Advanced Methods of Protein Crystallization

Abstract

This chapter provides a review of different advanced methods that help to increase the success rate of a crystallization project, by producing larger and higher quality single crystals for determination of macromolecular structures by crystallographic methods. For this purpose, the chapter is divided into three parts. The first part deals with the fundamentals for understanding the crystallization process through different strategies based on physical and chemical approaches. The second part presents new approaches involved in more sophisticated methods not only for growing protein crystals but also for controlling the size and orientation of crystals through utilization of electromagnetic fields and other advanced techniques. The last section deals with three different aspects: the importance of microgravity, the use of ligands to stabilize proteins, and the use of microfluidics to obtain protein crystals. All these advanced methods will allow the readers to obtain suitable crystalline samples for high-resolution X-ray and neutron crystallography.

N-Terminal Segment of TvCyP2 Cyclophilin from Trichomonas vaginalis Is Involved in Self-Association, Membrane Interaction, and Subcellular Localization

“…Initial crystallization screening of Tv CyP2 involved using commercial crystallization screen kits (Hampton Research, Jena Bioscience, and Qiagen, Aliso Viejo, CA, USA), 96-well Intelli-plates (Art Robbins Instruments, Sunnyvale, CA, USA) and a Phoenix robot (Art Robbins Instruments, Sunnyvale, CA, USA) at 298 K. The sitting drop vapor diffusion method [ 38 , 39 ] was used to grow crystals. The 0.6–3 µm crystals of Tv CyP2 appeared within 5 days in the commercial kit conditions.…”

Advanced Biocrystallogenesis

“…The principle of the method benefiting from electric fields relies on the separation of the nucleation and crystal growth; it can be applied in the electric as well as the electromagnetic field. The nucleation process takes place while the current is switched off [28]. In order to perform crystallization experiments in the electric field, the specially designed crystal growth cell has to be used.…”

“…It was reported that the current positively influences the size of protein crystals. Specifically, lower frequencies of alternating current provide less larger crystals compared to higher frequencies [28]. For the purposes of using electric current the e-crystallization cells based on vapour diffusion or batch method were made [29].…”

“…Moreno 2017 mentions the use of gels for obtaining large well-arranged crystals together with use of magnetic forces for at best two days and more days. For this type of experiment, the capillary glass pipettes were reported to be suitable for use of homogeneous or nonhomogeneous magnetic fields [28]. The best influence of magnetic field use is regulation of quality and crystal size [32].…”

“…Most widely used crystallization robots are from the Oryx series (Douglas Instruments Ltd.), the Mosquito (TTP Labtech, Royston, UK) and TOPAZ system (Fluidigm Corp., San Francisco CA, USA) [64]. Automation devices allow crystallization of nanovolumes into an array of plates for 96 or more conditions [28]. Each robot has its own specification, from which can be chosen preferred experimental method suitable for protein of interest.…”