Apr 25, 2025

Public workspaceCrystallisation of Enterovirus D68 3C protease apo structure and fragment screen in space group P212121 (PDB code 8CNX) V.2

DOI
dx.doi.org/10.17504/protocols.io.n92ldm6jnl5b/v2
  • 1XChem, Diamond Light Source;
  • 2Research complex at Harwell;
  • 3ASAP Discovery Consortium;
  • 4Centre of Medicines Discovery;
  • 5University of Oxford
  • Ryan Lithgo: The principle crystallographer on the Enterovirus 3C protease project.;
  • ASAP Discovery
Lizbé Koekemoer
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.n92ldm6jnl5b/v2
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-EVD68-3CLPRO
Protocol Citation: Ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon 2025. Crystallisation of Enterovirus D68 3C protease apo structure and fragment screen in space group P212121 (PDB code 8CNX). protocols.io https://dx.doi.org/10.17504/protocols.io.n92ldm6jnl5b/v2Version created by Mary-Ann Xavier
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 20, 2023
Last Modified: April 25, 2025
Protocol Integer ID: 119833
Keywords: crystallisation, 3C protease, 3CNY, XChem, ASAP, AViDD, Diamond Light Source, Enterovirus D68
Funders Acknowledgements:
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. The crystal structure was depostited to RCSB PDB with code 8CNX.
In this new version we added the Addgene ID and the protein expression and purification protocol. This construct gave us a crystal form suitable for fragment screen and initial compound soaking.
Materials
ReagentEV-D68 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #(Plasmid #204817)
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
Protocol materials
ReagentEV-D68 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #(Plasmid #204817)
ReagentEV-D68 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #(Plasmid #204817)
Protein expression and purification
Protein expression and purification
2d
2d
Small scale protein purification and expression protocols used for this assayReagentEV-D68 3C protease; aliases: Enterovirus A71 3C protease, non cleavable C-term 6xHis tagaddgeneCatalog #(Plasmid #204817)

Protocol
Enterovirus D68 3C protease small scale expression and purification protocol
NAME

Enterovirus D68 3C protease small scale expression and purification protocol

CREATED BY
Korvus Wang



Equipment needed
Equipment needed
Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
P100 multi-channel pipette
SwissCI 3 lens plate

Crystallisation experiment
Crystallisation experiment
1d
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 µL Concentration1 millimolar (mM) SampleSample to each lens using the SPT mosquito.
Dispense Amount200 µL Crystallisation screen to each lens using the SPT mosquito.
Dispense Amount50 µL 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.




Soaking tolerance
Soaking tolerance
2h 30m
2h 30m
Final condition idenitified: 20% DMSO, 2hr 30mins using the protocol bellow.

Protocol
XChem solvent test
NAME

XChem solvent test

CREATED BY
Peter Marples

2h 30m
Fragment and compound soaking
Fragment and compound soaking
2h 30m
2h 30m
20% fragment/compound, 2hr 30mins using the protocol bellow.

Protocol
XChem crystallographic fragment screening
NAME
XChem crystallographic fragment screening
CREATED BY
Peter Marples

2h 30m
Protocol references
N/A