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Viscoelastic characterization of the lipid cubic phase provides insights into high-viscosity extrusion injection for XFEL experiments

D. Zabelskii,E. Round,H. Han,D. von Stetten,R. Letrun,Chan Kim,Tokushi Sato,Diogo V M Melo,R. de Wijn,K. Kharitonov,Peter Smyth,K. Doerner,M. Kloos,Thomas Dietze,Luis Lopez Morillo,Richard J Bean,A. Round

Published 2025 in Scientific Reports

ABSTRACT

Serial crystallography (SX) is a powerful method for determining macromolecular structures, enabled by the advent of X-ray free-electron lasers (XFELs). SX experiments require the continuous delivery of microcrystals, achievable through liquid jets, high-viscosity extrusion (HVE) jets, and fixed-target methods. The lipid cubic phase (LCP) is a lipid-based medium commonly used for membrane protein crystallization and as a carrier medium for HVE injection. In this study, we present a rheometric characterization of LCP media and demonstrate that sample viscosity correlates well with the injection stability and, therefore, can effectively predict sample stability during HVE jetting. Using this approach, we determined the viscosity range corresponding to the stable, metastable, and unstable jetting regions. The critical viscosity values for the metastable and unstable jetting regions are 7 × 105 and 104 mPa⋅s, measured at the 0.3 s-1 shear rate. We show that ambient humidity is crucial for rheometric and fixed-target experiments involving LCP-embedded crystals. Specifically, LCP-embedded crystals rapidly lose diffraction quality when exposed to ambient humidity below 80%. Additionally, we demonstrate that sample viscosity measured in the rheometric experiment can help determine the necessary amount of stabilizing additive for HVE jet optimization. This approach was successfully tested on LCP mixed with long-chain polyethylene glycol and stabilized with Pluronic F-127 polymer.

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