3D-printed holders forin meso in situfixed-target serial X-ray crystallography

Abstract

The in meso in situ serial X-ray crystallography method was developed to ease the handling of small fragile crystals of membrane proteins and for rapid data collection on hundreds of microcrystals directly in the growth medium without the need for crystal harvesting. To facilitate mounting of these in situ samples on a goniometer at cryogenic or at room temperatures, two new 3D-printed holders have been developed. They provide for cubic and sponge phase sample stability in the X-ray beam and are compatible with sample-changing robots. The holders can accommodate a variety of window material types, as well as bespoke samples for diffraction screening and data collection at conventional macromolecular crystallography beamlines. They can be used for convenient postcrystallization treatments such as ligand and heavy-atom soaking. The design, assembly and application of the holders for in situ serial crystallography are described. Files for making the holders using a 3D printer are included as supporting information.

Implementation of wedged-serial protein crystallography at PROXIMA-1

“…The reduced thickness of the chip makes it highly bendable and therefore increases the need for a stiff handling structure, which takes the form of an interface between the chip and the goniometer to ensure effective manipulation. Such a procedure was used and reported previously for other in situ applications (Broecker et al, 2018;Huang et al, 2020). In the current work, a specific 3D-printed encapsulating frame was designed to give a rigid support that protects the chip from mechanical stress and deformation and permits the chip to be gripped and lifted either manually or by a sample-mounting robot (Fig.…”

“…S2). Though with a different design, use of a 3D-printed frame has already been reported (Broecker et al, 2018;Huang et al, 2020) and presents the advantage to rigidify the chip, which allows easier handling and eventually robot-assisted mounting. Additionally, and most importantly, using such a tool reduces the physical stress and, in turn, avoids liquid/sample movements inside the chip during manipulation.…”

The Crystal Structure of Bromide-BoundGtACR1 Reveals a Pre-Activated State in the Transmembrane Anion Tunnel

“…The most highly diffracting crystals were obtained from crystallization screen containing 15% 2-methyl-2,4-pentanediol (MPD), 0.1 M NaBr and buffer of 0.1 M MES, pH 5.5, or Na-acetate, pH 5.5. LCP crystals from glass plate were harvested using micromesh loops (MiTeGen, Ithaca, NY) and the wells with crystals-laden LCP in IMISX plate were retrieved using a glass cutter and scissors and mounted using 3D-printed holders (Huang et al, 2020). All the samples were flash-cooled in liquid nitrogen without any additional cryoprotectant and stored in uni-pucks (MiTeGen, Ithaca, NY) for X-ray diffraction.…”

3D-printed equipment to decouple (powder) X-ray diffraction sample preparation and measurement

“…Miller et al (2011) used 3D-printed models to make apertures for X-and -rays in single-photon emission computed tomography applications by cold casting (pouring a mixture of metal powder and resin into a mould). The existing technique of in meso in situ crystallographic characterization of proteins to determine their conformation within a liquid medium was recently improved by 3D-printed sample holders (Huang et al, 2020). Loops for crystal mounting can be 3Dprinted via digital light projection stereolithography.…”