THE IN VIVO MACROMOLECULAR CRYSTALLOGRAPHY PLATFORM AT NAGOYA UNIVERSITY

In recent years, a new branch of macromolecular crystallography (MX) is emerging that focuses on protein crystals naturally occurring inside cells and organisms. This field of research is in turn named in cellulo or in vivo MX (iMX), mostly depending on whether the crystals appear inside or outside cells. One of the motivations for studying these rare phenomena lies in their applicability to developing high throughput pipelines and revolutionizing the currently implemented workflows. The classical MX pipelines are composed of multiple interdependent steps. After overexpressing the protein of interest in heterologous expression systems, the samples are extensively purified to obtain a highly homogenous, concentrated, and stable sample solution. Before obtaining crystals of the purified protein, the sample solution is submitted to an empirical and semi-rational crystallization screening process that is time-consuming, costly, and with no insurance of success and crystallization. When considering iMX, all the steps of sample purification and crystallization are removed and left to be done by the organism overexpressing the protein of interest.  The iMX involves proteins that eventually present post-translational modifications, another difference compared to the in vitro MX that commonly clips out these modifications for homogeneity. Only a few modified protein structures prepared through in vitro pipelines are deposited in the PDB, although these modifications are often key to the proteins' functions. Numerous efforts from various groups worldwide have been provided to obtain deeper insights into the yet uncontrollable events dictating natural crystal growth. One of these efforts led to the initiation of a platform at Synchrotron SOLEIL (France). To facilitate the use of iMX by the larger community and in particular in Japan, milestones for the implementation of a complete pipeline have been set at Nagoya University and will be presented. The iMX platform is part of the Next-BINDS national project and is aimed to open to general users in the coming year.