DNA is renowned for its double helix structure and the base pairing that enables the recognition and highly selective binding of complementary DNA strands. These features, and the ability to create DNA strands with any desired sequence of bases, have led to the use of DNA rationally to design various nanostructures and even execute molecular computations. Of the wide range of self-assembled DNA nanostructures reported, most are one- or two-dimensional. Examples of three-dimensional DNA structures include cubes, truncated octahedra, octohedra and tetrahedra, which are all comprised of many different DNA strands with unique sequences. When aiming for large structures, the need to synthesize large numbers (hundreds) of unique DNA strands poses a challenging design problem. Here, we demonstrate a simple solution to this problem: the design of basic DNA building units in such a way that many copies of identical units assemble into larger three-dimensional structures. We test this hierarchical self-assembly concept with DNA molecules that form three-point-star motifs, or tiles. By controlling the flexibility and concentration of the tiles, the one-pot assembly yields tetrahedra, dodecahedra or buckyballs that are tens of nanometres in size and comprised of four, twenty or sixty individual tiles, respectively. We expect that our assembly strategy can be adapted to allow the fabrication of a range of relatively complex three-dimensional structures.

Hsp100 polypeptide translocases are conserved AAA+ machines that maintain proteostasis by unfolding aberrant and toxic proteins for refolding or proteolytic degradation. The Hsp104 disaggregase from S. cerevisiae solubilizes stress-induced amorphous aggregates and amyloid. The structural basis for substrate recognition and translocation is unknown. Using a model substrate (casein), we report cryo-EM structures at near-atomic resolution of Hsp104 in different translocation states. Substrate interactions are mediated by conserved, pore-loop tyrosines that contact an 80 Å-long unfolded polypeptide along the axial channel. Two protomers undergo a ratchet-like conformational change that advances pore-loop-substrate interactions by two-amino acids. These changes are coupled to activation of specific ATPase sites and, when transmitted around the hexamer, reveal a processive rotary translocation mechanism and a remarkable structural plasticity of Hsp104-catalyzed disaggregation.

G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy-terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins.

Ribosome assembly in eukaryotes requires approximately 200 essential assembly factors (AFs), and occurs via ordered events that initiate in the nucleolus and culminate in the cytoplasm. Here we present the cryo-electron microscopy (cryo-EM) structure of a late cytoplasmic 40S ribosome assembly intermediate from Saccharomyces cerevisiae. The positions of bound AFs were defined using cryo-EM reconstructions of pre-ribosomal complexes lacking individual components. All seven AFs are positioned to prevent each step in the translation initiation pathway by obstructing the binding sites for initiation factors, by preventing the opening of the mRNA channel, by blocking 60S subunit joining, and by disrupting the decoding site. We suggest that these highly redundant mechanisms ensure that pre-40S particles do not enter the translation pathway, which would result in their rapid degradation. Implications for the regulation of 40S maturation are also discussed.

Degeneracy in the genetic code, which enables a single protein to be encoded by a multitude of synonymous gene sequences, has an important role in regulating protein expression, but substantial uncertainty exists concerning the details of this phenomenon. Here we analyze the sequence features influencing protein expression levels in 6,348 experiments using bacteriophage T7 polymerase to synthesize messenger RNA in Escherichia coli. Logistic regression yields a new codon-influence metric that correlates only weakly with genomic codon-usage frequency, but strongly with global physiological protein concentrations and also mRNA concentrations and lifetimes in vivo. Overall, the codon content influences protein expression more strongly than mRNA-folding parameters, although the latter dominate in the initial ~16 codons. Genes redesigned based on our analyses are transcribed with unaltered efficiency but translated with higher efficiency in vitro. The less efficiently translated native sequences show greatly reduced mRNA levels in vivo. Our results suggest that codon content modulates a kinetic competition between protein elongation and mRNA degradation that is a central feature of the physiology and also possibly the regulation of translation in E. coli.

