Preparation and Delivery of Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Abstract

Membrane proteins (MPs) are important components of cellular membranes and primary drug targets. Rational drug design relies on the exact structural information about the protein, however MPs are difficult to handle and crystallize. Recent progress in MP structural determination has benefited greatly from the development of lipidic cubic phase (LCP) crystallization method, which yields well-diffracting but often small crystals that suffer from radiation damage during traditional crystallographic data collection at synchrotron sources. Introduction of the X-ray free-electron laser (XFEL) source that produces extremely bright femtosecond pulses enabled room temperature data collection from microcrystals with minimal, if any, radiation damage. Our recent efforts in combining LCP technology with serial femtosecond crystallography (LCP-SFX) have resulted in high-resolution structures of several human G protein-coupled receptors, the largest and the most important drug target superfamily, yet notoriously difficult for structural determination. In LCP-SFX technique, LCP is recruited as a matrix for both growth and delivery of membrane protein microcrystals to the intersection with an XFEL beam for crystallographic data collection. It has been demonstrated that LCP-SFX can significantly improve the diffraction resolution when only micron-sized crystals are available and when crystal quality is too poor to yield interpretable structural data at conventional synchrotron microfocus beamlines. Here we present the protocol for preparation, characterization and delivery of microcrystals in LCP for LCP-SFX experiments.