Dear Crystallographers,
Thank you all for responding! I will try to respond to the suggestions collectively. I did however have some questions for some of these suggestions...
@Herman Schreuder: I have performed refinement with alt conform as you suggested. I am not sure how to do group-occupancy of the inhibitor. Is that possible in Refmac?
@Colin Groom: Your suggestion for using maps from different dataset is very interesting and I am excited to chase this up! But I am not very competent with the exact terms and this procedure in general. Should I do this using FFT? I have the usual master MTZ from SCALA but not the other labels yet. How can I create them? I would appreciate if you could suggest to me which CCP4 MODULES (and their documentation) I should look at!
@David Mueller: apologies I wasn’t sure what details of the hydrophobic crown you asked from me... There were about 7 hydrophobic residues with Trp, Tyr and Phe.
Regarding the covalent modification, apo crystals are present with a protein residue chemically bonded to the co-factor. Our inhibitor (NOT derived from substrate) was expected to displace the protein residue and chemically bind itself to the co-factor. We had observed this somewhat in the density map as a proof of inhibitor binding. There was a significant red density (COOT) where the residue and co-factor forms the bond. However, when trying to bond the co-factor and the inhibitor, that too was unfavourable. So we have left the three components un-bonded at the moment.
I have tried alternate conformations for all the three components (protein residue, co-factor, inhibitor). At the moment I am playing with 50% occupancy to keep things simple. I defined the 'A' conformers when inhibitor is present and 'B' for the apo structure. (Question1: Can Refmac refine separately based on these two possibilities?). After incorporating the inhibitor in the structure and refinement of all components at 50% occupancy, we have not seen any negative density. The 2Fo-Fc (blue) map fits extremely well after refinement. But I am a little paranoid if it is due to phase bias introduced from the current model with inhibitor. Q2: How can I deduce the percentage occupancy of alt conformers? Should I look at the bfactors as a guide somehow?
I am ultimately concerned whether it is acceptable to publish this enzyme-inhibitor complex given the lower occupancy and poor initial density. We have tried a large number of experiments to optimize co-crystallization by varying all common strategies: seeding, soaking, co-crystallizing and crystallization of inhibited protein. The complex crystals were finally obtained from co-crystallization drops that were seeded with apo crystal nuclei. So these crystals were more like co-crystallization rather than pure soaking. The final inhibitor concentration in the co-crystallization drop was >500-fold more concentrated than the IC50. The cryo buffer also contained same concentration of inh as mother liquor.
Thank you again for your comments (I love CCP4BB)!
Naveed
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