Polyhedral 3D structure of human plasma very low density lipoproteins by individual particle cryo-electron tomography1
2016; Elsevier BV; Volume: 57; Issue: 10 Linguagem: Inglês
10.1194/jlr.m070375
ISSN1539-7262
AutoresYadong Yu, Yu‐Lin Kuang, Dongsheng Lei, Xiaobo Zhai, Meng Zhang, Ronald M. Krauss, Gang Ren,
Tópico(s)RNA modifications and cancer
ResumoHuman VLDLs assembled in the liver and secreted into the circulation supply energy to peripheral tissues. VLDL lipolysis yields atherogenic LDLs and VLDL remnants that strongly correlate with CVD. Although the composition of VLDL particles has been well-characterized, their 3D structure is elusive because of their variations in size, heterogeneity in composition, structural flexibility, and mobility in solution. Here, we employed cryo-electron microscopy and individual-particle electron tomography to study the 3D structure of individual VLDL particles (without averaging) at both below and above their lipid phase transition temperatures. The 3D reconstructions of VLDL and VLDL bound to antibodies revealed an unexpected polyhedral shape, in contrast to the generally accepted model of a spherical emulsion-like particle. The smaller curvature of surface lipids compared with HDL may also reduce surface hydrophobicity, resulting in lower binding affinity to the hydrophobic distal end of the N-terminal β-barrel domain of cholesteryl ester transfer protein (CETP) compared with HDL. The directional binding of CETP to HDL and VLDL may explain the function of CETP in transferring TGs and cholesteryl esters between these particles. This first visualization of the 3D structure of VLDL could improve our understanding of the role of VLDL in atherogenesis. Human VLDLs assembled in the liver and secreted into the circulation supply energy to peripheral tissues. VLDL lipolysis yields atherogenic LDLs and VLDL remnants that strongly correlate with CVD. Although the composition of VLDL particles has been well-characterized, their 3D structure is elusive because of their variations in size, heterogeneity in composition, structural flexibility, and mobility in solution. Here, we employed cryo-electron microscopy and individual-particle electron tomography to study the 3D structure of individual VLDL particles (without averaging) at both below and above their lipid phase transition temperatures. The 3D reconstructions of VLDL and VLDL bound to antibodies revealed an unexpected polyhedral shape, in contrast to the generally accepted model of a spherical emulsion-like particle. The smaller curvature of surface lipids compared with HDL may also reduce surface hydrophobicity, resulting in lower binding affinity to the hydrophobic distal end of the N-terminal β-barrel domain of cholesteryl ester transfer protein (CETP) compared with HDL. The directional binding of CETP to HDL and VLDL may explain the function of CETP in transferring TGs and cholesteryl esters between these particles. This first visualization of the 3D structure of VLDL could improve our understanding of the role of VLDL in atherogenesis. Lipoprotein particles are composed of amphipathic apolipoproteins, phospholipids, and cholesterol at their surfaces and neutral lipids, including TG and cholesteryl ester (CE), in their cores (1van der Vusse G.J. Glatz J.F. Stam H.C. Reneman R.S. Fatty acid homeostasis in the normoxic and ischemic heart.Physiol. Rev. 1992; 72: 881-940Crossref PubMed Scopus (444) Google Scholar, 2Davis R.A. Engelhorn S.C. Pangburn S.H. Weinstein D.B. Steinberg D. Very low-density lipoprotein synthesis and secretion by cultured rat hepatocytes.J. Biol. Chem. 1979; 254: 2010-2016Abstract Full Text PDF PubMed Google Scholar, 3McEneny J. O'Kane M.J. Moles K.W. McMaster C. McMaster D. Mercer C. Trimble E.R. Young I.S. Very low density lipoprotein subfractions in Type II diabetes mellitus: alterations in composition and susceptibility to oxidation.Diabetologia. 2000; 43: 485-493Crossref PubMed Scopus (39) Google Scholar). 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After further intracellular lipidation and processing in the endoplasmic reticulum and Golgi, VLDLs are secreted into the circulation, where additional apolipoproteins, including apoAs (apoAI, apoAII, and apoAIV), apoCs (apoCI, apoCII, and apoCIII), and apoE, are acquired (8Dominiczak M.H. Caslake M.J. Apolipoproteins: metabolic role and clinical biochemistry applications.Ann. Clin. Biochem. 