Oct 02, 2024

Public workspaceCrystallisation of Zika NS5 RdRp

Forked from a private protocol
DOI
dx.doi.org/10.17504/protocols.io.5jyl82746l2w/v1
  • 1Diamond Light Source;
  • 2Research Complex at Harwell
  • Anu V. Chandran: The principle crystallographer for the Zika NS5 RdRp polymerase project.;
  • ASAP Discovery
Peter Marples
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DOI: dx.doi.org/10.17504/protocols.io.5jyl82746l2w/v1
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-ZIKA-NS5-RDRP
Protocol Citation: Anu V. Chandran, Peter Marples, Martin austin Walsh 2024. Crystallisation of Zika NS5 RdRp. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82746l2w/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: October 02, 2024
Last Modified: October 02, 2024
Protocol Integer ID: 108828
Keywords: crystallisation, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, Zika NS5 RdRp, NS5 RdRp, Research complex at Harwell
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 main aim of this work was to identify small molecules that bind Zika NS5 RdRp (catalytic RNA-dependent RNA polymerase domain) through X-ray fragment-based screening. The Zika NS5 RDRP domain was cloned, expressed, purified, and crystallised. Suitable crystals for fragment screening were produced and optimised allowing an extensive fragment campaign to be performed. A native high-resolution structure was determined at 1.8Å and formed the basis for the fragment campaign. 
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/ Codes:
Midi: UVXPO-3LENS 3W96T-PS 3W96T-UVP

Morpheus HT-96 single reagent 250mL Catalog # MDSR-47-250-2-10

Purified Zika NS5 polymerase protein (Concentration5 mg/mL ) in Concentration20 millimolar (mM) HEPES Ph7.5 , Concentration300 millimolar (mM) NaCl, 2.5% Glycerol, Concentration10 micromolar (µM) ZnCl2, Concentration2 millimolar (mM) TCEP

306-903 residues- construct 2A1 (6 Hist sumo tag)
Safety warnings
Follow all handling warning for the chemicals used in the crystalllisation screen composition.
Zika NS5 RdRp expression and purification
Zika NS5 RdRp expression and purification
The protein used for crystallisation was expressed and purified using the following protocol.
Protocol
Zika NS5 RdRp His-SUMO construct small scale expression and purification protocol
NAME
Zika NS5 RdRp His-SUMO construct small scale expression and purification protocol
CREATED BY
Korvus Wang

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

Crystallisation experiment
Crystallisation experiment
1d
1d
Protein and buffer requirements:
Amount28.8 µL Concentration5 mg/mL SampleSample
Amount3.264 mL Crystallisation screen
Crystallisation screen composition:
Morpheous I E10 condition
Concentration0.12 Molarity (M) Ethylene glycols
Concentration0.1 Molarity (M) Buffer system 3 Ph8.5
30 % v/v Precipitant Mix 2
Stock solutions used:
Morpheous I E10 condition

Note
The crystallisation screen can be stored in a duran bottle.

For long term storage keep the crystallisation screen in the fridge at 4°C.

Dispense Amount34 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount100 undetermined Concentration5 mg/mL SampleSample to each lens using the SPT mosquito.
Dispense Amount50 undetermined Crystallisation screen to each lens using the SPT mosquito.

Drop ratio: 2:1 ratio (100 nl SampleSample : 50 nl reservoir solution)
Final drop volume: 150 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
Crystal form after ~24 h.
Expected result
The crystals reach their maximum size after 96 h and the precipitant has gone.

Crystals grown inconsistently to 2 sizes,one being half the size described below, but both sizes achieve the same results
Morphology: typically plates.
Size: ~ 250 μm in length and ~60 μm in width, depth of the crystals is ~2 μm
Appearance: glass shard.
Average resolution: 2.0 Å
Space group: P43212
Unit cell: 79 Å, 79 Å, 210 Å
90.00°, 90.00°, 90.00°



An example of a drop containing Zika NS5 RdRp ploymerase 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): 1.78
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