Stanford Synchrotron Radiation Lightsource (SSRL) ve Linac Coherent Light Source (LCLS) Users Meeting (23/09/2024) etkinliğine "Development of microfluidic mix-and-spray devices for time-resolved cryogenic microED" başlıklı sözlü sunum ile katıldık.
Abstract:
Cryogenic microcrystal Electron Diffraction (microED) has emerged as a crucial technique for determining high-resolution structures of macromolecules, particularly when established methods such as X-ray crystallography and serial femtosecond crystallography (SFX) are not feasible due to sample or resource limitations [1, 2]. The quality of microED data is highly dependent on sample preparation, which involves the deposition of microcrystals onto transmission electron microscopy (TEM) grids. In this work, we investigate the deposition and subsequent vitrification of enzyme microcrystals using gas dynamic virtual nozzle (GDVN) technology. We explore the effects of various GDVN parameters, including single and double GDVN setups, sheath gas pressure, and sample flow rate, on crystal deposition, ice thickness, and diffraction quality. Additionally, we examine the influence of different plunging speeds and environmental interactions on the degree of ice contamination present on the TEM grid. Furthermore, we discuss the possible use of microfluidic mixers [3] in combination with the proposed GDVN setup, enabling time-resolved ligand mixing studies. Preliminary results of cryogenic sample preparation and microED structure determination of proteinase K microcrystals are presented. Our findings suggest optimal GDVN parameters for microED sample preparation, presenting a path for diffusion-limited dynamics to be visualized by time-resolved microED.
References
[1] Hattne, J., Reyes, F. E., Nannenga, B. L., Shi, D., De La Cruz, M. J., Leslie, A. G., & Gonen, T. (2015). MicroED data collection and processing. Acta Crystallographica Section A: Foundations and Advances, 71(4), 353-360.
[2] Barends, T. R., Stauch, B., Cherezov, V., & Schlichting, I. (2022). Serial femtosecond crystallography. Nature reviews Methods primers, 2(1), 59.
[3] Mäeots, M. E., Lee, B., Nans, A., Jeong, S. G., Esfahani, M. M., Ding, S., ... & Enchev, R. I. (2020). Modular microfluidics enables kinetic insight from time-resolved cryo-EM. Nature communications, 11(1), 3465.
Yorumlar