ASAP protocol: Crystallization of Enterovirus D68 3C protease (PDB code 8CNX)

Formulatrix Rock Imager (or incubator of choice)

SPT mosquito

P100 multi-channel pipette

SwissCI 3 lens plate

Protein and buffer requirements:

32µL``1millimolar (mM) Sample

2.88mL

16µL Sample 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

Prepare seed stock: seed stock:

1: 100 000 dilution Sample seeds

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

Dispense 100nL``1millimolar (mM) Sample to each lens using the SPT mosquito.

Dispense 200nL to each lens using the SPT mosquito.

Dispense 50nL to each lens using the SPT mosquito.

Drop ratio: 1:2:05 ratio (100 nl Sample : 200 nl reservoir solution: 50 nl seeds)

Final drop volume: 350 nl

Incubate at 20°C for 24h 0m 0s 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.

Crystal form after ~12 h.

Citation

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 μmAppearance : glass shard.Average resolution: 1.5 ÅSpace group: P2₁2₁2₁Unit cell: 42.82 Å, 62.53 Å, 147.36 Å 90.00°, 90.00°, 90.00°