Biochemical and Biophysical Research Communications, Vol.493, No.1, 504-508, 2017
A hetero-micro-seeding strategy for readily crystallizing closely related protein variants
Protein crystallization remains difficult to rationalize and screening for optimal crystallization conditions is a tedious and time consuming procedure. Here, we report a hetero-micro-seeding strategy for producing high resolution crystals of closely related protein variants, where micro crystals from a readily crystallized variant are used as seeds to develop crystals of other variants less amenable to crystallization. We applied this strategy to Bovine Pancreatic Trypsin Inhibitor (BPTI) variants, which would not crystallize using standard crystallization practice. Out of six variants in our analysis, only one called BPTI[5,55]A14G formed well behaving crystals; and the remaining five (A14GA38G, AI4GA38V, A14GA381., A14GA38I, and AI4GA38K) could be crystallized only using micro-seeds from the BPTI-[5,55]A14G crystal. All hetero-seeded crystals diffracted at high resolution with minimum mosaicity, retaining the same space group and cell dimension. Moreover, hetero-micro-seeding did not introduce any biases into the mutant's structure toward the seed structure, as demonstrated by AI4GA38I structures solved using micro-seeds from AI4GA38G, AI4GA38L and A14GA38I. Though hetero-micro-seeding is a simple and almost naive strategy, this is the first direct demonstration of its workability. We believe that heteromicro-seeding, which is contrasting with the popular idea that crystallization requires highly purified proteins, could contribute a new tool for rapidly solving protein structures in mutational analysis studies. (C) 2017 Elsevier Inc. All rights reserved.