Apr 26, 2024

Public workspaceCrystallization of SARS-CoV-2 nsp15 NendoU

DOI
dx.doi.org/10.17504/protocols.io.36wgqn4yogk5/v1
  • 1Diamond Light Source;
  • 2Research Complex at Harwell;
  • 3Centre of Medicines Discovery, University of Oxford;
  • 4University of Sao Paulo
Lizbé Koekemoer
Open access
DOI: dx.doi.org/10.17504/protocols.io.36wgqn4yogk5/v1
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-SARS-COV-2-ENDORIBONUCLEASE
Protocol Citation: Peter Marples, Daren Fearon, Lizbé Koekemoer, Andre Schutzer de Godoy 2024. Crystallization of SARS-CoV-2 nsp15 NendoU. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqn4yogk5/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: April 26, 2024
Protocol Integer ID: 98842
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, SARS CoV-2 NSP15, NSP15
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
Sao Carlos Institute of Physics, University of Sao Paulo, Av. Joao Dagnone, 1100 - Jardim Santa Angelina, Sao Carlos, 13563-120, Brazil
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
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. This protocol was used to grow SARS CoV-2 NSP15 crystals that were applied high-throughput crystallographic fragment screening at XChem.
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) sodium citrate adjusted to Ph5 with HCl, Molecular Dimensions, Catalog # 133235
50% w/v PEG 6000, Molecular Dimensions, Catalog # MD2-250-12

Purified SARS CoV-2 NSP15 protein (Concentration3.4 mg/mL ) in Concentration20 millimolar (mM) HEPES Ph7.5 , Concentration150 millimolar (mM) NaCl, 5% (v/v) glycerol and Concentration0.5 millimolar (mM) TCEP


Safety warnings
Attention
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

Crystallization experiment
Crystallization experiment
1d
Prepare seed stock:
Protocol
Diamond XChem Seeding Protocol
NAME
Diamond XChem Seeding Protocol
CREATED BY
Peter Marples
No dilution SampleSample seeds
Protein and buffer requirements:
Amount86.4 µL Concentration3.4 mg/mL SampleSample
Amount2.88 mL Crystallization screen
Amount2.88 µL SampleSample seeds, no dilution

Crystallisation screen composition:
Concentration0.1 Molarity (M) sodium citrate Ph5
14% w/v PEG 6000

Stock solutions used:
Concentration1 Molarity (M) sodium citrate adjusted to Ph5 with HCl
50% w/v PEG 6000

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 Amount300 undetermined Concentration3.4 mg/mL SampleSample to each lens using the SPT mosquito.
Dispense Amount290 undetermined Crystallisation screen to each lens using the SPT mosquito.
Dispense Amount10 undetermined Seeds to each lens using the SPT mosquito.

Drop ratio: 30:29:1 ratio (300 nl SampleSample : 290 nl reservoir solution: 10 nl seeds)
Final drop volume: 600 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 ~96 h.
Expected result
The crystals reach their maximum size after 96 h.

Crystals typically form as single hexagonal crystals.

Morphology: hexagonal.
Size: ~100 μm in length and ~50 μm in width, depth of the crystals is ~10 μm
Appearance: glass shard.
Average resolution: 2.2 Å
Space group: P63


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: 0.9127 Å
Resolution (Å): 2.00
Beam Size (μm): 60 X 50
Number of images: 1500
Oscillation: 0.12°
Exposure (s): 0.0400
Transmission (%): 100
Flux (ph/s): 93.50e+11
Protocol references
N/A