Two polymorphic cholesterol monohydrate crystal structures form in macrophage culture models of atherosclerosis.

The formation of atherosclerotic plaques in the blood vessel walls is the result of LDL particle uptake, and consequently of cholesterol accumulation in macrophage cells. Excess cholesterol accumulation eventually results in cholesterol crystal deposition, the hallmark of mature atheromas. We followed the formation of cholesterol crystals in J774A.1 macrophage cells with time, during accumulation of LDL particles, using a previously developed correlative cryosoft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM) technique. We show, in the initial accumulation stages, formation of small quadrilateral crystal plates associated with the cell plasma membrane, which may subsequently assemble into large aggregates. These plates match crystals of the commonly observed cholesterol monohydrate triclinic structure. Large rod-like cholesterol crystals form at a later stage in intracellular locations. Using cryotransmission electron microscopy (cryo-TEM) and cryoelectron diffraction (cryo-ED), we show that the structure of the large elongated rods corresponds to that of monoclinic cholesterol monohydrate, a recently determined polymorph of the triclinic crystal structure. These monoclinic crystals form with an unusual hollow cylinder or helical architecture, which is preserved in the mature rod-like crystals. The rod-like morphology is akin to that observed in crystals isolated from atheromas. We suggest that the crystals in the atherosclerotic plaques preserve in their morphology the memory of the structure in which they were formed. The identification of the polymorph structure, besides explaining the different crystal morphologies, may serve to elucidate mechanisms of cholesterol segregation and precipitation in atherosclerotic plaques.

Helical Membrane Protein Crystallization in the New Era of Electron Cryo-Microscopy.

Helical assemblies of proteins, which consist of a two-dimensional lattice of identical subunits arranged with helical symmetry, are a common structural motif in nature. For membrane proteins, crystallization protocols can induce helical arrangements and take advantage of the symmetry found in these assemblies for the structural determination of target proteins. Modern advances in the field of electron cryo-microscopy (cryo-EM), in particular the advent of direct electron detectors, have opened the potential for structure determination of membrane proteins in such assemblies at high resolution. The nature of the symmetry in helical crystals of membrane proteins means that a single image potentially contains enough information for three-dimensional structural determination. With the current direct electron detectors, we have never been closer to making this a reality. Here, we present a protocol detailing the preparation of helical crystals, with an emphasis on further cryo-EM analysis and structural determination of the sarco(endo)plasmic reticulum Ca2+-ATPase in the presence of regulatory subunits such as phospholamban.