17504/protocols.io.x54v92jzzl3e/v1
External link: https://readdi-ac.org/
Protocol Citation: Jasmin Aschenbrenner, Peter Marples, michael fairhead, Andre Schutzer de Godoy, Daren Fearon 2024. READDI protocol: Crystallisation of CHIKV nsP3 macrodomain. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v92jzzl3e/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: April 26, 2024
Last Modified: July 02, 2024
Protocol Integer ID: 98846
Keywords: crystallisation, XChem, Diamond Light Source, i04-1, READDI, chikungunya virus, AViDD, Research complex at Harwell, CHIKV NS3
Funders Acknowledgement:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171292
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.
Guidelines
N/A
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP

Molecular Dimensions 'The BCS Screen Single Reagent' 2-44:
0.1 M Tris (pH 7.8), 0.1 M Potassium thiocyanate, 0.1 M Sodium bromide, 25 % v/v PEG Smear Broad, Catalog # MDSR-104-2-44

Purified CHIKV Mac protein (11 mg/mL) in 25 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 5 % Glycerol.
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
Ethics statement
N/A
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

Crystallization experiment
Crystallization 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
17m 40s
Protein and buffer requirements:
Amount21.6 µL Concentration11 mg/mL SampleSample
Amount2.88 mL Crystallisation screen
Amount10.08 µL SampleSample Seeds, dilution 1:100

Crystallisation screen composition:
Concentration0.1 Molarity (M) Tris-NaOH Ph7.8
Concentration0.1 Molarity (M) Potassium thiocyanate
Concentration0.1 Molarity (M) Sodium bromide
25 % v/v PEG Smear Broad

Stock solutions used:
Concentration1 Molarity (M) Tris adjusted to Ph7.8 with NaOH
Concentration1 Molarity (M) Potassium thiocyanate
Concentration1 Molarity (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 Amount30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount75 undetermined Concentration11 mg/mL SampleSample to each lens using the SPT mosquito.
Dispense Amount40 undetermined Crystallisation screen to each lens using the SPT mosquito.
Dispense Amount35 undetermined CHIKV Mac Seeds to each lens using the SPT mosquito.

Drop ratio: 15:8:7 ratio (75 nl SampleSample : 40 nl Crystallisation solution: 35 nl Seeds)
Final drop volume: 150 nl
10m
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

Expected result
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 μm
Appearance: glass shard.
Average resolution: 1.5 Å
Space group: P31
Unit 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
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
Protocol references
N/A