Single particle cryoelectron tomography characterization of the structure and structural variability of poliovirus-receptor-membrane complex at 30 A resolution

Abstract

As a long-term goal we want to use cryoelectron tomography to understand how non-enveloped viruses, such as picornaviruses, enter cells and translocate their genomes across membranes. To this end, we developed new image-processing tools using an in vitro system to model viral interactions with membranes. The complex of poliovirus with its membrane-bound receptors was reconstructed by averaging multiple sub-tomograms, thereby producing three-dimensional maps of surprisingly high-resolution (30 A). Recognizable images of the complex could be produced by averaging as few as 20 copies. Additionally, model-free reconstructions of free poliovirus particles, clearly showing the major surface features, could be calculated from 60 virions. All calculations were designed to avoid artifacts caused by missing information typical for tomographic data ("missing wedge"). To investigate structural and conformational variability we applied a principal component analysis classification to specific regions. We show that the missing wedge causes a bias in classification, and that this bias can be minimized by supplementation with data from the Fourier transform of the averaged structure. After classifying images of the receptor into groups with high similarity, we were able to see differences in receptor density consistent with the known variability in receptor glycosylation.

Figure 1

Cryoelectron tomography of poliovirus-receptor-liposome complexes. (A) A projection along the z-axis (beam direction), showing a portion of a 3D tomogram. This rectangle (~2 μm wide) includes about a quarter of the original tomogram. Liposomes (narrow arrowheads), virus particles (wide arrowheads), and gold fiducials are present. Scale bar: 10 nm. (B) Surface rendering, cropped for clarity, showing a few virus particles and liposomes. A representative virus particle (wide arrowhead), and liposome (narrow arrowhead) are indicated (C) Fourier shell correlation (FSC) indicates that the resolution of the reconstruction is around 3.0 nm, and is limited by the pixel size. (D) Reconstruction of the complex by single particle cryo-EM (Bubeck et al., 2005b). (E) Averaged tomographic structure: ~1500 copies of the complex were identified in the tomograms, boxed, and aligned to the reference by comparing Fourier terms. Terms in the missing wedge were not considered. Aligned particles were summed and five-fold averaged. The resulting reconstruction clearly shows the expected peaks and valleys on the viral surface, and prominent glycosylation sites on the middle domain of the receptor. As in (D), the membrane has a pronounced distortion in the vicinity of the five-fold axis. Panels (D) and (E) show the complex in the same orientation, and are calculated using the same pixel size (1 nm). Scale: the diameter of the virus is 30 nm.

Figure 2

Recognizable reconstructions can be produced by combining a surprisingly small number of individual sub-tomograms. Left-to-right: randomly chosen subsets of the data (consisting of 20, 40, 80 or 100 sub-tomograms) were summed and five-fold averaged. Densities for receptors and membrane are visible after averaging as few as 20 particles. Fourier shell correlation (vs. the ~1500-particle reconstruction) indicates resolutions of 5.0, 4.7, 4.4. and 4.2 nm for these four maps respectively.

Figure 3

Model-free icosahedral reconstruction of 160S poliovirions from tomographic images. (A) Views along its five-fold (left) and two-fold (right) axes. (B) Central slice through the reconstruction, shown as an iso-contour surface. Two-fold, five-fold, and three-fold axes are marked. Scale: the diameter of the virus is 30 nm. (C) Fourier shell correlation (FSC) indicates that a resolution of 3.6 nm was obtained by adding 60 virus particles.

Figure 4

The missing wedge causes a bias in classification: particles with similarly oriented missing wedges become distorted in similar ways, and tend to be grouped together. Filling in the missing wedge with Fourier coefficients from the averaged structure reduces the bias. For the largest classes, filled (blue) and unfilled (red), respectively, this figure illustrates how uniformly reciprocal space is sampled. (A) Angular coverage: the cavities in the surfaces correspond to under-sampled regions of reciprocal space after the members of each class are added together. The contour level is 1.1 standard deviations above the mean. (B) Simulated histogram showing how often points in reciprocal space are sampled for a random distribution of Euler angles. As in the tomograms, 72% of reciprocal space is occupied; a class with 100 members was assumed. (C, D) Actual histograms calculated from the two largest classes. Observe that leaving the missing wedge unfilled (red) causes a non-uniform sampling, reflecting a bias in classification. Restoring the missing wedge (blue) significantly reduces the bias. In panels B, C and D the horizontal axis represents the percentage (%) of boxed particles whose contribution to a reciprocal space direction lies outside of the missing wedge. The verical axis represents the number of reciprocal space directions having the indicated percentage; this value is on an arbitrary scale, depending on the finess of sampling of reciprocal space.

Figure 5

Conformational diversity, as revealed by classification. (A) The classification was based on pixels sampled from the membrane regions of 7500 views. Some of the classes differ markedly in the shape and inclination of the membrane. This example shows corresponding sections from a large class (2700 members, blue) and a small class (400 members, red). (B) Classification in the receptor region. The average receptor is shown in blue; the density in red represents the variability between the largest class averages. The large classes included in this calculation collectively represent 62% of the receptor population. The major differences coincide with the positions of the two large known glycosylation sites on the middle domain of the receptor. Scale bar: 5 nm.