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Microbial Ultrastructure, HIV, and the Development of Electron Cryotomography

Research Summary

Grant Jensen's laboratory uses state-of-the-art electron cryomicroscopy techniques to understand the structure and function of large protein machines and their arrangement within cells. Projects range from theoretical studies on the mathematics of three-dimensional reconstructions, to direct imaging of individual protein complexes, to tomography of viruses and cells.

Microbial Cell Biology
Although thousands of bacterial genomes have now been sequenced and a variety of high-throughput "omic" technologies are revealing gene expression patterns and how the various gene products mediate metabolism, our comparative ignorance about the more physical and mechanical processes that occur in a bacterial life cycle is surprising. We still do not know, for instance, how bacteria generate and maintain their characteristic shapes, establish polarity, organize their genomes, segregate their chromosomes, divide, and in some cases move. In some sense, the "omic" technologies are giving us lists of parts and reactions, but bacterial cells are not merely bags of enzymes—structural and mechanical details are also needed.

Fortunately, the emergence of electron cryotomography has now allowed us to obtain unprecedented three-dimensional (3D) views of proteins, viruses, and even whole bacterial cells in near-native states to molecular (~2- to 5-nm) resolution. In our lab we are both helping to develop electron cryotomography as a structural technique and also using it to study the structures and functions of the supramolecular complexes that, for example, shape bacterial cells, propel them forward, sense where to swim, organize metabolism, secrete signals, attack competitors, and drive growth and division.

The prokaryotic cytoskeleton. Because for many decades cytoskeletal filaments were only rarely seen in traditional thin-section electron microscopy (EM) images of bacteria, it was long thought that prokaryotes lacked a cytoskeleton and that this was one of the hallmark differences between pro- and eu-karyotes. More recently, bacterial homologs of actin, tubulin, and intermediate filament proteins have been identified, however, and fluorescent light microscopy (fLM) has confirmed that these and other proteins localize in filament-like patterns in vivo. We and others have now used cryotomography to directly visualize many different cytoskeletal filaments and filament bundles in a variety of species, confirming that bacteria do indeed have elaborate and extensive cytoskeletal systems. Seeing these filaments for the first time, however, produced a number of puzzles: while some of the filaments appeared in locations predicted by fLM, others did not, and sometimes filaments were not seen where they were expected. We are now working to identify each filament and gain insight into its function and mechanism.

Motility machines. Most motile bacteria propel themselves with flagella. We published the first structure of an entire flagellar motor, which showed among other things the number and shape of the stator "studs" in situ. We have now reconstructed motors from several different species; the comparison highlights how each species has adapted the motor to its purposes. The Treponema primitia motor, for example, is wider in diameter and has more stator studs, adaptations that could "gear" it down to produce the higher torque it might need as a spirochete to rotate the entire cell. Nonflagellar (gliding) motility mechanisms in bacteria have also been described. We imaged the attachment organelle of Mycoplasma pneumoniae and proposed that it is a conformationally dynamic engine driving motility in that species.

Chemotaxis. Given a means to move, bacteria must also know where to go. Bacterial chemotaxis is mediated by an array of chemoreceptors embedded in the cell membrane that sends signals to the flagellar motors. We used cryotomography to generate the first 3D structures of chemoreceptor arrays in intact, wild-type cells, revealing that the receptors are arranged as a hexagonal lattice of trimers of receptor dimers. We then showed that the basic architecture of chemoreceptor arrays is conserved across bacteria.

Enzymatic hyperstructures. Although some enzymes diffuse freely, others are tethered or packaged into hyperstructures that streamline certain metabolic pathways. We recently solved the quaternary structure of Escherichia coli's pyruvate dehydrogenase hyperstructure by cryotomography. More recently we have been analyzing the carboxysome, an organelle-like polyhedral body that facilitates carbon fixation by sequestering ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Our cryotomograms have revealed the number, size, shape, and positions of carboxysomes inside Halothiobacillus neapolitanus, as well as their surprising association with storage granules. The number, positions, and orientations of RuBisCOs inside purified carboxysomes are also visible.

Cryotomographic studies on other aspects of microbial ultrastructure are also ongoing.

Structural Biology of HIV
In addition to imaging small cells, we are also investigating the structural biology of the human immunodeficiency virus type 1 (HIV). HIV is unusual: although each virus has the same basic membrane and protein layers, they are all unique, making standard methods such as x-ray crystallography or EM-based single-particle analysis ineffective. Thus, while there are already hundreds of structures of individual HIV protein domains available in the Protein Data Bank, many questions remain about how these assemble to form a virus and how subsequent protein modifications drive maturation. In collaboration with Wesley Sundquist (University of Utah), we have now used cryotomography to determine the 3D structures of the immature and mature forms of HIV. This work confirmed previous conclusions from simpler projection images that the Gag lattice is organized into concentric spherical shells with hexagonal packing, but 3D images of these shells showed us that the shells are incomplete: the Gag lattice is a patchwork of loosely connected hexagonal nets separated by regions of disorder or vacancy. By computationally extracting individual unit cells from just the highly ordered patches, we produced an average structure of a Gag hexamer, which suggested that the SP1 (spacer peptide 1) forms a lattice-stabilizing six-helix bundle. This explains why proteolysis between capsid and SP1 during maturation triggers rearrangement, and why certain drugs that bind SP1 block maturation. Other tomograms showed that the cone angle of the mature capsid shell matches the predictions of the current fullerene cone model, and that some viruses have multiple and sometimes even nested capsid shells, arguing strongly against models of maturation that involve gradual collapse of a spherical immature shell.

Technology Development
Electron cryomicroscopy is a relatively young field, and we are working to improve sample preparation methods, the number and quality of images produced, and the algorithms used to process and analyze those images. We have, for example, described a new cryogen mixture that facilitates plunge-freezing, shown that liquid nitrogen is a better coolant than liquid helium, characterized the first stage for routine dual-axis tilting, begun testing a prototype lens-coupled CCD camera, automated sequential tilt-series acquisition, developed a model database for storing tomograms, and developed a new method for denoising images. We are currently developing methods to record fluorescent images of frozen samples en route to correlated light and electron microscopy.

This work is also supported by the National Institutes of Health, the Beckman Institute at Caltech, the Agouron Institute, and the Gordon and Betty Moore Foundation.

As of August 30, 2012

Scientist Profile

California Institute of Technology
Microbiology, Molecular Biology