Hi. Thanks for all of the helpful advice. Below is a summary of the suggestions, along with some things that I have tried and the results thus far.
1) Make sure that the crystals are protein and not salt. My crystals absorb Izit dye well and shooting some initial crystals did not produce any diffraction. I have yet to see them on a gel though, possibly due to too few crystals run, loss of crystals while washing, etc. 2) Try streak seeding using the hit conditions as well as new conditions. Streak seeding using two hit conditions and half of the protein concentration have thus far only produced crystals that are smaller than the initial crystals used to make the seed stock. This indicates that self nucleation is still occurs. I will try significantly lowering the precipitant conc. as well. Seeding into a few random conditions has produced some crystals in new conditions, but they look the same or worse that the previous ones. 3) Make sure that the protein is very pure. Include an additional purification step to your purification scheme, try thermal denaturation of contaminating proteins, crystallize out protein and the redissolve the crystals and use to set up new trays. I will try some of these. 4) Modify the protein to make it more amendable to crystallization. Try truncations, mutations, methylate lysines. I tried the FL version from two different organisms and neither crystallizes. When enzymatically proteolyzed, the protein from one organism crystallizes. However, when proteins containing similar truncations are purified recombinately, a ladder of bands appears after the major one, indicating protein instability. I have not tried any mutations or methylation yet. Thanks for all of the helpful suggestions, and if there are any more, please let me know. Matt On Wed, Oct 27, 2010 at 9:28 AM, Annie Hassell <[email protected]>wrote: > 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:[email protected]] *On Behalf Of > *Jürgen > Bosch > *Sent:* Tuesday, October 26, 2010 5:46 PM > > *To:* [email protected] > *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 > > >
