Dec 19, 2023
Version 1
ASAP protocol: Crystallization of Enterovirus A71 3C protease (PDB code 8CNY) V.1
- 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.;
Protocol Citation: ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon 2023. ASAP protocol: Crystallization of Enterovirus A71 3C protease (PDB code 8CNY). protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3e89l3p/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: September 07, 2023
Last Modified: December 19, 2023
Protocol Integer ID: 87512
Keywords: crystallisation, Enterovirus A71, 3C protease, 3CNY, XChem, ASAP, AViDD, CMD, Diamond Light Source, i04-1
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 A71 3C protease crystals. The crystal structure was depostited to RCSB PDB with code 8CNY.
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
Equipment needed
Equipment needed
Crystallization experiment
Crystallization experiment
1d
Protein and buffer requirements:
43.2 µL 1 millimolar (mM) Sample
2.88 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 30 µL Crystallisation screen into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 nL 1 millimolar (mM) Sample to each lens using the SPT mosquito.
Dispense 150 nL Crystallisation screen to each lens using the SPT mosquito.
Drop ratio: 1:1
Final drop volume: 300 nl
Incubate at 20 °C for 24:00:00 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.
Protocol references
N/A