2011; 48: 498-515Crossref PubMed Scopus (128) Google Scholar). A major function of VLDLs is to transport TGs from the liver to peripheral tissues for use as an energy source (8Dominiczak M.H. Caslake M.J. Apolipoproteins: metabolic role and clinical biochemistry applications.Ann. Clin. Biochem. 2011; 48: 498-515Crossref PubMed Scopus (128) Google Scholar, 15Niu Y.G. Evans R.D. Very-low-density lipoprotein: complex particles in cardiac energy metabolism.J. Lipids. 2011; 2011: 189876Crossref PubMed Google Scholar). The transfer process involves anchoring to endothelial surfaces by glycosylphosphatidylinositol-anchored HDL binding protein 1 (16Fang L. Choi S.H. Baek J.S. Liu C. Almazan F. Ulrich F. Wiesner P. Taleb A. Deer E. Pattison J. et al.Control of angiogenesis by AIBP-mediated cholesterol efflux.Nature. 2013; 498: 118-122Crossref PubMed Scopus (135) Google Scholar) and activation of LPL by apoCII, resulting in the hydrolysis of VLDL TG, the release of free fatty acids, and the formation of VLDL remnant particles (17Eisenberg S. Sehayek E. Remnant particles and their metabolism.Baillieres Clin. Endocrinol. Metab. 1995; 9: 739-753Abstract Full Text PDF PubMed Scopus (7) Google Scholar, 18Cohn J.S. Marcoux C. Davignon J. Detection, quantification, and characterization of potentially atherogenic triglyceride-rich remnant lipoproteins.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 2474-2486Crossref PubMed Scopus (140) Google Scholar). The remnants can be further hydrolyzed to form LDLs by hepatic lipase, and the LDLs can be internalized by several mechanisms, including interaction with the LDL receptor (8Dominiczak M.H. Caslake M.J. Apolipoproteins: metabolic role and clinical biochemistry applications.Ann. Clin. Biochem. 2011; 48: 498-515Crossref PubMed Scopus (128) Google Scholar). In plasma, VLDLs can also exchange their containing TGs with HDL CEs mediated by CE transfer protein (CETP) via a tunnel mechanism (19Tall A.R. Plasma lipid transfer proteins.J. Lipid Res. 1986; 27: 361-367Abstract Full Text PDF PubMed Google Scholar, 20Ihm J. Quinn D.M. Busch S.J. Chataing B. Harmony J.A. Kinetics of plasma protein-catalyzed exchange of phosphatidylcholine and cholesteryl ester between plasma lipoproteins.J. Lipid Res. 1982; 23: 1328-1341Abstract Full Text PDF PubMed Google Scholar, 21Zhang L. Yan F. Zhang S. Lei D. Charles M.A. Cavigiolio G. Oda M. Krauss R.M. Weisgraber K.H. Rye K.A. et al.Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein.Nat. Chem. Biol. 2012; 8: 342-349Crossref PubMed Scopus (108) Google Scholar) by which the hydrophobic distal end of the N-terminal β-barrel domain dominantly interacts with HDLs via a hydrophobic interaction (22Zhang M. Charles R. Tong H. Zhang L. Patel M. Wang F. Rames M.J. Ren A. Rye K.A. Qiu X. et al.HDL surface lipids mediate CETP binding as revealed by electron microscopy and molecular dynamics simulation.Sci. Rep. 2015; 5: 8741Crossref PubMed Scopus (40) Google Scholar). It is unclear why this hydrophobic distal end has less interaction with the same types of surface lipids of VLDL. The directional interaction of CETP with HDL and VLDL may relate to the directional transfer of TGs and CEs between VLDL and HDL. Cholesterol-enriched VLDL lipolytic remnants are associated with increased risk of CVD (23Mahley R.W. Huang Y. Atherogenic remnant lipoproteins: role for proteoglycans in trapping, transferring, and internalizing.J. Clin. Invest. 2007; 117: 94-98Crossref PubMed Scopus (122) Google Scholar). In plasma, VLDLs have highly complex compositions and the widest variation in particle size among the lipoprotein classes, with diameters ranging from 30 to 100 nm (7Vance D.E. Vance J.E. Assembly and secretion of lipoproteins.In Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier, Amsterdam. 2002; : 505-526Crossref Google Scholar, 24Zhang L. Song J. Cavigiolio G. Ishida B.Y. Zhang S. Kane J.P. Weisgraber K.H. Oda M.N. Rye K.A. Pownall H.J. et al.Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy.J. Lipid Res. 2011; 52: 175-184Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 25van Antwerpen R. La Belle M. Navratilova E. Krauss R.M. Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy.J. Lipid Res. 1999; 40: 1827-1836Abstract Full Text Full Text PDF PubMed Google Scholar). Their heterogeneity poses a great challenge in studying their 3D structure via current structural biology methods, such as X-ray crystallography, nuclear magnetic resonance, or cryo-electron microscopy (cryo-EM) single particle reconstruction, which require either a 3D lattice or mono-dispersed particles of repeating structure. Although recent developments have enabled single particle reconstruction to classify the 3D structures of a few different conformations in silico, the numerous combinations of proteins and lipids among VLDL particles preclude a simple solution. To understand the general 3D structure of VLDL particles and the variations among them, we imaged human plasma VLDL particles under near native conditions by cryo-electron tomography (cryo-ET) and then reconstructed the 3D density maps of each VLDL particle by the individual-particle electron tomography (IPET) reconstruction method (26Zhang L. Ren G. IPET and FETR: experimental approach for studying molecular structure dynamics by cryo-electron tomography of a single-molecule structure.PLoS One. 2012; 7: e30249Crossref PubMed Scopus (61) Google Scholar). To confirm the 3D structures, we also examined the VLDLs below and above their lipid phase transition temperature and reconstructed the 3D density maps of the complexes of VLDLs bound to an anti-apoB100 antibody. Comparison of the structures indicated that VLDL surface proteins and phospholipids cooperate with each other to form a polyhedral structure. Human VLDLs were isolated from the plasma of a healthy individual by standard overnight ultracentrifugation at d = 1.006 g/ml, 40,000 rpm, and 10°C. Mouse anti-human apoB monoclonal antibody, mAB012, was obtained from Chemicon (EMD Millipore Corporation, Temecula, CA). The epitope of mAB012 is the first 20 N-terminal amino acid residues of human apoB100. To make the antigen-antibody complex, VLDLs and mAB012 were mixed at a 1:1 molar ratio and incubated overnight at 4°C before plunge-freezing the cryo-EM grids. Plunge-freezing of the cryo-EM grids was conducted using a Leica EM GP (Leica, Buffalo Grove, IL) that incorporates a chamber to control the humidity and temperature for blotting and evaporation. VLDL was first preincubated in a water-bath at 4, 40, and 45°C, the first temperature below and the latter two temperatures above the phase transition temperature (20–40°C), for more than 30 min. For each preincubation, the chamber of the plunge-freezer was set to the preincubation temperature, and the relative humidity in the chamber was maintained at 85%. Preincubated VLDL solution (3 μl) was applied to a glow-discharged lacey-carbon grid on a 200-mesh grid (EMS, Hatfield, PA) and kept in the chamber for 5 min prior to blotting and plunge-freezing. Cryo-EM imaging was conducted using a Zeiss Libra 120 transmission electron microscope (Carl Zeiss SMT GmbH, Oberkochen, Germany) equipped with a LaB6 gun (operating at 120 kV), an in-column Ω energy filter, and a 4 k × 4 k Gatan UltraScan 4000 CCD camera. The opening angle of the electron source was set to 100 μrad to select only the central portion. The electron source was further constrained by a 75 μm-diameter condenser aperture, which shines on an area twice the size of the CCD at 50 k× (2.4 Å/pixel). A 50 μm-diameter objective aperture after the specimen was used to increase contrast. The in-elastically diffracted electrons were removed by using an in-column Ω energy filter (window width set to 20 eV). A single-axis tilt series of frozen hydrated VLDLs alone was collected from −63° to +63° in steps of 1.5° at a nominal magnification of 50 k× (2.4 Å/pixel). The defocuses of the tilted views were set to 2 μm. The data were collected in low-dose mode, and the total dose was ∼150 e−/Å2. A single-axis tilt series of the VLDL-mAB012 complex was collected under a scheme similar to that of the VLDLs alone. To preserve the delicate structure of the antibodies, the total dose was reduced to ∼80 e−/Å2, and the micrographs were 2× binned during image acquisition (4.8 Å/pixel). Low-dose data acquisition was conducted by using the transmission electron microscope (TEM) tomography software (Gatan Inc., Pleasanton, CA) in advanced tomography mode. The tilt series of the micrographs was initially aligned using the software package, IMOD (27Kremer J.R. Mastronarde D.N. McIntosh J.R. Computer visualization of three-dimensional image data using IMOD.J. Struct. Biol. 1996; 116: 71-76Crossref PubMed Scopus (3601) Google Scholar). The defocus values of the tilt series were calculated by using the programs tomops.exe and tomoctffind.exe in TomoCTF (28Fernández J.J. Li S. Crowther R.A. CTF determination and correction in electron cryotomography.Ultramicroscopy. 