Crystallization of Zika NS5 RdRp

Peter Marples, Anu V. Chandran, Martin austin Walsh

Published: 2024-04-26 DOI: 10.17504/protocols.io.n2bvjnxjngk5/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 https://doi.org/10.1107/S2059798320014114

Abstract

The main aim of this work was to identify small molecules that bind Zika NS5 RdRp (catalytic R NA- d ependent R NA p olymerase 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.

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:

28.8µL``5mg/mL Sample

3.264mL

Crystallisation screen composition:

Morpheous I E10 condition

0.12Molarity (M) Ethylene glycols

0.1Molarity (M) Buffer system 3 8.5

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 34µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.

Dispense 100``5mg/mL Sample to each lens using the SPT mosquito.

Dispense 50 to each lens using the SPT mosquito.

Drop ratio: 2:1 ratio (100 nl Sample : 50 nl reservoir solution)

Final drop volume: 150 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.

Crystal form after ~24 h.

Citation

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 resultsMorphology: typically plates.Size: ~ 250 μm in length and ~60 μm in width, depth of the crystals is ~2 μmAppearance : glass shard.Average resolution: 2.0 ÅSpace group: P4₃2₁2Unit 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

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