Structure of a heterogeneous, glycosylated, lipid-bound,in vivo-grown protein crystal at atomic resolution from the viviparous cockroachDiploptera punctata

Abstract

Macromolecular crystals for X-ray diffraction studies are typically grownin vitrofrom pure and homogeneous samples; however, there are examples of protein crystals that have been identifiedin vivo. Recent developments in micro-crystallography techniques and the advent of X-ray free-electron lasers have allowed the determination of several protein structures from crystals grownin cellulo. Here, an atomic resolution (1.2 Å) crystal structure is reported of heterogeneous milk proteins grown inside a living organism in their functional niche. Thesein vivo-grown crystals were isolated from the midgut of an embryo within the only known viviparous cockroach,Diploptera punctata. The milk proteins crystallized in space groupP1, and a structure was determined by anomalous dispersion from the native S atoms. The data revealed glycosylated proteins that adopt a lipocalin fold, bind lipids and organize to form a tightly packed crystalline lattice. A single crystal is estimated to contain more than three times the energy of an equivalent mass of dairy milk. This unique storage form of nourishment for developing embryos allows access to a constant supply of complete nutrients. Notably, the crystalline cockroach-milk proteins are highly heterogeneous with respect to amino-acid sequence, glycosylation and bound fatty-acid composition. These data present a unique example of protein heterogeneity within a singlein vivo-grown crystal of a natural protein in its native environment at atomic resolution.