Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

Spherical high density lipoproteins (HDL) † predominate in human plasma. However, little information exists on the structure of the most common HDL protein, apolipoprotein (apo) A-I, in spheres vs. better studied discoidal forms. We produced spherical HDL by incubating reconstituted discoidal HDL with physiological plasmaremodeling enzymes and compared apoA-I structure in discs and spheres of comparable diameter (79 -80 and 93-96 Å). Using cross-linking chemistry and mass spectrometry, we determined that the general structural organization of apoA-I was overall similar between discs and spheres, regardless of diameter. This was the case despite the fact that the 93 Å spheres contained three molecules of apoA-I per particle compared with only two in the discs. Thus, apoA-I adopts a consistent general structural framework in HDL particles-irrespective of shape, size and the number of apoA-Is present. Furthermore, a similar cross-linking pattern was demonstrated in HDL particles isolated from human serum. We propose the first experiment-based molecular model of apoA-I in spherical HDL particles. This model provides a new foundation for understanding how apoA-I structure modulates HDL function and metabolism.sphere ͉ disk G iven the inverse correlation between high density lipoprotein (HDL) levels and cardiovascular disease, a key question in vascular biology relates to how apolipoproteins modulate the metabolism and function of HDL. Significant evidence supports a role for HDL in the process of reverse cholesterol transport whereby lipids and cholesterol in the vessel wall are transported to the liver for catabolism. However, because of a lack of information on HDL structure and the molecular basis of its interactions with other proteins, our understanding of HDL metabolism and function is at a basic stage.The ''glue'' that holds most HDL particles together is apolipoprotein (apo)A-I, a highly ␣-helical, 28-kDa polypeptide. It comprises some 70% of HDL protein mass, making it the clear starting point for deriving a basic understanding of HDL structure. In humans, apoA-I is primarily present in two major spherical HDL species, HDL 2 (d ϭ 1.063-1.125 g/ml) and HDL 3 (d ϭ 1.125-1.210 g/ml) with diameters ranging from 70 to 120 Å. More minor, but clearly important, HDL subspecies include lipid-poor apoA-I and nascent discoidal particles (reviewed in ref. 1). Highly reactive but low abundance discoidal HDLs are critical intermediates between lipid-poor apoA-I and mature spherical HDL. Easily produced in vitro, they have been heavily used for structural studies (2). Despite some debates on details of certain regions of apoA-I in discs, the majority of recent theoretical and experimental data supports the so-called ''double belt'' model (3). In this scheme, each of two ring-shaped apoA-I molecules wrap around a leaflet of a disk-like patch of lipid bilayer in an anti-parallel orientation.Despite their abundance in plasma, much less is known about the structure of apoA-I in spherical particles. They contain a neutral lipid...

The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.

Apolipoprotein E (apoE), one of the major protein components of lipoproteins in the peripheral and central nervous systems, regulates cholesterol metabolism through its interaction with members of the low density lipoprotein receptor family. One key to understanding apoE function is determining the structure of lipid-bound forms of apoE. Negative-staining (NS) electron microscopy (EM) is an easy and rapid approach for studying the structure and morphology of lipid-bound forms of apoE. However, an artifact of using the conventional NS protocol is that the apoE•phospholipid particles form rouleaux. In this study, we used cryo-electron microscopy (cryo-EM) to examine apoE4•palmitoyl-oleoylphosphatidylcholine (POPC) particles in a frozen-hydrated native state. By comparing the particle sizes and shapes produced by different NS protocols to those produced by cryo-EM, we propose an optimized protocol to examine apoE4•POPC particles. Statistical analysis demonstrated that the particle sizes differ by less than 5% between the optimized protocol and the cryo-EM method, with similar shapes. The high contrast and fine detail of particle images produced using this optimized protocol lend themselves to the structural study of lipid-bound forms of apoE.

Three-dimensional (3D) structural analysis is essential to understand the relationship between the structure and function of an object. Many analytical techniques, such as X-ray diffraction, neutron spectroscopy, and electron microscopy imaging, are used to provide structural information. Transmission electron microscopy (TEM), one of the most popular analytic tools, has been widely used for structural analysis in both physical and biological sciences for many decades, in which 3D objects are projected into two-dimensional (2D) images. In many cases, 2D-projection images are insufficient to understand the relationship between the 3D structure and the function of nanoscale objects. Electron tomography (ET) is a technique that retrieves 3D structural information from a tilt series of 2D projections, and is gradually becoming a mature technology with sub-nanometer resolution. Distinct methods to overcome sample-based limitations have been separately developed in both physical and biological science, although they share some basic concepts of ET. This review discusses the common basis for 3D characterization, and specifies difficulties and solutions regarding both hard and soft materials research. It is hoped that novel solutions based on current state-of-the-art techniques for advanced applications in hybrid matter systems can be motivated.

Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

Cholesterol has been proposed to play a critical role in regulating neurotransmitter release and synaptic plasticity. The neuronal porosome/fusion pore, the secretory machinery at the nerve terminal, is a 12−17 nm cup-shaped lipoprotein structure composed of cholesterol and a number of proteins, among them calcium channels, and the t-SNARE proteins syntaxin-1 and SNAP-25. During neurotransmission, synaptic vesicles dock and fuse at the porosome via interaction of their v-SNARE protein with t-SNAREs at the porosome base. Membrane-associated neuronal t-SNAREs interact in a circular array with liposome-associated neuronal v-SNARE, to form the t-/v-SNARE ring complex. The SNARE complex along with calcium is required for the establishment of continuity between opposing bilayers. Here we show that although cholesterol is an integral component of the neuronal porosome and is required for maintaining its physical integrity and function, it has no influence on the conformation of the SNARE ring complex.