Importance of the DNA “bond” in programmable nanoparticle crystallization

If a solution of DNA-coated nanoparticles is allowed to crystallize, the thermodynamic structure can be predicted by a set of structural design rules analogous to Pauling’s rules for ionic crystallization. The details of the crystallization process, however, have proved more difficult to characterize as they depend on a complex interplay of many factors. Here, we report that this crystallization process is dictated by the individual DNA bonds and that the effect of changing structural or environmental conditions can be understood by considering the effect of these parameters on free oligonucleotides. Specifically, we observed the reorganization of nanoparticle superlattices using time-resolved synchrotron small-angle X-ray scattering in systems with different DNA sequences, salt concentrations, and densities of DNA linkers on the surface of the nanoparticles. The agreement between bulk crystallization and the behavior of free oligonucleotides may bear important consequences for constructing novel classes of crystals and incorporating new interparticle bonds in a rational manner.Materials scientists have accomplished much by studying the way atoms and molecules crystallize. In these systems, however, the identity of the atom and its bonding behavior cannot be independently controlled, limiting our ability to tune material properties at will. In contrast, when a nanoparticle is modified with a dense shell of upright, oriented DNA, it can behave as a programmable atom equivalent (PAE) (1, 2) that can be used to synthesize diverse crystal structures with independent control over composition, scale, and lattice symmetry (3–14). The thermodynamic product of this crystallization process has been extensively studied by both experimental and theoretical means, and thus a series of design rules has been proposed and validated with a simple geometric model known as the complementary contact model (CCM). These rules allow one to predict the thermodynamically favored structure as the arrangement of particles that maximizes complementary contacts and therefore DNA hybridization (2, 6). These efforts have been very successful in predicting the thermodynamically favored product; recent studies have even demonstrated that PAEs can form single-crystal Wulff polyhedra that are analogous to those formed in atomic systems with the same crystallographic symmetry (15). However, the fact that there is a crystalline thermodynamic product does not mean that any choice of DNA and nanoparticles will result in crystalline systems in practice (3, 4). For example, crystallization has been observed for a relatively narrow class of PAEs (16) and in a manner that is primarily dependent upon the length of the DNA linker and temperature at which assembly occurs (8). Thus, absent from our understanding of these systems is a connection between the crystallization process and the properties of the DNA bonds that form the foundation of these structures.Here, we study the crystallization process and find that the complexity of the polyvalent DNA interactions can be simply understood by considering the behavior of a single DNA bond. By systematically studying the roles of nucleobase sequence, solution ionic strength, DNA density, and temperature on crystallization, we find that the effects of these factors are mirrored by the rates of hybridization and dehybridization of free DNA. In addition to examining steady-state structures, we evaluate the formation and reorganization of these crystals in a time-resolved manner using small-angle X-ray scattering (SAXS) to study how crystallization dynamics are affected by each design variable. Finally, we develop a predictive model that allows one to compare the range of temperatures over which crystallization will occur for different conditions. In addition to providing an avenue for improving PAE crystallization and realizing new architectures, the effectiveness of this reductionist model suggests that this approach can be applied to study crystallization in a broader class of systems, thus making an impact in the materials by design community.     

