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Matt- You might want to try heating your protein to get rid of unfolded/improperly folded protein. We have used 37C for 10 min with good success, but a time course at different temperatures is the best way to determine which parameters are optimum for your protein. Heat-chill it on ice-centrifuge--& then set up your crystallization trays. It's a pretty quick test to see if this will work for your protein. Do you have any ligands for your protein? These have often been the key to getting good crystals in our lab. If you do have good ligands, you may want to express and/or purify your protein in the presence of these compounds. Good Luck! annie From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Jürgen Bosch Sent: Tuesday, October 26, 2010 5:46 PM To: [log in to unmask] Subject: Re: [ccp4bb] Help with Optimizing Crystals Hi. Here is some additional information. 1. The purification method that I used included Ni, tag cleavage, and SEC as a final step. I have tried samples from three different purification batches that range in purity, and even the batch with the worst purity seems to produce crystals. Resource Q ? two or more species perhaps ? Does it run as a monomer dimer multimer on your SEC ? 2. The protein is a proteolyzed fragment since the full length version did not crystallize. Mutagenesis and methylation, however, may be techniques to consider since the protein contains quite a few lysines. 3. There are not any detergents in the buffer, so these are not detergent crystals. The protein buffer just contains Tris at pH 8, NaCl, and DTT. 4. Some experiments that I have done thus far seem to suggest that the crystals are protein. Izit dye soaks well into the crystals, and the few crystals that I shot previously did not produce any diffraction pattern whatsoever. However, I have had difficulty seeming them on a gel and they are a bit tough to break. Do they float or do they sink quickly when you try to mount them ? 5. I tried seeding previously as follows: I broke some crystals, made a seed stock, dipped in a hair, and did serial streak seeding. After seeding, I usually saw small disks or clusters along the path of the hair but nothing larger or better looking. I also had one more question. Has anyone had an instance where changing the precipitation condition or including an additive improved diffraction but did not drastically change the shape of the protein? If so, I may just try further optimization with the current conditions and shoot some more crystals. The additive screen from Hampton is not bad and can make a big difference. A different topic is it a direct cryo what you are using as a condition ? If not what do you use a s a cryo ? Have you tried the old-fashioned way of shooting at crystals at room temperature using capillaries (WTHIT ?) You might be killing your crystal by trying to cryo it is what I'm trying to say here. Jürgen Thanks for all the helpful advice thus far, Matt