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Are you using the same columns for purifying both WT and mutant? Could you have the tiniest fraction of WT contaminating your mutant batches which would then turnover you substrate in crystallo over the couple of weeks? You could try washing the columns/systems with NaOH or pepsin to remove WT, or just use separate columns. Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 -----Original Message----- From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Pierre Nioche Sent: Thursday, April 13, 2017 11:28 AM To: [log in to unmask] Subject: [ccp4bb] in crystallo enzymatic activity Dear CCP4bb, We work on an enzyme that we crystallized with two substrates bound in the active site (the reaction transform two substrates into two products). We have also the structure with the two products. We are able to see densities for the substrates when we collect data at different time point post-crystallization (days or weeks later). There is no change over time and no in crystallo enzymatic reaction despite the fact that in solution using the same crystallization solution, the reaction occurs readily. This is not surprising and there are already many examples in the literature. However, when we crystallize a single amino acid variant (mutant within the active site) with the same two substrates, we initially see the substrates but we then observe in crystallo enzymatic activity and formation of the final products over time. This structure is identical to the one determined with the two products co-crystallized with the enzyme. The crystal packing does not seem to be at play here. I understand that in crystallo activities are well documented in the literature and can be induced by addition of ligands, X-rays, change in oxidative environment, etc? Here, the substrates are present from the beginning of the crystallization experiments with the same concentration. Nothing is added to the crystals later on. Only the time differentiate the two type of crystals: after a couple of weeks, one has the substrates in the active site (wt) while the other has the products (variant). Is anyone aware of similar examples where a variant induce in crystallo enzymatic activity without perturbation of the crystal? Thanks, Pierre Dept of Pharmacology, Toxicology and cellular signaling Paris Descartes University