Microseed matrix-screening combined with random screens (rMMS) is a significant recent breakthrough in protein crystallization. In this study, a very reproducible assay for crystal seeds was set up that allowed the following recommendations to be made: (1) the suitability of a solution for suspending seed crystals can be predicted by incubating (uncrushed) crystals in it for one day and observing crystal stability. (2) For routine rMMS, seed crystals should be suspended in the crystallization cocktail that gave the original crystals. (3) Seed crystals can be suspended in PEG or NaCl solutions to reduce the prevalence of salt crystals. (4) Protein complexes can be seeded with seed crystals suspended in PEG. If necessary, seed crystals can also be suspended in the original crystallization cocktail with any individual ingredients that destabilize the complex removed. ( 5) "Preseeding" of the protein stock should not be used if rMMS is available, because it is less effective. ( 6) Seed crystals can be harvested from microfluidic devices. (7) Heterogeneous nucleants and cross-seeding are less effective than rMMS, but they can be used if seed crystals cannot be obtained. A theoretical case and practical suggestions are also put forward for producing crystals with different space groups.
Arylamine N-acetyltransferase from Mycobacterium tuberculosis (TBNAT) plays an important role in the intracellular survival of the microorganism inside macrophages. Medicinal chemistry efforts to optimize inhibitors of the TBNAT enzyme have been hampered by the lack of a three-dimensional structure of the enzyme. In this paper, the first structure of TBNAT, determined using a lone crystal produced using cross-seeding with the homologous protein from M. marinum, is reported. Despite the similarity between the two enzymes (74% sequence identity), they show distinct physical and biochemical characteristics. The structure elegantly reveals the characteristic features of the protein surface as well as details of the active site of TBNAT relevant to drug-discovery efforts. The crystallographic analysis of the diffraction data presented many challenges, since the crystal was twinned and the habit possessed pseudo-translational symmetry.
A new method of increasing the success rate in protein crystallization screening experiments by combining microseeding with counter-diffusion crystallization in capillaries (SCD) is presented. We have investigated the number of crystallization hits obtained with and without microseeding with 10 model proteins. For the cases studied, SCD generally increases the number of hits and is particularly useful when only relatively low protein concentration stocks are available, either because the stocks were prepared for, e.g., vapor diffusion experiments, or because the protein is poorly soluble. In either case, the addition of seeds becomes necessary to overcome the nucleation energy barrier so that crystal growth can take place even when the wave of supersaturation that passes along the capillary is insufficient to promote nucleation.
Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.
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