Protein crystal structure obtained at 2.9 Å resolution from injecting bacterial cells into an X-ray free-electron laser beam

Significance In vivo microcrystals have been observed in prokaryotic and eukaryotic cells. With rare exception, however, the ∼100,000 biological structures determined by X-ray crystallography to date have required the macromolecule under study to be extracted from the cells that produced it and crystallized in vitro. In vivo crystals present a challenge for structure determination and pose the question of the extent to which in vivo macromolecular structures are similar to those of extracted and recrystallized macromolecules. Here we show that serial femtosecond crystallography enabled by a free-electron laser yields the structure of in vivo crystals, as they exist in a living cell, and in this case the in vivo structure is essentially identical to the structure of extracted and recrystallized protein. It has long been known that toxins produced by Bacillus thuringiensis (Bt) are stored in the bacterial cells in crystalline form. Here we describe the structure determination of the Cry3A toxin found naturally crystallized within Bt cells. When whole Bt cells were streamed into an X-ray free-electron laser beam we found that scattering from other cell components did not obscure diffraction from the crystals. The resolution limits of the best diffraction images collected from cells were the same as from isolated crystals. The integrity of the cells at the moment of diffraction is unclear; however, given the short time (∼5 µs) between exiting the injector to intersecting with the X-ray beam, our result is a 2.9-Å-resolution structure of a crystalline protein as it exists in a living cell. The study suggests that authentic in vivo diffraction studies can produce atomic-level structural information.