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Abstract: Algorithms for three-dimensional (3D) reconstruction of objects based on their projections are essential in various biological and medical imaging modalities. In cryo-electron tomography (CET) a major challenge for reconstruction is the limited range of projection angles, which manifests itself as a “missing wedge” of data in Fourier space making the reconstruction problem ill-posed. Here, we apply an iterative reconstruction method that makes use of nonuniform fast Fourier transform (NUFFT) to the reconstruction of cryo-electron tomograms. According to several measures the reconstructions are superior to those obtained using conventional methods, most notably weighted backprojection. Most importantly, we show that it is possible to fill in partially the unsampled region in Fourier space with meaningful information without making assumptions about the data or applying prior knowledge. As a consequence, particles of known structure can be localized with higher confidence in cryotomograms and subtomogram averaging yields higher resolution densities. [Copyright &y& Elsevier]

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 (30Å). 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. [Copyright &y& Elsevier]

Abstract: The structure determination of macromolecular complexes is entering a new era. The methods of optical microscopy, electron microscopy, X-ray crystallography, and nuclear magnetic resonance increasingly are being combined in hybrid method approaches to achieve an integrated view of macromolecular complexes that span from cellular context to atomic detail. A particularly important application of these hybrid method approaches is the structural analysis of the Human Immunodeficiency Virus (HIV) proteins with their cellular binding partners. High resolution structure determination of essential HIV – host cell protein complexes and correlative analysis of these complexes in the live cell can serve as critical guides in the design of a broad, new class of therapeutics that function by disrupting such complexes. Here, with the hope of stimulating some discussion, we will briefly review some of the literature in the context of what could be done to further apply structural methods to HIV research. We have chosen to focus our attention on certain aspects of the HIV replication cycle where we think that structural information would contribute substantially to the development of new therapeutic and vaccine targets for HIV. [Copyright &y& Elsevier]

Abstract: Cryoelectron tomography (CET) combines the potential of three-dimensional (3D) imaging with a close-to-life preservation of biological samples. It allows the examination of large and stochastically variable structures, such as organelles or whole cells. At the current resolution it becomes possible to visualize large macromolecular complexes in their functional cellular environments. Pattern recognition methods can be used for a systematic interpretation of the tomograms; target molecules are identified and located based on their structural signature and their correspondence with a template. Here, we demonstrate that such an approach can be used to map 70S ribosomes in an intact prokaryotic cell (Spiroplasma melliferum) with high fidelity, in spite of the low signal-to-noise ratio (SNR) of the tomograms. At a resolution of 4.7nm the average generated from the 236 ribosomes found in a tomogram is in good agreement with high resolution structures of isolated ribosomes as obtained by X-ray crystallography or cryoelectron microscopy. Under the conditions of the experiment (logarithmic growth phase) the ribosomes are evenly distributed throughout the cytosol, occupying approximately 5% of the cellular volume. A subset of about 15% is found in close proximity to and with a distinct orientation with respect to the plasma membrane. This study represents a first step towards generating a more comprehensive cellular atlas of macromolecular complexes. [Copyright &y& Elsevier]

Large nuclear ribonucleoprotein particles, which can be viewed as the naturally assembled precursor messenger RNA (pre-mRNA) processing machine, were analyzed in frozen–hydrated preparations by cryoelectron microscopy. A general and reproducible strategy for preparing ice-embedded large nuclear ribonucleoprotein (lnRNP) particles at sufficiently high concentration was developed. Taking advantage of their negatively charged components, the lnRNP particles are adsorbed and thus concentrated on a positively charged lipid monolayer while preserving their native structure. Using this approach we carried out cryoelectron tomography and three-dimensional image reconstruction of individual lnRNP particles. The study revealed a structure similar to that of negatively stained particles studied previously, yet with additional features. The small additional domain visualized in negative stain appeared to be larger in the ice preparations. In addition, using image restoration from focus series of ice-embedded lnRNP particles, new features such as holes within the subunits were visualized in two dimensions, and it was shown that the subunits are interconnected via a fiber, very likely formed by the pre-mRNA. This finding supports the model that each subunit represents a spliceosome that splices out the intron wound around it. [Copyright &y& Elsevier]

Using a 400-kV cryoelectron microscope, we have obtained tomographic reconstructions of frozen-hydrated sea urchin axonemes with 8–10-nm resolution, as assessed by detection of characteristic components including doublet microtubules, radial spokes, central sheath projections, and outer dynein arms. We did not detect the inner dynein arms or the microtubule lattice. The 1/(8 nm) and 1/(16 nm) layer lines are consistently present in power spectra of both projection images and tomographic reconstructions. Strength and detection of the layer lines are dependent upon total electron dose and defocus. Both layer lines are surprisingly resistant to electron doses of up to 11 000 electrons/nm2. We present a summary of resolution considerations in cryoelectron tomography and conclude that the fundamental limitation is the total electron dose required for statistical significance. The electron dose can be fractionated among the numerous angular views in a tomographic data set, but there is an unavoidable fourth-power dependence of total dose on target resolution. Since higher-resolution features are more beam-sensitive, this dose requirement places an ultimate limit on the resolution of individual tomographic reconstructions. Instrumental and computational strategies to circumvent this limitation are discussed. [Copyright &y& Elsevier]