2006; 106: 587-596Crossref PubMed Scopus (135) Google Scholar). The tilt views were phase-flipped by using the ctfcorrect.exe program of TomoCTF. To determine the 3D structures of individual particles, we boxed the images of the particle from each tilt series of micrographs and submitted the tilt series of the particle for reference-free alignment and 3D reconstruction by IPET (26Zhang L. Ren G. IPET and FETR: experimental approach for studying molecular structure dynamics by cryo-electron tomography of a single-molecule structure.PLoS One. 2012; 7: e30249Crossref PubMed Scopus (61) Google Scholar). To validate the 3D reconstructions from IPET, a popular 3D reconstruction method, IMOD, was also used for reconstruction. In the absence of nano-gold particles, the centers of the small VLDL particles were used as fiducial markers to track and align the raw views of the tilt series. The fine alignment using these markers yielded an accuracy of 3 nm or better. A 3D whole tomogram was constructed from the aligned views using the weighted back-projection implemented in IMOD. The resolution of the refined 3D model was estimated by the Fourier shell correlation (FSC) between two 3D density maps reconstructed independently from odd and even number tilt series, respectively. The resolution based on the 0.143 threshold or the 0.5 threshold is reported. The 3D structure was displayed by using UCSF Chimera (29Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. UCSF Chimera–a visualization system for exploratory research and analysis.J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (28178) Google Scholar). To gain the overall shape of each particle, the VLDL particles were simplified to polyhedrons by manually marking the vertices on the surface of 5.0 nm low-pass-filtered maps, connecting the vertices to represent the observed edges, and grouping the edges to represent the particle faces. These structural markers were chosen at a scale near 5.0 nm, leaving out smaller features for the sake of simplicity. Slight curvature of the faces was tolerated by only marking edges where dihedral angles were larger than 20° (the dihedral angle is defined as the angle between two nearby intersecting surface planes). A plasma VLDL sample prepared from a healthy person with a normal TG level of 127 mg/dl was examined by both optimized negative stain (OpNS) (30Rames M. Yu Y. Ren G. Optimized negative staining: a high-throughput protocol for examining small and asymmetric protein structure by electron microscopy.J. Vis. Exp. 2014; 90: e51087Google Scholar, 31Zhang L. Song J. Newhouse Y. Zhang S. Weisgraber K.H. Ren G. An optimized negative-staining protocol of electron microscopy for apoE4 POPC lipoprotein.J. Lipid Res. 2010; 51: 1228-1236Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) EM and cryo-EM techniques. OpNS was refined from conventional negative staining to prevent lipoprotein particles, especially HDLs, from stacking together (24Zhang L. Song J. Cavigiolio G. Ishida B.Y. Zhang S. Kane J.P. Weisgraber K.H. Oda M.N. Rye K.A. Pownall H.J. et al.Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy.J. Lipid Res. 2011; 52: 175-184Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 30Rames M. Yu Y. Ren G. Optimized negative staining: a high-throughput protocol for examining small and asymmetric protein structure by electron microscopy.J. Vis. Exp. 2014; 90: e51087Google Scholar, 31Zhang L. Song J. Newhouse Y. Zhang S. Weisgraber K.H. Ren G. An optimized negative-staining protocol of electron microscopy for apoE4 POPC lipoprotein.J. Lipid Res. 2010; 51: 1228-1236Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Cryo-EM is a cutting-edge technique used to examine proteins at near native state via imaging the proteins embedded in vitreous ice (32Gilkey J.C. Staehelin L.A. Advances in ultrarapid freezing for the preservation of cellular ultrastructure.J. Electron Microsc. Tech. 1986; 3: 177-210Crossref Scopus (320) Google Scholar, 33Bailey S.M. Chiruvolu S. Longo M.L. Zasadzinski J.A. Design and operation of a simple environmental chamber for rapid freezing fixation.J. Electron Microsc. Tech. 1991; 19: 118-126Crossref PubMed Scopus (11) Google Scholar, 34McLellan M.R. Day J.G. Cryopreservation and freeze-drying protocols. Introduction.Methods Mol. Biol. 1995; 38: 1-5PubMed Google Scholar), which can avoid potential artifacts associated with negative staining, such as dehydration and flattening. However, the image contrasts are significantly lower than the contrasts of negative staining. Survey OpNS-EM micrographs (Fig. 1A) and selected particle views (Fig. 1B) displayed roundish shapes