Caroline J. Fu

Mechanism of Folding Chamber Closure in a Group II Chaperonin

Group II chaperonins are essential mediators of cellular protein folding in eukaryotes and archaea. These oligomeric protein machines, ∼1 megadalton, consist of two back-to-back rings encompassing a central cavity that accommodates polypeptide substrates. Chaperonin-mediated protein folding is critically dependent on the closure of a built-in lid, which is triggered by ATP hydrolysis. The structural rearrangements and molecular events leading to lid closure are still unknown. Here we report four single particle cryo-electron microscopy (cryo-EM) structures of Mm-cpn, an archaeal group II chaperonin, in the nucleotide-free (open) and nucleotide-induced (closed) states. The 4.3 Å resolution of the closed conformation allowed building of the first ever atomic model directly from the single particle cryo-EM density map, in which we were able to visualize the nucleotide and more than 70% of the side chains. The model of the open conformation was obtained by using the deformable elastic network modelling with the 8 Å resolution open-state cryo-EM density restraints. Together, the open and closed structures show how local conformational changes triggered by ATP hydrolysis lead to an alteration of intersubunit contacts within and across the rings, ultimately causing a rocking motion that closes the ring. Our analyses show that there is an intricate and unforeseen set of interactions controlling allosteric communication and inter-ring signalling, driving the conformational cycle of group II chaperonins. Beyond this, we anticipate that our methodology of combining single particle cryo-EM and computational modelling will become a powerful tool in the determination of atomic details involved in the dynamic processes of macromolecular machines in solution.

Visualizing virus assembly intermediates inside marine cyanobacteria

Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET). This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.

Effect of the Peptide Secondary Structure on the Peptide Amphiphile Supramolecular Structure and Interactions

Bottom-up fabrication of self-assembled nanomaterials requires control over forces and interactions between building blocks. We report here on the formation and architecture of supramolecular structures constructed from two different peptide amphiphiles. Inclusion of four alanines between a 16-mer peptide and a 16 carbon long aliphatic tail resulted in a secondary structure shift of the peptide headgroups from α helices to β sheets. A concomitant shift in self-assembled morphology from nanoribbons to core-shell worm-like micelles was observed by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). In the presence of divalent magnesium ions, these a priori formed supramolecular structures interacted in distinct manners, highlighting the importance of peptide amphiphile design in self-assembly.

Filamentous, mixed micelles of triblock copolymers enhance tumor localization of indocyanine green in a murine xenograft model

Polymeric micelles formed by the self-assembly of amphiphilic block copolymers can be used to encapsulate hydrophobic drugs for tumor-delivery applications. Filamentous carriers with high aspect ratios offer potential advantages over spherical carriers, including prolonged circulation times. In this work, mixed micelles composed of poly(ethylene oxide)-poly[(R)-3-hydroxybutyrate]-poly(ethylene oxide) (PEO-PHB-PEO) and Pluronic F-127 (PF-127) were used to encapsulate a near-infrared fluorophore. The micelle formulations were assessed for tumor accumulation after tail vein injection to xenograft tumor-bearing mice by noninvasive optical imaging. The mixed micelle formulation that facilitated the highest tumor accumulation was shown by cryo-electron microscopy to be filamentous in structure compared to spherical structures of pure PF-127 micelles. In addition, increased dye loading efficiency and dye stability were attained in this mixed micelle formulation compared to pure PEO-PHB-PEO micelles. Therefore, the optimized PEO-PHB-PEO/PF-127 mixed micelle formulation offers advantages for cancer delivery over micelles formed from the individual copolymer components.

