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Hi Nicola, I would suggest that you keep increasing the concentration of the peptide as long as it results in crystals appearing in your drop. If your peptide has low affinity for the protein, it may take a lot of it to see it in the crystal structure. Try 10, 20, 50, 100 fold higher concentration of the peptide. Also, shoot your 1:5 ratio crystals, it may already be there. In some cases, if the crystals have large enough solvent channels, the morphology of the crystals may not change significantly upon binding of the peptide. Congratulations on having a fast crystallizing protein at hand. That makes it much easier to try new things and test your hypothesis at a faster pace. Also, you can try variation in length of your peptide, if increasing the peptide concentration doesn't give you the complex. May be, addition or deletion of a residue or two will result in better binding and you will have your complex. with best wishes, Gyan On Fri, Apr 1, 2016 at 10:58 AM, Nicola Evans <[log in to unmask]> wrote: > Hello all, I have a question regarding co-crystals with peptides, please > do share any experiences you may have had. In my group we are trying to > crystalise 15KDa protein with a ~1KDa peptide (related to a binding > partner). We initially set up apo crystallisation and protein:peptide at a > 1:1 molar ratio. We had crystals in both with very similar morphology > (plates), the protein:peptide mix crystals diffracted well and gave us a > good structure, but no peptide was bound, however the purely apo tray gave > crystals with much poorer anomalous diffraction. It looks therefore like > the peptide is helping the protein to crystalise in a more ordered form, > but maybe the concentration isn't high enough to reach saturation. ITC > indicates that this peptide does bind to our protein. > > We have set up further crystallisation with a protein:peptide ratio of 1:5 > and have again grown crystals, however the morphology and plate profiles > look exactly the same as before. In my experience with compounds this is > quite common (and of course plates are a common morphology) but would you > expect with the larger peptides to see this? Or would you expect something > different? If not in morphology in the cell dimensions of the crystal? Is > it worth collecting a dataset for these crystals to see if the peptide is > bound? > > Some more information is that the crystallisation is very fast > (concentration dependant), within minutes to hours. The peptide is also > co-purified with the protein. > > Thanks in advance for any help. > -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839