miniaturized-sample-preparation-for-transmission-electron-microscopy

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

University of Basel

Phospholipases are abundant in various types of cells and compartments, where they play key roles in physiological processes as diverse as digestion, cell proliferation, and neural activation. In Gram-negative bacteria, outer membrane phospholipase A (OmpLA) is involved in outer-membrane lipid homeostasis and bacterial virulence. Although the enzymatic activity of OmpLA can be probed with an assay relying on an artificial monoacyl thioester substrate, only little is known about its activity on diacyl phospholipids. Here, we used high-speed atomic force microscopy (HS-AFM) to directly image enzymatic phospholipid degradation by OmpLA in real time. In the absence of Ca, reconstituted OmpLA diffused within a phospholipid bilayer without revealing any signs of phospholipase activity. Upon the addition of Ca, OmpLA was activated and degraded the membrane with a turnover of ~2 phospholipid molecules per second and per OmpLA dimer until most of the membrane phospholipids were hydrolyzed and the protein became tightly packed.

Real-time Visualization of Phospholipid Degradation by Outer Membrane Phospholipase A using High-Speed Atomic Force Microscopy.

This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies.

Article	Year
Real-time Visualization of Phospholipid Degradation by Outer Membrane Phospholipase A using High-Speed Atomic Force Microscopy. Journal of molecular biology\| PubMed ID: 28283404	2017
Miniaturizing EM Sample Preparation: Opportunities, Challenges, and "Visual Proteomics". Proteomics\| PubMed ID: 29441686	2018

Luca Rima

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