Joel Quispe

A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron

Molecular self-assembly offers a means of spontaneously forming complex and well-defined structures from simple components. The specific bonding between DNA base pairs has been used in this way to create DNA-based nanostructures and to direct the assembly of material on the subnanometre to micrometre scale. In principle, large-scale clonal production of suitable DNA sequences and the directed evolution of sequence lineages towards optimized behaviour can be realized through exponential DNA amplification by polymerases. But known examples of three-dimensional geometric DNA objects are not amenable to cloning because they contain topologies that prevent copying by polymerases. Here we report the design and synthesis of a 1,669-nucleotide, single-stranded DNA molecule that is readily amplified by polymerases and that, in the presence of five 40-mer synthetic oligodeoxynucleotides, folds into an octahedron structure by a simple denaturation–renaturation procedure. We use cryo-electron microscopy to show that the DNA strands fold successfully, with 12 struts or edges joined at six four-way junctions to form hollow octahedra approximately 22 nanometres in diameter. Because the base-pair sequence of individual struts is not repeated in a given octahedron, each strut is uniquely addressable by the appropriate sequence-specific DNA binder.

Unnatural Amino Acid Incorporation into Virus-Like Particles

Virus-like particles composed of hepatitis B virus (HBV) or bacteriophage Qbeta capsid proteins have been labeled with azide- or alkyne-containing unnatural amino acids by expression in a methionine auxotrophic strain of E. coli. The substitution does not affect the ability of the particles to self-assemble into icosahedral structures indistinguishable from native forms. The azide and alkyne groups were addressed by Cu(I)-catalyzed [3 + 2] cycloaddition: HBV particles were decomposed by the formation of more than 120 triazole linkages per capsid in a location-dependent manner, whereas Qbeta suffered no such instability. The marriage of these well-known techniques of sense-codon reassignment and bioorthogonal chemical coupling provides the capability to construct polyvalent particles displaying a wide variety of functional groups with near-perfect control of spacing.

ATP-Triggered Conformational Changes Delineate Substrate-Binding and -Folding Mechanics of the Groel Chaperonin.

Summary The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the “power stroke” that ejects substrate into the folding chamber.

Structure of the chloroplast ribosome: novel domains for translation regulation.

Gene expression in chloroplasts is controlled primarily through the regulation of translation. This regulation allows coordinate expression between the plastid and nuclear genomes, and is responsive to environmental conditions. Despite common ancestry with bacterial translation, chloroplast translation is more complex and involves positive regulatory mRNA elements and a host of requisite protein translation factors that do not have counterparts in bacteria. Previous proteomic analyses of the chloroplast ribosome identified a significant number of chloroplast-unique ribosomal proteins that expand upon a basic bacterial 70S-like composition. In this study, cryo-electron microscopy and single-particle reconstruction were used to calculate the structure of the chloroplast ribosome to a resolution of 15.5 Å. Chloroplast-unique proteins are visualized as novel structural additions to a basic bacterial ribosome core. These structures are located at optimal positions on the chloroplast ribosome for interaction with mRNAs during translation initiation. Visualization of these chloroplast-unique structures on the ribosome, combined with mRNA cross-linking, allows us to propose a model for translation initiation in chloroplasts in which chloroplast-unique ribosomal proteins interact with plastid-specific translation factors and RNA elements to facilitate regulated translation of chloroplast mRNAs.

Supramolecular Architecture of Severe Acute Respiratory Syndrome Coronavirus Revealed by Electron Cryomicroscopy

ABSTRACT Coronavirus particles are enveloped and pleomorphic and are thus refractory to crystallization and symmetry-assisted reconstruction. A novel methodology of single-particle image analysis was applied to selected virus features to obtain a detailed model of the oligomeric state and spatial relationships among viral structural proteins. Two-dimensional images of the S, M, and N structural proteins of severe acute respiratory syndrome coronavirus and two other coronaviruses were refined to a resolution of ∼4 nm. Proteins near the viral membrane were arranged in overlapping lattices surrounding a disordered core. Trimeric glycoprotein spikes were in register with four underlying ribonucleoprotein densities. However, the spikes were dispensable for ribonucleoprotein lattice formation. The ribonucleoprotein particles displayed coiled shapes when released from the viral membrane. Our results contribute to the understanding of the assembly pathway used by coronaviruses and other pleomorphic viruses and provide the first detailed view of coronavirus ultrastructure.

Multiple states of a nucleotide-bound group 2 chaperonin.

