Crystallization of SARS-CoV-2 nsp15 NendoU
Peter Marples, Lizbé Koekemoer, Daren Fearon, Andre Schutzer de Godoy
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 http://doi.org/10.1107/S2059798320014114
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). The NSP15 endoribonuclease enzyme, known as NendoU, is highly conserved and plays a critical role in the ability of the virus to evade the immune system. NendoU is a promising target for the development of new antiviral drugs. This protocol was used to grow SARS CoV-2 NSP15 crystals that were applied high-throughput crystallographic fragment screening at XChem.
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
Prepare seed stock: seed stock:
Diamond XChem Seeding ProtocolNo dilution Sample seeds
Protein and buffer requirements:
86.4µL``3.4mg/mL Sample
2.88mL
2.88µL Sample seeds, no dilution
Crystallisation screen composition:
0.1Molarity (M) sodium citrate 5
14% w/v PEG 6000
Stock solutions used:
1Molarity (M) sodium citrate adjusted to 5 with HCl
50% w/v PEG 6000
Dispense 30µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 300``3.4mg/mL Sample to each lens using the SPT mosquito.
Dispense 290 to each lens using the SPT mosquito.
Dispense 10 to each lens using the SPT mosquito.
Drop ratio: 30:29:1 ratio (300 nl Sample : 290 nl reservoir solution: 10 nl seeds)
Final drop volume: 600 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 ~96 h.
Data collection at Synchrotron
Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength: 0.9127 Å
Resolution (Å): 2.00
Beam Size (μm): 60 X 50
Number of images: 1500
Oscillation: 0.12°
Exposure (s): 0.0400
Transmission (%): 100
Flux (ph/s): 93.50e+11
