UV and phase separation

Here is the image without UV.

UV and phase separation

With the UV, it is much easier to spot the crystals inside the phase separation.

See tutorial 3 for more information about phase separation.

UV and phase separation

But what to do about crystals like this? They are too small to put in the beam, but they can be used as seeds. This is exactly what I did. These crystals grew in one of the conditions in the Morpheus screen. I made a seed stock of them, then reseeded all 96 conditions of the Morpheus screen. This is known as matrix microseeding (see the tutorial on seeding for more details.)

After optimization

Here are the crystals that grew 24 hours later in a single-step optimization. The structure, Pseudomonas aeruginosa IspC, was solved with the crystals shown here.

After optimization

UV image of the above crystals.

False positives

The intrinsic fluorescence of tryptophans when excited at 340 nm can be used to distinguish salt from protein crystals. This presupposes that your protein has tryptophans of course. However, it is still possible to get false positives, i.e., salt crystals which emit fluorescence.

Take a look at these crystals. Notice anything strange about them? The fluorescence is only on the edges of the crystals. What has happened here is that protein has precipitated on the salt crystals. (We confirmed this by putting the crystals into the x-ray beam.)

Tips, tricks, and pitfalls  with UV: read  Janet Newman's excellent paper.