Aug 19, 2024
Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P1 21 1
Forked from Crystallization of SARS-CoV-2 Mpro
- 1Diamond Light Source;
- 2Research Complex at Harwell;
- 3Centre of Medicines Discovery, University of Oxford
- Jasmin Aschenbrenner: The principle crystallographer on the SARS-CoV-2 nsp3 macrodomain project;
- ASAP Discovery
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-SARS-COV-2-NSP3-MAC1
Protocol Citation: Jasmin Aschenbrenner, Peter Marples, Lizbé Koekemoer, Charlie Tomlinson, Daren Fearon 2024. Crystallisation protocol for SARS-CoV-2 nsp3 macrodomain in P1 21 1. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5ej7g47/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: June 24, 2024
Last Modified: August 19, 2024
Protocol Integer ID: 102306
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, macrodomain, nsp3, SARS-CoV-2
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 COVID-19 pandemic has demonstrated the need for novel therapeutic interventions and improved pandemic preparedness strategies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This protocol details an optimized crystallization method for the SARS-CoV-2 nsp3 macrodomain, a potential drug target. Using sitting drop vapor diffusion with seeding, we describe specific buffer conditions and procedures to consistently produce high-quality crystals suitable for XChem fragment screening. The method yields crystals that diffract to an average resolution of 1.5 Å, enabling high-resolution structural studies and can also be used for compound development through co-crystallization experiments.
All structures solved during the development of tool compounds for the SARS-CoV-2 nsp3 macrodomain are deposited on the PDB (Group deposition: G_1002283).
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP
1 Molarity (M) MES adjusted to 6.5 with HCl, Molecular Dimensions, Catalog # MD2-013-PH 6.5
50% w/v PEG 3000, Molecular Dimensions, Catalog # MD2-100-8
Purified SARS-CoV-2 nsp3 macrodomain protein (21.6 mg/mL ) in 20 millimolar (mM) Tris, 7.5 , 150 millimolar (mM) NaCl, 5% glycerol, 1 millimolar (mM) TCEP
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
SARS-CoV-2 nsp3 macrodomain expression and purification
SARS-CoV-2 nsp3 macrodomain expression and purification
5d
5d
The protein used for crystallisation was expressed and purified using the following protocol.
Protocol
NAME
SARS-CoV-2 nsp3 macrodomain His-SUMO tagged expression and purification protocol for crystallization (c004) CREATED BY
Korvus Wang
Equipment needed
Equipment needed
Formulatrix Rock Imager (or incubator of choice)
Equipment
Mosquito HV
NAME
High Volume 16-Channel Robotic Liquid Handler
TYPE
SPT LabTech
BRAND
3097-01057
SKU
LINK
P100 8 multi-channel pipette
Crystallization experiment
Crystallization experiment
1d
1d
Protocol
NAME
Diamond XChem Seeding ProtocolCREATED BY
Peter Marples
1: 2 dilution Sample seeds
17m 40s
Protein and buffer requirements:
115.2 µL 21.6 mg/mL Sample
2.88 mL Crystallization screen
28.8 µL Sample seeds, dilution 1:2
Crystallisation screen composition:
0.1 Molarity (M) MES 6.5
30% w/v PEG 3000
Stock solutions used:
1 Molarity (M) MES adjusted to 6.5 with HCl
50% w/v PEG 3000
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 30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 400 nL 21.6 mg/mL Sample to each lens using the SPT mosquito.
Dispense 300 nL Crystallisation screen to each lens using the SPT mosquito.
Dispense 100 nL Seeds to each lens using the SPT mosquito.
Drop ratio: 4:3:1 ratio (400 nl Sample : 300 nl reservoir solution: 100 nl seeds)
Final drop volume: 800 nl
10m
Incubate at 20 °C for 24:00:00 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 either as plates or as long rods.
Morphology: plates / rods.
Size: ~50 μm in length and ~50 μm in width, depth of the crystals is ~20 μm / ~70 μm in length and ~10 μm in width, depth of the crystals is ~10 μm
Appearance: rectangular or rods.
Average resolution: 1.5 Å
Space group: P1211
Unit cell: 37 Å, 33 Å, 61 Å
90.00°, 96.00°, 90.00°
An example of a drop containing SARS-CoV-2 nsp3 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): 1.62
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