Nearly 150,000 Americans younger than the retirement benefit age (<65 years) were killed each year by cardiovascular disease (CAD) according to the latest statistics from the American Heart Association. Major risk factors for CVD are the plasma lipoprotein levels. Lipoproteins are classified according to their densities as high , low-, intermediate- and very low-density lipoproteins (HDL, LDL, IDL and VLDL respectively), as well as chylomicrons; HDL and LDL are major players in plasma cholesterol metabolism. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in CVD. LDL can undergo oxidative modifications that mediate the accretion of LDL-cholesterol in the arterial wall. LDL particles vary in size, shape, and composition, and comprise large LDL (LDL1-2) and small, dense LDL (LDL3-7) subclasses; the latter are more prone to oxidation. Each LDL particle contains one molecule of apolipoprotein B-100 (apoB-100), a ligand for hepatic clearance of plasma cholesterol via LDL receptors. HDL sequesters cholesterol from peripheral tissues, including the arterial wall, and transports it to the liver fo recycling and disposal, a process called reverse cholesterol transport (RCT). HDL subspecies comprise particles that vary in size, shape, and composition. They distribute according to size and lipid amount into lipid- poor, nascent and spherical HDL. Plasma HDL particles contain multiple apolipoproteins, but the most abundant is apoA-I. ApoA-I mediates cholesterol efflux via the cellular ATP-binding cassette transporter A1 (ABCA1), and produces nascent discoidal HDL particles that are then converted to spherical HDL by lecithin- cholesterol acyltransferase (LCAT). Spherical HDL is the dominant form of HDL in plasma and is hepatically removed by scavenger receptor class B, type I (SR-BI), which mediates selective cholesteryl ester uptake. Structural determination of lipoprotein particles has been frustrated by conventional techniques (X-ray and NMR) because lipoproteins vary in size, shape, components, and biological functions and are dynamic in nature. Electron microscopy (EM), as a novel technique, allows direct visualization of individual particles. We have successfully viewed frozen-hydrated lipoproteins without distorting stains or fixatives, but the contrast is limited. Although the contrast can be enhanced by conventional cryoEM classification and averaging methods in which thousands of images from different particles are grouped and averaged based on similarity (cross- correlation coefficient) between each two images, this strategy fails for heterogeneous particle populations. Thus, we invented an individual particle electron tomography (IPET) technique that allows us to obtain a 3D cryoET density map using intermediate resolution (~3nm) based images from one targeted single-molecule (PLoS One, 2012, 7:e30249, 1-19). Although the approach sounds ambitious and aggressive, considering that very limited lipoprotein structure information has been discovered even after NIH funding for four decades, our aggressive approach has revealed more than a hundred 3D density maps from HDL particles that vary in size from 7nm to 20nm in the last two years. Thus, it is worthy to expect a significant exploration in lipoprotein structure by our IPET approach supported by NIH funding. It is necessary for the review committee to have an open mind in considering a funding opportunity for a totally new approach that has never been used before. Although there may be unexpected difficulties in using this new approach, considering my rich cryoEM experience and achievement in various lipoprotein structure studies in the last few years (12 peer-reviewed articles,leading5 articles in high impact journals under no major funding condition), my experience should be sufficient for troubleshooting to reproduce and even improve our achieved resolution shown in 17nm HDL that is already sufficient to fit the helical bundle domain in apoA-I/HDL particles, provide an overall frame of lipoprotein structure and answer the major biological questions in lipoprotein mechanism (more details in proposal). As a backup approach, if the resolution from low-contrast cryoET images is unexpectedly too low to provide a useful structure of lipoprotein, we will apply the IPET reconstruction on the high-contrast NS images by using our reported optimized negative-staining (OpNS) protocol. We have successfully achieved near ~1nm resolution maps from high-contrast OpNS images: for example, a ~1.4 nm resolution map of a well-known dynamic molecule, a single antibody (PLoS One, 2012) and a ~1.1 nm resolution map of a smaller single molecule, 53kDa CETP structure (PLoS One, 2012, 7:e30249, 1-19). This resolution is sufficient to provide the helical bundle framework and ligand spacing. Our reported OpNS is developed to eliminate the rouleau artifact represented in the conventional NS EM (NS-EM). As an additional backup approach, concerning the flatness artifact from the drying procedure of OpNS, we will apply the IPET reconstruction on our reported high-contrast cryo-positive-staining (cryo-PS) images. Our cryo- PS images can provide high-contrast and amazing structural details of small proteins, such as CETP (NCB, 2012) and spherical HDL (JLR, 2011). It is reasonable to expect a high-resolution no-flatness 3D map. Four specific aims are proposed: 1) To test the structural model of LDL by IPET and anti-apoB antibodies; 2) To test eight structural models of nascent HDL by IPET 3) To test two structure models of spherical HDL by IPET; 4) To validate the structural model of HDL by IPET and apoA-I ligands (LCAT and anti-ApoA-I antibodies).