ASAP protocol: Crystallization of Enterovirus A71 3C protease (PDB code 8CNY)

Formulatrix Rock Imager (or incubator of choice)

SPT mosquito

P100 multi-channel pipette

SwissCI 3 lens plate

Protein and buffer requirements:

43.2µL``1millimolar (mM)``Sample

2.88mL

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

Dispense 30µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.

Dispense 150nL``1millimolar (mM)``Sample to each lens using the SPT mosquito.

Dispense 150nL to each lens using the SPT mosquito.

Drop ratio: 1:1

Final drop volume: 300 nl

Incubate at 20°C for 24h 0m 0s 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.

Crystal typically form after ~24hrs

Citation

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 aglass shard appearanceAverage resolution: 1.5 ÅSpace group: C222₁Unit cell: 64.90 Å, 65.73 Å, 76.27 Å 90.00°, 90.00°, 90.00°