A method to produce microseed stock for use in the crystallization of biological macromolecules

Abstract

A method is presented for producing a seed-stock mixture for macromolecular crystallization. A PTFE bead and micro-centrifuge tube act as mortar and pestle for pulverizing seed crystals of macromolecules. Energy for the bead's motion is supplied by a vortex mixer or an ultrasonic bath. The crushed crystal is serially diluted to prepare a seed-stock mixture of the desired concentration for crystallization. Crystals produced using both hanging-drop vapor diffusion and a capillary microbatch method show expected dilution behavior. This technique of producing seed stock is compared with traditional means and advantages over the standard protocol are demonstrated.

Structural Determinants for Binding of Sorting Nexin 17 (SNX17) to the Cytoplasmic Adaptor Protein Krev Interaction Trapped 1 (KRIT1)

“…Initial crystals were obtained by vapor diffusion in sitting drops at room temperature with a 1:1 (v/v) ratio of purified protein complex to reservoir solution containing 2.0 M ammonium sulfate, 0.1 M HEPES, pH 7.5, and 2% PEG 400. Optimization of crystal singularity and size was achieved by preparing a serially diluted seed stock of microcrystals in mother liquor similar to the method described in (37) and adjusting the concentration of ammonium sulfate to 1.9 M and PEG 400 to 5%. The best crystals were obtained by adding seed stock at 1:10 (v/v) ratio to a drop containing 1:1 ratio of protein complex stock and mother liquor.…”

Crystallization of an apo form of human arginase: using all the tools in the toolbox simultaneously

“…The seed stock was created using a 3 mm Teflon sphere in a 1.6 ml Eppendorf tube according to the method described by Luft & DeTitta (1999). In order to produce a very robust seed stock, we created one batch of cross-linked seeds: in this case, the MMT buffer was omitted from the seed solution as the Tris component of the MMT buffer would swamp the glutaraldehyde cross-linking reaction.…”

Time‐Controlled Microfluidic Seeding in nL‐Volume Droplets To Separate Nucleation and Growth Stages of Protein Crystallization

“…[10,17,18] Seeding eliminates the need for nucleation and growth to occur in the same solution and separates the two stages of protein crystallization in trials that use microliter volumes and larger. [14,15,[19][20][21] However, seeding using small (nL) volumes can be more difficult or not possible at all, because seeds are too small to be seen [22,23] or are too delicate to be handled. [17] To overcome these challenges, we have developed a plug-based microfluidic system to perform seeding and bridge the supersaturation gap in nL volumes.…”