Dec 19, 2023

Public workspaceASAP protocol: Crystallization of Enterovirus D68 3C protease (PDB code 8CNX) V.1

DOI
dx.doi.org/10.17504/protocols.io.n92ldm6jnl5b/v1
  • 1XChem, Diamond Light Source;
  • 2Research complex at Harwell;
  • 3Centre of Medicines Discovery, University of Oxford;
  • 4Diamond Light Source
  • ryan Lithgo: The principle crystallographer on the Enterovirus 3C protease project.;
Lizbé Koekemoer
Open access
DOI: dx.doi.org/10.17504/protocols.io.n92ldm6jnl5b/v1
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-EVD68-3CLPRO
Protocol Citation: ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon 2023. ASAP protocol: Crystallization of Enterovirus D68 3C protease (PDB code 8CNX). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm6jnl5b/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: December 19, 2023
Last Modified: December 19, 2023
Protocol Integer ID: 92511
Keywords: crystallisation, 3C protease, 3CNY, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1, Enterovirus D68
Funders Acknowledgement:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: Grant ID: U19AI171399
Abstract
The crystallization protocol and buffer conditions used to obtain Enterovirus D68 3C protease crystals suitable for XChem fragment screening. The crystal structure was depostited to RCSB PDB with code 8CNX.
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/
1 M Tris-HCl pH 8.14
1 M Ammonium acetate
50% w/v PEG 3350
Equipment needed
Equipment needed
Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
P100 multi-channel pipette
SwissCI 3 lens plate

Crystallization experiment
Crystallization experiment
1d
Protein and buffer requirements:
Amount32 µL Concentration1 millimolar (mM) SampleSample
Amount2.88 mL Crystallization screen
Amount16 µL SampleSample seeds, dilution 1:100 000

Crystallisation screen composition:
0.1 M Tris-HCl (pH 8.14)
0.2 M Ammonium acetate
25% w/v PEG 3350

Stock solutions used:
1 M Tris-HCl adjusted to pH 8.14 with HCl
1 M Ammonium acetate
50% w/v PEG 3350

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.

Prepare seed stock:
1: 100 000 dilution SampleSample seeds
Dispense Amount30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount100 nL Concentration1 millimolar (mM) SampleSample to each lens using the SPT mosquito.
Dispense Amount200 nL Crystallisation screen to each lens using the SPT mosquito.
Dispense Amount50 nL Seeds to each lens using the SPT mosquito.

Drop ratio: 1:2:05 ratio (100 nl SampleSample : 200 nl reservoir solution: 50 nl seeds)
Final drop volume: 350 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 ~12 h.
Expected result
The crystals reach their maximum size after 24 h and most of the precipitant has gone.

Crystals typically form either as single crystals or in small clusters containng 4-6 crystals in a flower-like arrangement. Upon mounting a gentle nudge dissociates the cluster.

Morphology: typically thin rectangles with pointed ends.
Size: ~100 μm in length and ~50 μm in width, depth of the crystals is ~10 μm
Appearance: glass shard.
Average resolution: 1.5 Å
Space group: P212121
Unit cell: 42.82 Å, 62.53 Å, 147.36 Å
90.00°, 90.00°, 90.00°
An example of a drop containing EV D68 3C protease crystals.




Protocol references
N/A