Apr 17, 2025
Version 2
Crystallization of Zika virus NS2B-NS3 protease (closed conformation in space group P4322 | PDB ID: 8PN6) V.2
Version 1 is forked from ASAP protocol: Crystallization of Enterovirus A71 3C protease (PDB code 8CNY)
- Xiaomin Ni1,
- Peter Marples2,3,
- Daren Fearon2,3,
- Lizbé Koekemoer1
- 1Centre of Medicines Discovery, University of Oxford, ASAP Discovery Consortium;
- 2Diamond Light Source;
- 3Research Complex at Harwell, ASAP Discovery Consortium
- Xiaomin Ni: The principle crystallographer on the Zika NS2B-NS3 protease project.;
- ASAP Discovery
Protocol Citation: Xiaomin Ni, Peter Marples, Daren Fearon, Lizbé Koekemoer 2025. Crystallization of Zika virus NS2B-NS3 protease (closed conformation in space group P4322 | PDB ID: 8PN6). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyj51mlx9/v2Version created by Xiaomin Ni
Manuscript citation:
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: October 31, 2024
Last Modified: April 17, 2025
Protocol Integer ID: 111352
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, Research complex at Harwell, Zika NS2BNS3, NS2BNS3, 8PN6, crystallization of zika virus ns2b, zika polyprotein precursor, zika virus ns2b, zika virus, ns3 protease, protein, protein production protocol, ns3 structure, ns2b cofactor, crystallization, ns2b, pdb code, robust crystal system, conformation in space group p4322, virus, pdb
Funders Acknowledgements:
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
Zika virus (ZIKV) NS3 protease with its NS2B cofactor is essential for the cleavage of Zika polyprotein precursor into individual structural and non-structural proteins and is therefore an attractive drug target. We optimized a robust crystal system of co-expressed NS3 protease with its NS2B cofactor. The crystals appeared within 24 hours and diffracted to 1.7Å in average. The NS2B-NS3 structure is in closed conformation and has been deposited to PDB (PDB code: 8PN6). In this version we added the addgene id and the protein production protocol.
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) Ammonium sulfate, Molecular Dimensions, Catalog # MD2-250-35
1 Molarity (M) Sodium acetate 4.8 , Molecular Dimensions, Catalog # 133225
50% w/v PEG 2000, Molecular Dimensions, Catalog # MD2-250-17
Purified Zika NS2BNS3 protein (15 mg/mL ) in 10 millimolar (mM) HEPES, 7.5 , 0.5 Molarity (M) NaCl, 5% glycerol, 0.5 millimolar (mM) TCEP
Protein construct https://www.addgene.org/204791/
Protocol materials
Zika virus portion of NS2B protease coexpressed with NS3 proteaseaddgeneCatalog #228663
Troubleshooting
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
Protein expression and purification
The protein used for this assay was expressed and purified following the protocol below. The plasmid used was:Zika virus portion of NS2B protease coexpressed with NS3 proteaseaddgeneCatalog #228663
Protocol
CREATED BY
Korvus Wang
Equipment needed
Crystallization experiment
1d
Detailed information of the construct used for crystallization is in publication: Zika NS2B-NS3 protease co-expression construct small scale expression and purification protocol
Crystallisation screen composition:
30% w/v PEG 2000
0.2 Molarity (M) Ammonium sulfate
0.1 Molarity (M) sodium acetate 4.8
Stock solutions used:
1 Molarity (M) Ammonium sulfate
1 Molarity (M) Sodium acetate 4.8
50% w/v PEG 2000
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.
Protein and buffer requirements:
43.2 µL 15 mg/mL Sample
2.88 mL Crystallization screen
Dispense 30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 µL nl 15 mg/mL Sample to each lens using the SPT mosquito.
Dispense 150 µL Crystallisation screen to each lens using the SPT mosquito.
Drop ratio: 1:1
Final drop volume: 300 nl
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
Expected result
Crystals typically form within 12 h, within 24 h they have reached their maximum size with slight precipitant. Crystals form on their own and have cubic appearance. (see image below)
Morphology: cubic
Size: ~50 μm in length, width and depth
Average resolution: 1.7 Å
Space group: P4322
Unit cell: 43 Å, 43 Å, 217 Å
90.00°, 90.00°, 90.00°
An example of a drop containing Zika NS2B-NS3 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): 1.62
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
Protocol references
N/A