Apr 26, 2024
Crystallization of Zika virus NS3 helicase
- 1University of Sao Paulo;
- 2Diamond Light Source;
- 3Research Complex at Harwell;
- 4Centre of Medicines Discovery, University of Oxford
Protocol Citation: Andre Schutzer de Godoy, Peter Marples, Lizbé Koekemoer 2024. Crystallization of Zika virus NS3 helicase . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw152dlmk/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: 98838
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, Research complex at Harwell, Zika NS3, NS3
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
The crystallization protocol and buffer conditions used to obtain Zika NS3 helicase crystals suitable for XChem fragment screening. The Zika virus (ZIKV), discovered in Africa in 1947, swiftly spread across continents, causing significant concern due to its recent association with microcephaly in newborns and Guillain-Barré syndrome in adults. Despite a decrease in prevalence, the potential for a resurgence remains, necessitating urgent therapeutic interventions. Like other flaviviruses, ZIKV presents promising drug targets within its replication machinery, notably the NS3 helicase (NS3Hel) protein, which plays critical roles in viral replication. However, a lack of structural information impedes the development of specific inhibitors targeting NS3Hel. This protocol was used to grow Zika NS3 crystals that were applied high-throughput crystallographic fragment screening on ZIKV NS3 Helicase.
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) NPS Mix (consisting of 0.3 M Sodium phosphate dibasic dihydrate, 0.3 M Ammonium sulphate, and 0.3 M Sodium nitrate from Molecular Dimensions), Catalog # MD2-250-72
1 Molarity (M) MES, Molecular Dimensions, Catalog # MD2-013-PH
Precipitant Mix 4 (11% MPD, 11% PEG 1,000, and 11% PEG 3,350 from Molecular Dimensions), Catalog # MD2-250-84
Purified Zika NS3 protein (5 mg/mL ) in 20 millimolar (mM) Bis-Tris 7 , 500 millimolar (mM) NaCl, 10% glycerol
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
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
Protocol
NAME
Diamond XChem Seeding ProtocolCREATED BY
Peter Marples
1: 100 dilution Sample seeds
Protein and buffer requirements:
43.2 µL 5 mg/mL Sample
4.8 mL Crystallization screen
4.8 mL seeds, dilution 1:100
Crystallisation screen composition:
0.12 Molarity (M) NPS Mix
0.1 Molarity (M) MES 6.5
33% Prcipitant Mix 4
Stock solutions used:
1 Molarity (M) NPS Mix (consisting of 0.3 M Sodium phosphate dibasic dihydrate, 0.3 M Ammonium sulphate, and 0.3 M Sodium nitrate from Molecular Dimensions)
1 Molarity (M) MES (Molecular Dimensions)
Precipitant Mix 4 (11% MPD, 11% PEG 1,000, and 11% PEG 3,350 from Sigma Aldrich)
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 50 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 undetermined 5 mg/mL Sample to each lens using the SPT mosquito.
Dispense 100 undetermined Crystallisation screen to each lens using the SPT mosquito.
Dispense 50 undetermined Seeds to each lens using the SPT mosquito.
Drop ratio: 3:2:1 ratio (150 nL Sample : 100 nL reservoir solution: 50 nL seeds)
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
The crystals reach their maximum size after 48 h.
Crystals typically form either as single crystals or overlapping thin plates
Morphology: typically thin square plates
Size: ~100 μm in length and ~100 μm in width, depth of the crystals is ~10 μm
Appearance: glass shard.
Average resolution: 1.8 Å
Space group: P121
Unit cell: 53 Å, 69 Å, 57 Å
90.00°, 92.00°, 90.00°
An example of a drop containing Zika NS3 helicase 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