Apr 30, 2025

Public workspaceCrystallization of Enterovirus A71 3C protease in C2221 apo structure (PDB 8CNY) V.2

DOI
dx.doi.org/10.17504/protocols.io.yxmvm3e89l3p/v2
  • 1XChem;
  • 2Diamond Light Source;
  • 3Research complex at Harwell;
  • 4ASAP Discovery Consortium;
  • 5Centre of Medicines Discovery;
  • 6University 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.yxmvm3e89l3p/v2
External link: https://asapdiscovery.org/outputs/target-enabling-packages/#ASAP-EVA71-3CLPRO
Protocol Citation: Ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon 2025. Crystallization of Enterovirus A71 3C protease in C2221 apo structure (PDB 8CNY). protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3e89l3p/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: In development
We are still developing and optimizing this protocol
Created: September 07, 2023
Last Modified: April 30, 2025
Protocol Integer ID: 162060
Keywords: crystallisation, Enterovirus A71, 3C protease, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1
Funders Acknowledgements:
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.
Abstract
The crystallization protocol and buffer conditions used to obtain Enterovirus A71 3C protease crystals. The crystal structure was depostited to RCSB PDB with code 8CNY. In this version we added: protein production, solvent tolerance test protocols and the Addgene ID of the contruct used.
Materials
SwissCI 3 lens crystallization plates https://swissci.com/product/3-lens-crystallisation-plate/
1 M Tris-HCl pH 8.5
1 M Magnesium Chloride Hexahydrate supplier
50% PEG 4000
Protocol materials
ReagentEnterovirus A71 3C proteaseaddgeneCatalog #204816
Protein expression and purification
Protein expression and purification
The below protocol was used for protein expression and purification of the plasmid:ReagentEnterovirus A71 3C proteaseaddgeneCatalog #204816

Protocol
Enterovirus A71 3C protease small scale expression and purification protocol
NAME

Enterovirus A71 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

Crystallization experiment
Crystallization experiment
1d
1d
Protein and buffer requirements:
Amount43.2 µL Concentration1 millimolar (mM) SampleSample
Amount2.88 mL Crystallization screen
Crystallisation screen composition:
0.1 M Tris-HCl (pH 8.50)
0.2 M Magnesium chloride hexahydrate
30% w/v PEG 4000

Stock solutions used:
1 M Tris-HCl adjusted to pH 8.5 with HCl
1 M MgCl2.6H20
50% w/v PEG 4000

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.

Dispense Amount30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense Amount150 µL Concentration1 millimolar (mM) SampleSample to each lens using the SPT mosquito.
Dispense Amount150 µL Crystallisation screen to each lens using the SPT mosquito.

Drop ratio: 1:1
Final drop volume: 300 nl
Incubate at Temperature20 °C for Duration24:00:00 h in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12hrs and the imaging schedule follows a Fibonacci sequence of days for further collections.
1d
Crystal typically form after ~24hrs
Expected result
Crystals typically reach their maximum size after ~24 h with precipitation often remaining.
They typically form without seeding and are sturdy enough to be manipulated in solution readily.

Morphology: typically thin rectangles with pointed ends.
Size: ~50 μm in length and ~25 μm in width, depth of the crystals is ~5 μm, giving a glass shard appearance
Average resolution: 1.5 Å
Space group: C2221
Unit cell: 64.90 Å, 65.73 Å, 76.27 Å
90.00°, 90.00°, 90.00°

An example of a drop containing EV A71 3C protease crystals.



Soaking tolerance
Soaking tolerance
2h 30m
2h 30m
Final condition idenitified: 20% DMSO Duration Duration02:30:00

Protocol
XChem solvent test
NAME

XChem solvent test

CREATED BY
Peter Marples

2h 30m
Protocol references
N/A