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James, I would have a look at the paper by Judge et al: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1300446/pdf/10465769.pdf Specifically, in this paper you will find that the crystallization behavior of lysozyme changes drastically with pH. At the time the paper wasn't really written to manipulate for small crystal size, but looking back at the paper (specifically Fig 5), it appears that you can read the conditions that will give you crystals around the size you want. Not re-reading the paper, quoting from memory (which we all think is better than it really is), it is important to use good quality lysozyme to get reproducible results. Good quality probably means freshly purified from fresh (farm-acquired) eggs. I am not kidding you, it makes a big difference. Also, I am going out on a limb to say (I know you know this) that the buffer preparation method matters a lot. Taking sodium acetate solution and pH-ing it with HCl will give very different results from taking acetic acid and pH-ing it with NaOH (because the ionic strength of the buffer is not the same). Lysozyme crystallizes so easily that we tend to forget tedious details. Hope this helps. This paper will probably give you some ideas in the right direction. Mark van der Woerd -----Original Message----- From: James Holton <[log in to unmask]> To: [log in to unmask] Sent: Tue, Jul 26, 2011 11:55 am Subject: [ccp4bb] small lysozyme crystals? Does anyone out there have a protocol of growing HEWL crystals that are all 50-100 microns wide? I gave this project to a summer student recently, thinking it would be easy, but it is turning out to be more difficult than I thought. Keep getting sphereulites instead of small crystals. Yes, I know you can smash a large lysozyme crystal with a hammer, but that is not exactly what I was going for. What I was hoping for was a well-defined protocol for growing "reference" crystals that stay evenly illuminated in our x-ray beams as they rotate. The beam is 100 um wide. I'm sure someone has done this before? -James Holton MAD Scientist