Evaluation of the data-collection strategy for room-temperature micro-crystallography studied by serial synchrotron rotation crystallography combined with the humid air and glue-coating method

Abstract

Synchrotron serial crystallography (SSX) is an emerging data-collection method for micro-crystallography on synchrotron macromolecular (MX) crystallography beamlines. At SPring-8, the feasibility of the fixed-target approach was examined by collecting data using a 2D raster scan combined with goniometer rotation. Results at cryogenic temperatures demonstrated that rotation is effective for efficient data collection in SSX and the method was named serial synchrotron rotation crystallography (SS-ROX). To use this method for room-temperature (RT) data collection, a humid air and glue-coating (HAG) method was developed in which data were collected from polyvinyl alcohol-coated microcrystals fixed on a loop under humidity-controlled air. The performance and the RT data-collection strategy for micro-crystallography were evaluated using microcrystals of lysozyme. Although a change in unit-cell dimensions of up to 1% was observed during data collection, the impact on data quality was marginal. A comparison of data obtained at various absorbed doses revealed that absorbed doses of up to 210 kGy were tolerable in both global and local damage. Although this limits the number of photons deposited on each crystal, increasing the number of merged images improved the resolution. On the basis of these results, an equation was proposed that relates the achievable resolution to the total photon flux used to obtain a data set.

In situ crystal data-collection and ligand-screening system at SPring-8

“…4(b), the resolution of the merged data improves as the number of crystals for each protein increases in the in situ diffraction measurements using crystallization plates. This result has an analogy to the correlation between the number of crystals and the resolution in SS-ROX measurements (Hasegawa et al, 2017(Hasegawa et al, , 2021. In situ diffraction measurements from multiple crystals using a microfluidic channel have also reportedly obtained a higher resolution and completeness of the data by increasing the number of crystals measured (Maeki et al, 2020).…”

“…Furthermore, synchrotron microbeam technology enables small-wedge data collection from multiple tiny crystals or multiple domains within each crystal (Cherezov et al, 2007;Rasmussen et al, 2011;Rosenbaum et al, 2011). This technique has been further developed into serial synchrotron rotation crystallography (SS-ROX; Gati et al, 2014;Hasegawa et al, 2017Hasegawa et al, , 2021. The small-wedge data-collection method is useful in the case of multiple tiny crystals, which limit the X-ray dose due to the small volume of each crystal.…”

Approach of Serial Crystallography II

“…During the drafting of this Special Issue, various SX technologies have continued to further develop. Although these studies are not addressed in this Special Issue, they provide useful information for maintaining an optimal environment specific to each facility [14][15][16][17][18][19] or target sample delivery method [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35]. All articles in this Special Issue discussing SX approaches and recently developed SX techniques will provide great opportunities to better understand macromolecular functions in greater depth.…”