of the VLDL particles ranging from 30 to 60 nm in diameter. However, the smaller particles displayed more significant surface vertices (indicated by arrows in Fig. 1A, B), which differs from the generally accepted idea of spherical emulsion-like particles. To confirm that the surface vertices were not induced by an artifact of the negative-staining method, the cryo-EM technique was used to examine the same sample frozen from a starting temperature of 4°C. The survey cryo-EM micrographs (Fig. 1C) and selected particle views (Fig. 1D) of VLDL embedded in vitreous ice showed that the particles have a similar diameter range to those observed with negative staining, i.e., from 30 to 60 nm. Also consistent was the finding that VLDL displayed angular shapes with several vertices on each particle, especially for small VLDL (indicated by arrows in Fig. 1C, D). We next sought to investigate whether the angular shape is an intrinsic structural feature of VLDL or an artifact of the crystalline core of TGs induced by low temperature when the sample was frozen from 4°C, which is below the lipid phase transition temperature (∼20–40°C) (35Deckelbaum R.J. Shipley G.G. Small D.M. Lees R.S. George P.K. Thermal transitions in human plasma low density lipoproteins.Science. 1975; 190: 392-394Crossref PubMed Scopus (134) Google Scholar). To do this, we repeated the above experiment after incubating the same sample at a temperature above the phase transition temperature (40–45°C for 30 min), directly flash freezing the sample from the above phase transition temperature into liquid nitrogen temperature by the cryo-EM technique, and examining the sample under the same cryo-EM operation conditions. The freezing speed is too fast (on the order of 104–105 K/s) for the water molecules to form a crystal (36Robards A.W. Sleytr U.B. Low temperature methods in biological electron microscopy.in: Glauert A.M. In Practical Methods in Electron Microscopy. Elsevier, Amsterdam1985: 5-133Google Scholar, 37Cheng D. Mitchell D.R.G. Shieh D-B. Braet F. Practical considerations in the successful preparation of specimens for thin-film cryo-transmission electron microscopy.in: Mendez-Vilas A. In Current Microscopy Contributions to Advances in Science and Technology. FORMATEX, Badajoz, Spain2012: 880-890Google Scholar), and it is reasonable to assume that molecules larger than water, such as lipids, have insufficient time to crystallize during rapid freezing. Thus, structural changes, such as crystallization of TGs and CEs, during the freezing process are unlikely. The cryo-EM rapid freezing technique has been utilized since the early 1980s to preserve biological specimens in their native state for TEM examination, and to our knowledge, there are no reported freezing related artifacts (32Gilkey J.C. Staehelin L.A. Advances in ultrarapid freezing for the preservation of cellular ultrastructure.J. Electron Microsc. Tech. 1986; 3: 177-210Crossref Scopus (320) Google Scholar, 33Bailey S.M. Chiruvolu S. Longo M.L. Zasadzinski J.A. Design and operation of a simple environmental chamber for rapid freezing fixation.J. Electron Microsc. Tech. 1991; 19: 118-126Crossref PubMed Scopus (11) Google Scholar, 34McLellan M.R. Day J.G. Cryopreservation and freeze-drying protocols. Introduction.Methods Mol. Biol. 1995; 38: 1-5PubMed Google Scholar). Thus, VLDL visualized by cryo-EM should be in its native structure and conformation. The survey cryo-EM micrographs of VLDL particles frozen from above the phase transition temperature exhibited essentially the same particle diameter range and confirmed the angular morphologies as those frozen from below the phase transition temperature (Fig. 1E, F), i.e., the angular shape was more pronounced in smaller rather than larger VLDL particles. The consistency of the findings in the above experiments suggests that the angular shape is an inherent feature of the VLDL structure. Statistical analysis of ∼600 cryo-EM images of VLDL particles showed that the particle diameter is linearly related to the surface angle. For particles within four size groups (30–39 nm, 40–49 nm, 50–59 nm, and 60–69 nm), the average smallest angles were 67, 60, 55, and 46°, respectively (Fig. 1G). The high heterogeneity of plasma VLDLs was consistent with previous results from negative-staining (NS) EM (7Vance D.E. Vance J.E. Assembly and secretion of lipoproteins.In Biochemistry of Lipids, Lipoproteins, and Membranes. Elsevier, Amsterdam. 