X-ray Diffraction of Protein Crystal Grown in a Nano-liter Scale Droplet in a Microchannel and Evaluation of Its Applicability

Abstract

We describe the technical aspects of the in-situ X-ray diffraction of a protein crystal prepared by a nanodroplet-based crystallization method. We were able to obtain diffraction patterns from a crystal grown in a capillary without any manipulation. Especially in our experimental approach, the crystals that moved to the nanodroplet interface were fixed strongly enough to carry out X-ray diffraction measurements that could be attributed to the high surface tension of the nanodroplet. The crystal was damaged by an indirect action of the X-rays because our in-situ X-ray diffraction measurement was carried out in the liquid phase without freezing the crystal; however, the obtained several diffraction patterns were of sufficiently fine quality for the crystal structure factors to be generated. We consider the technical examination presented in this paper to represent a seamless coupling of crystallization to X-ray analysis.

In meso in situ serial X-ray crystallography of soluble and membrane proteins

“…In situ data collection is not new. It has been demonstrated on a number of other crystallization platforms that include vapour-diffusion plates (Bingel-Erlenmeyer et al, 2011;Axford et al, 2012;Cipriani et al, 2012), microfluidic chips (Pinker et al, 2013;Heymann et al, 2014;Perry et al, 2014), acoustically loaded pins (Yin et al, 2014) and microcapillaries (Pineda-Molina et al, 2012), and with nanodroplets (Maeki et al, 2012). However, to date there are but a few examples in which the in situ approach has been implemented with in meso crystallogenesis.…”

Microfluidic Approaches for Protein Crystal Structure Analysis

“…The microfluidic devices also provide many advantages for X-ray crystal structure analysis, such as allowing on-chip cryoprotection and X-ray diffraction measurement. [26][27][28][29][30][31] Several microfluidic-based platforms, techniques, and approaches for protein crystallization have been studied. Notably, protein crystallization coupled with crystal structure analysis is the most significant methodology for novice researchers involved in protein crystallography.…”

“…We demonstrated in situ X-ray diffraction measurements coupled with droplet-based protein crystallization, and examined the effect of the wall thickness and the material of the capillary used in the X-ray diffraction measurement. 27 Glass and PFA capillaries were used for in situ X-ray analysis. The thicknesses of the capillary walls were 10 and 80 μm for glass and PFA capillaries, respectively.…”

A microfluidic-based protein crystallization method in 10 micrometer-sized crystallization space

“…Recently, several in situ X-ray analysis methods have been reported that improve the efficiency of diffraction data collection and can be applied to membrane protein crystallography studies. [12][13][14] We have also demonstrated the potential for droplet-based microfluidics to obtain a protein single crystal. 15,16 We found that the droplet size was an essential parameter for controlling protein crystallization.…”