Apr 26, 2024

Public workspaceCrystallization of Zika virus NS2B-NS3 protease

 Forked from ASAP protocol: Crystallization of Enterovirus A71 3C protease (PDB code 8CNY)
DOI
dx.doi.org/10.17504/protocols.io.eq2lyj51mlx9/v1
  • 1Centre of Medicines Discovery, University of Oxford;
  • 2Diamond Light Source;
  • 3Research Complex at Harwell
  • xiaomin.ni Ni: The principle crystallographer on the Zika NS2BNS3 project.;
xiaomin.ni Ni
Open access
DOI: dx.doi.org/10.17504/protocols.io.eq2lyj51mlx9/v1
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-ZIKA-NS2B-NS3
Protocol Citation: xiaomin.ni Ni, Peter Marples, Daren Fearon, Lizbé Koekemoer 2024. Crystallization of Zika virus NS2B-NS3 protease. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyj51mlx9/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: January 08, 2024
Last Modified: April 26, 2024
Protocol Integer ID: 93071
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, Research complex at Harwell, Zika NS2BNS3, NS2BNS3, 8PN6
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
Zika virus (ZIKV) infections causes microcephaly in new-borns and Guillain-Barre syndrome in adults raising a global public health concern, yet no vaccines or antiviral drugs are available to treat or prevent ZIKV infections. The viral 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 and used this in a crystallographic fragment screening campaign.
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) Ammonium sulfate, Molecular Dimensions, Catalog # MD2-250-35
Concentration1 Molarity (M) Sodium acetate Ph4.8 , Molecular Dimensions, Catalog # 133225
50% w/v PEG 2000, Molecular Dimensions, Catalog # MD2-250-17

Purified Zika NS2BNS3 protein (Concentration15 mg/mL ) in Concentration10 millimolar (mM) HEPES, Ph7.5 , Concentration0.5 Molarity (M) NaCl, 5% glycerol, Concentration0.5 millimolar (mM) TCEP

Protein construct https://www.addgene.org/204791/
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
Protein and buffer requirements:
Amount43.2 µL Concentration15 mg/mL SampleSample
Amount2.88 mL Crystallization screen
Crystallisation screen composition:
30% w/v PEG 2000
Concentration0.2 Molarity (M) Ammonium sulfate
Concentration0.1 Molarity (M) sodium acetate Ph4.8

Stock solutions used:
Concentration1 Molarity (M) Ammonium sulfate
Concentration1 Molarity (M) Sodium acetate Ph4.8
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.

Dispense Amount30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount150 nL nl Concentration15 mg/mL SampleSample to each lens using the SPT mosquito.
Dispense Amount150 nL Crystallisation screen to each lens using the SPT mosquito.

Drop ratio: 1:1
Final drop volume: 300 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


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 NS2BNS3 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): 9.50e+11
Protocol references
N/A