Three-dimensional cryoEM reconstruction of native LDL particles to 16Å resolution at physiological body temperature

Abstract

Background: Low-density lipoprotein (LDL) particles, the major carriers of cholesterol in the human circulation, have a key role in cholesterol physiology and in the development of atherosclerosis. The most prominent structural components in LDL are the core-forming cholesteryl esters (CE) and the particle-encircling single copy of a huge, non-exchangeable protein, the apolipoprotein B-100 (apoB-100). The shape of native LDL particles and the conformation of native apoB-100 on the particles remain incompletely characterized at the physiological human body temperature (37 °C).

Methodology/principal findings: To study native LDL particles, we applied cryo-electron microscopy to calculate 3D reconstructions of LDL particles in their hydrated state. Images of the particles vitrified at 6 °C and 37 °C resulted in reconstructions at ~16 Å resolution at both temperatures. 3D variance map analysis revealed rigid and flexible domains of lipids and apoB-100 at both temperatures. The reconstructions showed less variability at 6 °C than at 37 °C, which reflected increased order of the core CE molecules, rather than decreased mobility of the apoB-100. Compact molecular packing of the core and order in a lipid-binding domain of apoB-100 were observed at 6 °C, but not at 37 °C. At 37 °C we were able to highlight features in the LDL particles that are not clearly separable in 3D maps at 6 °C. Segmentation of apoB-100 density, fitting of lipovitellin X-ray structure, and antibody mapping, jointly revealed the approximate locations of the individual domains of apoB-100 on the surface of native LDL particles.

Conclusions/significance: Our study provides molecular background for further understanding of the link between structure and function of native LDL particles at physiological body temperature.

Figure 1. Comparison of raw and averaged data for LDL particles at 6°C and at 37°C.

The first rows show projections from the reconstructed volume in different views, the second rows show the corresponding class averages. The third rows show one of the original images from each class average and the bottom rows show the corresponding denoised image.

Figure 2. Scaled radial intensity profiles of the reconstructed volumes of LDL particles.

The overall intensity of the interior of the LDL particle reconstruction is higher at 6°C than at 37°C. This indicates a pseudo-random structure of lipids in the LDL core at 37°C, while at 6°C the organization is more rigid. The 2D averages are shown in the insets.

Figure 3. Consecutive sections through the 3D reconstructions of LDL particles at 6°C and at 37°C.

Each slice is as thick as a pixel whose size is 1.4 Å. The slice number is shown in the bottom right corner of each image. The slice with number s128 is the central slice of the 3D volume, while s78 marks the slice which is 50 pixels (70 Å) behind the central slice. Similarly s188 marks the slice which is 60 pixels (84 Å) in front of the central slice. The transparent violet isosurfaces for the corresponding 3D maps are made at threshold of µ+0.7σ (showing mainly the lipid surface of the particles) and the opaque brown high-density isosurfaces are made at threshold of µ+3σ (showing mainly rigid structures of apoB-100). These 3D reconstructions were made in the final iterations of the single particle reconstructions using non-filtered 2D cryo-EM images after determining the classes of their filtered versions.

Figure 4. Main regions of variance within the LDL particle reconstructions.

A boot-strap method was used to produce three dimensional variance maps (red) superimposed on average maps (green) of the 6°C and 37°C particles. The average volume is shown at threshold of µ+2σ intensity level. The regions of high variance for LDL at 6°C are labeled as L1, L2, and L3 and for LDL at 37°C as H1, H2, H3 and H4. For the rightmost illustration at 6°C the threshold for the average map was lower (at µ+σ intensity level) and the volume was cut in the middle to visualize the inner core structure.

Figure 5. Results of the fitting of the backbone of lipovitellin PDB structure (PDB code: 1LSH) with the LDL reconstruction at 37°C.

The isosurface shown here is from a 3D volume which has been low pass filtered to 17 Å after thresholding at 1.7σ above the mean intensity. The PDB structure of the backbone has been shown with different colors representing their rank assigned by the docking tool ADP_EM. The magenta color is for rank 1 and cyan for rank 2. Both of the two best results are docked similarly although there is slight rotational difference between the best and second-best docked results. The boxed lipovitellin region is shown enlarged on the left.

