Lard Injection Matrix for Serial Crystallography

Abstract

Serial crystallography (SX) using X-ray free electron laser or synchrotron X-ray allows for the determination of structures, at room temperature, with reduced radiation damage. Moreover, it allows for the study of structural dynamics of macromolecules using a time-resolved pump-probe, as well as mix-and-inject experiments. Delivering a crystal sample using a viscous medium decreases sample consumption by lowering the flow rate while being extruded from the injector or syringe as compared to a liquid jet injector. Since the environment of crystal samples varies, continuous development of the delivery medium is important for extended SX applications. Herein, I report the preparation and characterization of a lard-based sample delivery medium for SX. This material was obtained using heat treatment, and then the soluble impurities were removed through phase separation. The lard injection medium was highly stable and could be injected via a syringe needle extruded at room temperature with a flow rate < 200 nL/min. Serial millisecond crystallography experiments were performed using lard, and the room temperature structures of lysozyme and glucose isomerase embedded in lard at 1.75 and 1.80 Å, respectively, were determined. The lard medium showed X-ray background scattering similar or relatively lower than shortenings and lipidic cubic phase; therefore, it can be used as sample delivery medium in SX experiments.

Room-Temperature Structure of Xylitol-Bound Glucose Isomerase by Serial Crystallography: Xylitol Binding in the M1 Site Induces Release of Metal Bound in the M2 Site

“…The monomer subunit further forms the functional tetrameric assembly observed in the crystal symmetry ( Figure 2 b). The subunit topology and tetrameric assembly of SruGIs were identical with previously reported crystal structures of SruGI [ 10 , 11 , 13 , 29 , 30 ]. The structure of native SruGI was highly similar to other room-temperature crystal structures of native SruGI determined by serial crystallography (PDB codes: 6KCA, 6KD2, 7BVL, and 7CK0) [ 10 , 11 , 29 , 30 ], with r.m.s.d.…”

“…These images were further indexed and scaled using the CrystFEL program [ 35 ]. The phasing problem was solved using the molecular-replacement method, as implemented in MOLREP [ 36 ], and the crystal structure of SruGI (PDB code: 7CK0) [ 29 ] as the search model. Model building was performed using the COOT program [ 37 ], and structural refinement was performed using REFMAC5 [ 38 ].…”

Polyimide mesh-based sample holder with irregular crystal mounting holes for fixed-target serial crystallography

“…Accordingly, to reduce the efforts in sample preparation, minimizing the consumption of crystal samples during SX data collection is one of the key factors. Various sample delivery techniques such as injectors 11 – 13 , syringes 14 , 15 , injection with viscous medium 12 , 14 , 16 – 21 , fixed target scanning 22 – 28 , microfluidic devices 29 , capillaries 5 , 30 , and conveyer belts 31 have been developed and applied to SX experiments at XFEL facilities or synchrotrons. Among these, the fixed target sample scanning method has a great advantage for low sample consumption and for minimizing the physical damage to the crystal during the data collection when compared with other sample delivery methods 22 , 25 , 26 .…”

Serial X-ray Crystallography

“…Injectors that have been recently developed are evolving to enable time-resolved studies by internally mixing substrates or inhibitors with protein crystals [25][26][27]. Additionally, after the development of lipid cubic phase (LCP) injectors [28,29], which could reduce sample consumption at the XFEL facility with a low repetition rate or synchrotron, various viscous materials, such as different types of grease [30][31][32][33], Vaseline [34], agarose [35], hyaluronic acid [32], hydroxyethyl cellulose [31], carboxymethyl cellulose sodium salt [36], pluronic F-127 [36], polyethylene oxide [37], polyacrylamide [38], wheat starch [39], alginate [39], shortening [40], and lard [41], were developed and applied. This provided an opportunity to deliver samples according to the characteristics of crystal samples [23].…”