Abstract
Large Stokes shift (LSS) fluorescent proteins (FPs) are important for dual-color fluorescence cross-correlation spectroscopy and multicolor imaging. tKeima is a tetrameric LSS FP from the stony coral Montipora sp. Analyzing the tetrameric interface of tKeima is necessary to understand the oligomeric state of the Keima family and to provide insights into engineering oligomeric FPs to generate monomeric FPs, which are useful for FP-based molecular and cell biology studies. Here, detailed experimental procedures for tKeima were reported, including spontaneous crystal growth, data collection for X-ray diffraction, and structure determination. This information can be used for future experiments to obtain the high-resolution structure of tKeima, providing accurate structural information to comprehensively understand the molecular function of tKeima and the protein engineering of tetrameric FPs.