Figure 6. ApoB-100 domains and antibody locations in reconstruction of LDL at 37°C.

An LDL particle at 37°C (white mesh isosurface at the threshold of µ+2σ) with different segments (colored), docked lipovitellin (N-terminal amino acids 1–680 shown in yellow), and fitted antibodies' locations. These segments were found automatically by 3D segmentation and their unfiltered versions are shown in different colors at threshold at which the whole reconstructed 3D volume represents a protein mass of 550 kDa. The pentapartite model of apoB-100 is shown in the bottom together with the epitope locations of the antibodies (MB19, 71; MB24, 405–539; MB11, 995–1082; 2D8, 1438–1480; B4, 1854–1878; B3, 2239–2331; 4G3, 2980– 3084; MB47, 3429–3453 and 3507–3523; MB43, 4027–4081; Bsol16, 4154–4189; Bsol7, 4517–4536). Different colors are used for different supra-molecular structures of apoB-100 in all subfigures. The rectangular segment shown with cyan color, between antibody locations MB11, 2D8 and B4, most likely represent the β1 region (approximately from residue 827 to 2000). The segment, shown in magenta, surrounded by antibody locations B3, B4 and 4G3, likely refers to the α2 region (approximately from residue 2075 to 2570). The largest segment surrounded by Bsol7, MB19, Bsol16 and 4G3, shown in red color, appears to be consisting of the β2- (approximately from residue 2571 to 4050) as well as the α3-region (approximately from residue 4050 to 4500). The segmentation tool could not separate the β2- and α3-regions, possibly due to their overlapping locations. However, the docked antibodies clearly point to the possible location of the α3-domain shown shaded and encircled with a dashed line.

Figure 7. Similarity between 6°C LDL reconstruction and previously published low-resolution structure of LDL-LDLr complex at 4°C.

The result of automatic alignment (Align3d in EMAN) of our 6°C LDL reconstruction in yellow with that of LDL in complex with the LDL-receptor (LDL-LDLr) in violet at 4°C (23). The published LDL-LDLr reconstruction (23) was downloaded from the EMdatabank (emdatabank.org accession code EMD-5158). The isosurfaces were generated at a threshold of µ+σ. The view from one side is shown in (a) and the view from the same orientation is shown in (b) as a thick central section from both volumes. Notice that nearly all the regions of volumes of 6°C LDL and LDL-LDLr overlap except the heterogeneous region L2. The 3D volume of 6°C LDL is low-pass filtered to 15 Å resolution and LDL-LDLr is low-pass filtered to 28Å.

Figure 8. Similar orientation views show LDL-receptor binding site in reconstructions of LDL at 6°C and 37°C and LDL-LDLr complex.

The published 3D reconstruction LDL in complex with the LDL-receptor (LDL-LDLr) (23) EMdatabank accession code EMDB-5158. The orientation of the 3D reconstruction of the LDL-LDLr complex was determined by using automatic alignment with 6°C LDL map. For LDL at 37°C and LDL at 6°C the transparent violet iso-surfaces are made at threshold of µ+0.7σ (representing lipid) and opaque brown high density iso-surfaces are made at threshold of µ+3σ (representing rigid structures of protein). For LDL-LDLr complex the transparent violet isosurface is made at threshold of µ+0.25σ and opaque pink high density isosurface is made using a threshold of µ+2.5σ. The 3D volumes of 6°C LDL and 37°C LDL are low-pass filtered to 15Å resolution and 3D map of LDL-LDLr is low-pass filtered to 28Å. In (a) LDL at 37°C is also shown with fitted antibodies locations. The shown antibodies with their epitope locations on the apoB-100 are MB19 (71), MB24 (405–539), 4G3 (2980–3084), MB47 (3429–3453, 3507–3523), MB43 (4027–4081), Bsol16 (4154–4189) and Bsol7 (4517–4536). In (b) the rib like structures in 6°C LDL are indicated by arrows. In (c) the LDL receptor is visible in LDL-LDLr complex. The visible location of LDL-receptor in LDL-LDLr complex corresponds to the region close to MB47 lying in between 4G3 and MB47 mAb locations in LDL at 37°C.