Molecular self-assembly is a promising approach to the preparation of nanostructures. DNA, in particular, shows great potential to be a superb molecular system. Synthetic DNA molecules have been programmed to assemble into a wide range of nanostructures. It is generally believed that rigidities of DNA nanomotifs (tiles) are essential for programmable self-assembly of well defined nanostructures. Recently, we have shown that adequate conformational flexibility could be exploited for assembling 3D objects, including tetrahedra, dodecahedra, and buckyballs, out of DNA three-point star motifs. In the current study, we have integrated tensegrity principle into this concept to assemble well defined, complex nanostructures in both 2D and 3D. A symmetric five-pointstar motif (tile) has been designed to assemble into icosahedra or large nanocages depending on the concentration and flexibility of the DNA tiles. In both cases, the DNA tiles exhibit significant flexibilities and undergo substantial conformational changes, either symmetrically bending out of the plane or asymmetrically bending in the plane. In contrast to the complicated natures of the assembled structures, the approach presented here is simple and only requires three different component DNA strands. These results demonstrate that conformational flexibility could be explored to generate complex DNA nanostructures. The basic concept might be further extended to other biomacromolecular systems, such as RNA and proteins.icosahedron ͉ three-dimensional ͉ polyhedron ͉ cryo-EM ͉ molecular cages M olecular self-assembly provides a bottom-up approach to the preparation of nanostructures (1-3). DNA, in particular, shows great potential to be a superb molecular system (4). In the last 20 years, DNA has been explored as building blocks for nanoconstructions, including preparation of periodic and aperiodic 2D nanopatterns (5-8) and 3D polyhedra (9-14). Most of the branched DNA structures are intrinsically flexible and are not suitable building blocks for construction of well defined geometric structures. How to overcome the conformational flexibility of branched DNA structures is a major challenge in structural DNA nanotechnology. In the last decade, a series of rigid structural motifs have been successfully engineered that lead to the rapid evolution of structural DNA nanotechnology (4). However, with more experience and knowledge, it is possible to controllably introduce the conformational flexibility to prepare complex DNA nanostructures (15). In our recent study of 3D self-assembly of DNA three-point-star tiles (16), we found that DNA tetrahedra could be readily assembled, and the tetrahedra are well behaved during sample characterizations. In contrast, DNA dodecahedra and buckyballs have significantly lower assembly yields and are prone to deformation. This phenomenon can be explained by the geometrical differences of these structures. Tetrahedra consist of triangular faces, but others do not. According to tensegrity principle, triangular faces will lead to rigid s...

ClpB and Hsp104 are conserved AAA+ protein disaggregases that promote survival during cellular stress. Hsp104 acts on amyloids, supporting prion propagation in yeast, and can solubilize toxic oligomers connected to neurodegenerative diseases. A definitive structural mechanism, however, has remained elusive. We have determined the cryo-EM structure of Hsp104 in the ATP state, revealing a near-helical hexamer architecture that coordinates the mechanical power of the twelve AAA+ domains for disaggregation. An unprecedented heteromeric AAA+ interaction defines an asymmetric seam in an apparent catalytic arrangement that aligns the domains in a two-turn spiral. N-terminal domains interact to form a broad channel entrance for substrate engagement and Hsp70 interaction. Middle-domain helices bridge adjacent protomers across the nucleotide pocket, explaining roles in hydrolysis and disaggregation. Remarkably, substrate-binding pore loops line the channel in a continuous spiral that appears optimized for substrate transfer across the AAA+ domains, establishing a directional path for polypeptide translocation.

The active-state complex between an agonist-bound receptor and a guanine nucleotide-free G protein represents the fundamental signaling assembly for the majority of hormone and neurotransmitter signaling. We applied single-particle electron microscopy (EM) analysis to examine the architecture of agonist-occupied β 2 -adrenoceptor (β 2 AR) in complex with the heterotrimeric G protein Gs (Gαsβγ). EM 2D averages and 3D reconstructions of the detergent-solubilized complex reveal an overall architecture that is in very good agreement with the crystal structure of the active-state ternary complex. Strikingly however, the α-helical domain of Gαs appears highly flexible in the absence of nucleotide. In contrast, the presence of the pyrophosphate mimic foscarnet (phosphonoformate), and also the presence of GDP, favor the stabilization of the α-helical domain on the Ras-like domain of Gαs. Molecular modeling of the α-helical domain in the 3D EM maps suggests that in its stabilized form it assumes a conformation reminiscent to the one observed in the crystal structure of Gαs-GTPγS. These data argue that the α-helical domain undergoes a nucleotidedependent transition from a flexible to a conformationally stabilized state.G protein-coupled receptor | negative stain electron microscopy | random conical tilt T he majority of hormones and neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs), which instigate intracellular signaling by activating their cognate heterotrimeric G proteins on the cytoplasmic side. GPCRs constitute the largest family of membrane proteins and play essential roles in regulating every aspect of normal physiology, thereby representing major pharmacological targets. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G-protein activation remain elusive. The β 2 -adrenergic receptor (β 2 AR) and its complex with heterotrimeric stimulatory G-protein Gs (Gαsβγ) represent an ideal model system for the large family of GPCRs activated by diffusible ligands. Agonist binding to the β 2 AR promotes interactions with GDP-bound Gsαβγ heterotrimer, leading to the exchange of GDP for GTP, and the functional dissociation of Gs into Gα-GTP and Gβγ subunits. To examine the architecture of agonist occupied β 2 AR in complex with Gαsβγ under different conditions, we used electron microscopy (EM) and single-particle analysis. Because of the limited size of the protein complex (∼148 kDa), we visualized specimens embedded in negative stain, which provides sufficient contrast from relatively small protein assemblies (1). This approach allowed us to obtain 2D projection averages and 3D reconstructions that provided new insights into dynamic features of the β 2 AR-Gs complex, and helped guide a successful approach to crystallize the complex enabling a high-resolution structure (2). Results and DiscussionIn a first step, we sought to examine the architecture of complexes in the nucleot...