Lemon-shaped halo archaeal virus His1 with uniform tail but variable capsid structure

Significance Many lemon-shaped double-stranded DNA viruses have been observed to infect archaeal cells in both extreme and moderate environments. We used cryo-electron tomography with subtomogram classification and averaging to reveal the three-dimensional structures of a lemon-shaped haloarchaeal virus, His1. Although the His1 exhibited size and shape heterogeneity, its tail structure was found to be constant. Extensive biochemical studies show that, while extremely stable, under certain conditions the capsid can transform into a tube without the genome. These observations demonstrate that the capsid proteins are able to perform a remarkable surface lattice transformation. Lemon-shaped viruses are common in nature but so far have been observed to infect only archaea. Due to their unusual shape, the structures of these viruses are challenging to study and therefore poorly characterized. Here, we have studied haloarchaeal virus His1 using cryo-electron tomography as well as biochemical dissociation. The virions have different sizes, but prove to be extremely stable under various biochemical treatments. Subtomogram averaging of the computationally extracted virions resolved a tail-like structure with a central tail hub density and six tail spikes. Inside the tail there are two cavities and a plug density that separates the tail hub from the interior genome. His1 most likely uses the tail spikes to anchor to host cells and the tail hub to eject the genome, analogous to classic tailed bacteriophages. Upon biochemical treatment that releases the genome, the lemon-shaped virion transforms into an empty tube. Such a dramatic transformation demonstrates that the capsid proteins are capable of undergoing substantial quaternary structural changes, which may occur at different stages of the virus life cycle.

Zernike phase-contrast electron cryotomography applied to marine cyanobacteria infected with cyanophages

Advances in electron cryotomography have provided new opportunities to visualize the internal 3D structures of a bacterium. An electron microscope equipped with Zernike phase-contrast optics produces images with markedly increased contrast compared with images obtained by conventional electron microscopy. Here we describe a protocol to apply Zernike phase plate technology for acquiring electron tomographic tilt series of cyanophage-infected cyanobacterial cells embedded in ice, without staining or chemical fixation. We detail the procedures for aligning and assessing phase plates for data collection, and methods for obtaining 3D structures of cyanophage assembly intermediates in the host by subtomogram alignment, classification and averaging. Acquiring three or four tomographic tilt series takes ∼12 h on a JEM2200FS electron microscope. We expect this time requirement to decrease substantially as the technique matures. The time required for annotation and subtomogram averaging varies widely depending on the project goals and data volume.

Visualizing virus assembly intermediates inside marine cyanobacteria by zernike phase contrast electron cryo-tomography

1. National Center for Macromolecular Imaging, Verna and Marrs Mclean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA. 2. Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX, USA. 3. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. 4. Department of Biology, Northeastern University, Boston, MA, USA. 5. National Institute for Physiological Sciences, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Japan † Present address: FEI, 5350 Dawson Creek Drive, Hillsboro, OR, USA

Zernike Phase Plate Configuration at Intermediate Lens Position on JEM2200FS

Cryo-electron microscopy is an emerging tool for structural biology to study biological specimens in their native conformational states. High contrast images with high signal-to-noise ratio are always beneficial for extracting features out of subcellular components in a cell tomogram or visualizing small macromolecules in single particle cryoEM. There are recent advances in enhancing image contrast using direct electron detectors and Zernike phase plate. The application of direct electron detector has demonstrated its usefulness in enhancing the low and high resolution contrast to facilitate the near atomic resolution structure determination of single particles. Zernike phase plate offers impressive low resolution image contrast to reveal structural details of subcellular components in a crowded environment of cell tomograms.

Structure of Trypanosoma Brucei Flagellum Accounts for its Bihelical Motion

Trypanosoma brucei is a parasitic protozoan that causes African sleeping sickness. It contains a flagellum required for locomotion and viability. In addition to a microtubular axoneme, the flagellum contains a crystalline paraflagellar rod (PFR) and connecting proteins. We show here, by cryoelectron tomography, the structure of the flagellum in three bending states. The PFR lattice in straight flagella repeats every 56 nm along the length of the axoneme, matching the spacing of the connecting proteins. During flagellar bending, the PFR crystallographic unit cell lengths remain constant while the interaxial angles vary, similar to a jackscrew. The axoneme drives the expansion and compression of the PFR lattice. We propose that the PFR modifies the in-plane axoneme motion to produce the characteristic trypanosome bihelical motility as captured by high-speed light microscope videography.