2002; : 505-526Crossref Google Scholar, 24Zhang L. Song J. Cavigiolio G. Ishida B.Y. Zhang S. Kane J.P. Weisgraber K.H. Oda M.N. Rye K.A. Pownall H.J. et al.Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy.J. Lipid Res. 2011; 52: 175-184Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) and cryo-EM (25van Antwerpen R. La Belle M. Navratilova E. Krauss R.M. Structural heterogeneity of apoB-containing serum lipoproteins visualized using cryo-electron microscopy.J. Lipid Res. 1999; 40: 1827-1836Abstract Full Text Full Text PDF PubMed Google Scholar). The opaque appearance of VLDLs in cryo-EM might be attributable to high density matter on their surfaces, such as apolipoproteins. To confirm the observation of the angular shape of VLDL in 3D, we imaged the samples from a series of tilting angles by cryo-ET under low-dose mode with a total dose of ∼150 e−/Å2 and a magnification of 50 k× (corresponding to 2.4 Å/pixel) (supplemental Video S1). The survey of cryo-ET micrographs at three representative angles displayed eight VLDL particles whose diameters and shapes differed substantially from each other, such that they could not be averaged for 3D reconstruction (Fig. 2A); therefore, we used the IPET reconstruction method we developed to reconstruct the 3D density maps for single VLDL particles. For IPET 3D reconstruction, a series of tilted images of each targeted particle was boxed from the tilted whole cryo-ET micrographs after contrast transfer function correction. The selected tilt images of a representative targeted particle were iteratively aligned to their global center to achieve a final ab initio 3D reconstruction (Fig. 2B, left panel). The step-by-step refinement procedures and the intermediate results are shown in Fig. 2B. The final 3D density map (after low-pass filtering to 5.0 nm) displayed from two perpendicular viewing directions showed the particle having a diameter of 35 nm with an overall polyhedral structure (Fig. 2C), which was confirmed by the corresponding 2D projections (Fig. 2D). The FSC analysis showed that the 3D resolution is ∼3.5 nm based on a 0.143 criteria or ∼5.0 nm based on a 0.5 criteria (Fig. 2E) (details given in the Materials and Methods). The projections of the slices of 3D density maps at different heights (Fig. 2F, G) showed that the density of the core was generally lower than that of the shell and that the shell was ∼3 nm thick on average, consistent with the thickness of a phospholipid monolayer. The shell was uneven, even when low-pass filtered at 5.0 nm, indicating an irregular distribution of protein densities in the lipid membrane. The achieved resolution of 3.5–5 nm seems to be incompatible with the observation of a phospholipid monolayer (∼3 nm). Several reasons may be related to this phenomenon: i) The resolution of 3.5–5 nm is an estimated resolution based on the FSC analysis of two 3D reconstructions in which each of the 3D reconstructions was reconstructed from half of the tilt images. Thus, the qualities of these two 3D reconstructions could be poorer than the final 3D reconstruction that incorporates the full set of tilted images, especially when the total number of tilt images is less than 100, which leads to an underestimation of the final 3D resolution. ii) The resolution estimated from the FSC analysis is defined differently from that of the standard optical resolution, i.e., the distance between two distinguishable radiating points. The FSC resolution is a rough estimated resolution. iii) Identifying a single line object (lipid monolayer) is easier than identifying two distinct points. Identifying the location of the single line object is dependent only on the intensity of the object and not on the dimension of the object. Based on the above reasons, the lipid monolayer, which forms a line object, can be observed under the current resolution. By repeating the above IPET process, we reconstructed a second 3D density map from another individual VLDL particle (Fig. 2H–M). The representative tilt images showed that the VLDL was still visible (Fig. 2H, left panel). Through IPET reconstruction processing, the tilt images were gradually and iteratively aligned to the global center (Fig. 2H). The two perpendicular views of the final 3D density maps of the second VLDL particle showed that it is a near roundish structure of 40 nm in diameter, but still has a visible polyhedral structure (Fig. 2I). The shape can also be displayed by its corresponding projections (Fig. 2J). The observed high density shell and low den
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