Crystallization of Zika virus NS2B-NS3 protease

Peter Marples, Lizbé Koekemoer, Daren Fearon, xiaomin.ni Ni

Published: 2024-04-26 DOI: 10.17504/protocols.io.eq2lyj51mlx9/v1

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 http://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.

Steps

Equipment needed

Formulatrix Rock Imager (or incubator of choice)

SPT mosquito

Equipment

Value	Label
Mosquito HV	NAME
High Volume 16-Channel Robotic Liquid Handler	TYPE
SPT LabTech	BRAND
3097-01057	SKU

P100 8 multi-channel pipette

SwissCI 3 lens plate

Crystallization experiment

Protein and buffer requirements:

43.2µL``15mg/mL``Sample

2.88mL

Crystallisation screen composition:

30% w/v PEG 2000

0.2Molarity (M) Ammonium sulfate

0.1Molarity (M) sodium acetate 4.8

Stock solutions used:

1Molarity (M) Ammonium sulfate

1Molarity (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.

Dispense 30µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.

Dispense 150nL``15mg/mL``Sample to each lens using the SPT mosquito.

Dispense 150nL to each lens using the SPT mosquito.

Drop ratio: 1:1

Final drop volume: 300 nl

Incubate at 20°C for 24h 0m 0s 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.

Citation

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: cubicSize: ~50 μm in length, width and depthAverage resolution: 1.7 ÅSpace group: P4₃22Unit 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

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