Chaperonin action is controlled by cycles of nucleotide binding and hydrolysis. Here, we examine the effects of nucleotide binding on an archaeal group 2 chaperonin. In contrast to the ordered apo state of the group 1 chaperonin GroEL, the unliganded form of the homo-16-mer Methanococcus maripaludis group 2 chaperonin is very open and flexible, with intersubunit contacts only in the central double belt of equatorial domains. The intermediate and apical domains are free of contacts and deviate significantly from the overall 8-fold symmetry. Nucleotide binding results in three distinct, ordered 8-fold symmetric conformations--open, partially closed, and fully closed. The partially closed ring encloses a 40% larger volume than does the GroEL-GroES folding chamber, enabling it to encapsulate proteins up to 80 kDa, in contrast to the fully closed form, whose cavities are 20% smaller than those of the GroEL-GroES chamber.

An Improved Holey Carbon Film for Cryo-Electron Microscopy

Two issues that often impact the cryo-electron microscopy (cryoEM) specimen preparation process are agglomeration of particles near hole edges in holey carbon films and variations in vitreous ice thickness. In many cases, the source of these issues was identified to be the residues and topography often seen in commercially available films. To study and minimize their impact during specimen preparation, an improved holey carbon film has been developed. Rather than using a consumable template based on soft materials that must be removed prior to grid assembly, a method was developed that uses a hard template and a water-soluble release layer to replicate the template pattern into the carbon films. The advantages of this method are the improved purity and flatness of the carbon films, and these attributes are shown to have a dramatic improvement on the distribution of single particles embedded in vitreous ice suspended across the holes. Improving particle distribution is an enabling factor toward increasing the throughput of data collection for cryoEM.

Block Liposomes from Curvature-Stabilizing Lipids: Connected Nanotubes, -rods or -spheres

We report on the discovery of block liposomes, a new class of chain-melted (liquid) vesicles, with membranes comprised of mixtures of the membrane-curvature-stabilizing multivalent lipid MVLBG2 of colossal charge +16 e and neutral 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). In a narrow MVLBG2 composition range (8-10 mol%), cryo-TEM revealed block liposomes consisting of distinctly shaped, yet connected, nanoscale spheres, pears, tubes, or rods. Unlike typical liposome systems, where spherical vesicles, tubular vesicles, and cylindrical micelles are separated on the macroscopic scale, within a block liposome, shapes are separated on the nanometer scale. Diblock (pear-tube) and triblock (pear-tube-pear) liposomes contain nanotubes with inner lumen diameter of 10-50 nm. Diblock (sphere-rod) liposomes were found to contain micellar nanorods approximately 4 nm in diameter and several micrometers in length, analogous to cytoskeletal filaments of eukaryotic cells. Block liposomes may find a range of applications in chemical and nucleic acid delivery and as building blocks in the design of templates for hierarchical structures.

Rapid routine structure determination of macromolecular assemblies using electron microscopy: current progress and further challenges.

Although the methodology of molecular microscopy has enormous potential, it is time consuming and labor intensive. The techniques required to produce a three-dimensional (3D) electron density map of a macromolecular structure normally require manual operation of an electron microscope by a skilled operator and manual supervision of the sometimes complex software needed for analysis and calculation of 3D maps. Systems to automate the process of data acquisition from an electron microscope are being developing and these systems are being integrated with specimen handling operations and post acquisition data processing. Here, the current performance of our existing systems and the future challenges involved in substantially improving both the sustained throughput and the yield of automated data collection and analysis are reported.

Automation in Single-Particle Electron Microscopy: Connecting the Pieces

Throughout the history of single-particle electron microscopy (EM), automated technologies have seen varying degrees of emphasis and development, usually depending upon the contemporary demands of the field. We are currently faced with increasingly sophisticated devices for specimen preparation, vast increases in the size of collected data sets, comprehensive algorithms for image processing, sophisticated tools for quality assessment, and an influx of interested scientists from outside the field who might lack the skills of experienced microscopists. This situation places automated techniques in high demand. In this chapter, we provide a generic definition of and discuss some of the most important advances in automated approaches to specimen preparation, grid handling, robotic screening, microscope calibrations, data acquisition, image processing, and computational infrastructure. Each section describes the general problem and then provides examples of how that problem has been addressed through automation, highlighting available processing packages, and sometimes describing the particular approach at the National Resource for Automated Molecular Microscopy (NRAMM). We contrast the more familiar manual procedures with automated approaches, emphasizing breakthroughs as well as current limitations. Finally, we speculate on future directions and improvements in automated technologies. Our overall goal is to present automation as more than simply a tool to save time. Rather, we aim to illustrate that automation is a comprehensive and versatile strategy that can deliver biological information on an unprecedented scale beyond the scope available with classical manual approaches.