Mikko J Hynönen, Rajaram Venkatesan
Obtaining reproducible and high-quality crystals is a prerequisite in X-ray crystallography to determine high resolution protein structures. Often, protein crystallization is sensitive to several factors leading to difficulties in their reproducibility and diffraction quality. A number of methods have been developed to improve crystallization. Here, we report on the strategies applied to overcome the challenges encountered during the crystallization and structure determination of a truncated soluble variant of OmamC (Rv1363c; OmamC129-261). OmamC is a homolog of mammalian cell entry (Mce) associated membrane (Mam) proteins of the lipid importing Mce complexes. OmamC is essential for the storage and utilization of lipids under starvation in mycobacteria. Heat treatment of OmamC129-261 prior to crystallization setup to remove partially unfolded protein and using the reservoir solution from the wells which successfully produced crystals as precipitant in subsequent crystallization trials (precipitant salvaging) enhanced the reproducibility and diffraction quality of OmamC129-261 crystals significantly. Heat treatment combined with precipitant salvaging is easily adaptable to any protein of interest and therefore, a valuable addition to the existing toolkit of protein crystallization improvement strategies. Furthermore, despite the AlphaFold2 predicted model providing an initial molecular replacement solution, experimental phasing was necessary highlighting its importance in determining the structure of OmamC129-261.