Shape-sensitive crystallization in colloidal superball fluids

Guiding the self-assembly of materials by controlling the shape of the individual particle constituents is a powerful approach to material design. We show that colloidal silica superballs crystallize into canted phases in the presence of depletants. Some of these phases are consistent with the so-called “Λ₁” lattice that was recently predicted as the densest packing of superdisks. As the size of the depletant is reduced, however, we observe a transition to a square phase. The differences in these entropically stabilized phases result from an interplay between the size of the depletants and the fine structure of the superball shape. We find qualitative agreement of our experimental results both with a phase diagram computed on the basis of the volume accessible to the depletants and with simulations. By using a mixture of depletants, one of which is thermosensitive, we induce solid-to-solid phase transitions between square and canted structures. The use of depletant size to leverage fine features of the shape of particles in driving their self-assembly demonstrates a general and powerful mechanism for engineering novel materials.Determining the relationship between the macroscopic structure of a material and the properties of its microscopic constituents is a fundamental problem in condensed matter science. A particularly interesting aspect of this problem is to understand how the self-assembly of a collection of particles is determined by their shape. These so-called “packing problems” have long interested physicists, mathematicians, and chemists alike and have been used to understand the structures of many condensed phases of matter (1–3). Computational and experimental advances continue to enable new explorations into fundamental aspects of these problems today (4–13). Recent discoveries include dense packings of tetrahedra into disordered, crystalline, and quasi-crystalline structures (14, 15), as well as the singular dense packings of ellipsoids (16).Technologically speaking, these discoveries are becoming increasingly crucial as new synthesis techniques are allowing for the creation of more and more complex shaped nanoscopic and microscopic particles (17, 18). The self-assembly of these particles into ordered structures creates new possibilities for the fabrication of novel materials (19–23). Moreover, advances in synthesis techniques have created new capabilities for experimentally investigating how the shapes of particles can be exploited in their self-assembly (24–26).Here, we experimentally and computationally explore the self-assembly of colloidal superballs interacting with depletion forces. We find that monolayers of superballs can be tuned to equilibrate into both their densest known packings—so-called “Λ₀” and “Λ₁” lattices (12)—as well as into less dense structures of different symmetries depending on an interplay between the subtle features of the particle shapes and the size of the depletants. The family of superballs can smoothly interpolate shapes between spheres and cubes (Fig. 1E) and is modeled as(x)^m + (y)^m + (z)^m ≤ 1, [1]where m is the shape parameter. For m = 2, this parameterization describes a purely isotropic sphere. As m is increased, the shape increasingly resembles a cube, as shown in Fig. 1. The amorphous colloidal superballs were prepared via controlled deposition of silica on the surface of hematite templates, using a synthetic technique (27) that yields high amounts of monodisperse (3% polydispersity) particles. Each batch of particles, which were made from the same initial hematite cores, contains superballs of comparable sizes (∼1.3 μm), but differing shape parameters as a result of differing amounts of silica precipitated on the surface. Size and shape of superballs were analyzed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM) micrographs (Fig. 1). Analyzing the particle shape from TEM images, we find agreement between the contour of the particle and the superball shape as shown in Fig. 1B, in which the red contours correspond to the superball fits. More information on the fitting procedure and shape polydispersity can be found in SI Text.Open in a separate windowFig. 1.(A) SEM images of sample m = 3.9. The particles have a cubic shape with rounded edges. (B–D) TEM micrographs of samples with m = 3.5, m = 3.0, and m = 2.0, respectively. All samples are uniform with a size polydispersity as low as 3%. (Scale bars: 1 μm.) In B the particles are shown with their corresponding superball fit highlighted in red (see also Fig. S1). (E, Top) Computer-generated models of colloidal superballs with different shape parameters m. A gradual increase of the absolute value of the shape parameter from m = 2 (spheres) results in a gradual alteration of the particle shape to resemble more cube-like particles. (E, Bottom) TEM images of silica superballs with different m values.Fig. 1 shows SEM and TEM images of the silica superballs used for the experiments. Although the particles still possess a distinct cubic symmetry, they have rounded edges whose curvatures are consistent with superballs of shape parameters m = 2.0, m = 3.0, m = 3.5, and m = 3.9. The spherical particles with shape parameters m = 2 were purchased from Bangs Laboratoires.To perform the experiments, silica superballs were dispersed in slightly alkaline water (pH = 9) and were stabilized against aggregation by surface charges. Sodium chloride (10 mM, final concentration) was added to the dispersion to screen the charges and lower the Debye length down to a thickness of about 3 nm, small enough to allow the particles to fully experience their anisotropic shape. Attractive forces between superballs arise by addition of depletion agents with gyration radii of R_g =  57 nm, 65 nm, 70 nm, 210 nm, 228 nm, and 329 nm. Flat optical capillaries were filled with aqueous mixtures of superballs and depletants and were monitored in time with bright-field microscopy. More experimental details as well as information on depletants and sample preparation can be found in SI Text.At low particle concentration, the superballs first sediment to the bottom of the capillary where they are attracted to the glass wall by depletion forces. While diffusing in the plane, the particles cluster together into monolayers. Once clusters are formed, time-lapsed images are collected and analyzed. The images show the appearance of several qualitatively distinct phases (Fig. 2). The particles are found to arrange into crystallite islands, often possessing grain boundaries, which we separate by orientation and analyze independently. We do not exclude a priori the possibility that a cluster does not have a coherent crystal structure.Open in a separate windowFig. 2.Representative optical microscope images showing three different ordered structures found in superball samples. (A–C, Right) Histogram of the relative positions of nearest neighbors for each particle in a crystallite (Top) and a histogram of the interparticle bond angles (Bottom) (see also Fig. S2). The structures of the crystallites are characterized by bond angels of 54° (A), 90° (B), and 60° (C). Note that the superballs in A and B have the same shape. The different lattice structures in these two samples result from different depletant sizes.To characterize the structure of each cluster, the positions of the constituent particles are identified for every time-lapsed image. The relative positions of nearest neighbors are then computed for each particle. For spherical superballs the distribution of these positions are found to be consistent with triangular lattices (Fig. 2C). For superballs with intermediate shape parameters (2 < m < ∞), however, the behavior becomes more interesting. Experimentally, we observe that the particles often form canted structures (Fig. 2A) characterized by interparticle bond angles distinct from 60°, indicative of triangular lattices, and 90°, which are characteristic of square lattices. Recently, the densest packings of superdisks with these intermediate shape parameters were predicted to fall into two families of lattices, referred to as Λ₀ and Λ₁ packings (12). Testing the distribution of relative nearest-neighbor positions in the experiment for consistency with the lattice vectors of these structures confirms, for the first time to our knowledge, the observation of an equilibrium Λ₁ lattice of superballs in experiments (Fig. 2). We also find that, for superballs with these same intermediate shape parameters (m = 3.5 and m = 3.9), the equilibrium structure transitions to a square lattice as the depletant size is decreased, suggesting that the resulting phases are determined by an interplay between the shape of the particle and the size ratio q = 2R_g/L between the depletant and the superball.To understand this interplay, we look at the depletion interactions between the superballs. Each superball is surrounded by an exclusion zone of thickness R_g that is unavailable for the centers of the depletants to occupy. Superball configurations that minimize the volume excluded from the depletants by overlapping exclusion zones increase the overall entropy of the system. To understand the favorability of the three lattices for a given choice of parameters, we compute the free energy of a depletion-stabilized bound state of a particle for each crystal type. For a number density n of depletants, this energy is given by U = ?nK_BTΔV_ex, where ΔV_ex is the change in volume excluded when a particle is removed from the interior of an otherwise filled lattice. By computing and comparing ΔV_ex for the Λ₀, Λ₁, and square lattices, we estimate which lattice is energetically favorable for a particular value of m (Fig. 3). In this model, the magnitude of ΔV_ex, and thus the overall bound state energy, will generally scale with R_g. We note that this model neglects the entropy of the superballs. It has been suggested that the role of rotational entropy of the particles can be significant in stabilizing canted phases (25), although the relative importance of this effect is debated (13). Fig. 3B shows that, for fixed-sized depletants and superballs, ΔV_ex varies smoothly for each lattice type as m is varied. For a particular combination of m and q, the lattice with the highest value of ΔV_ex represents the preferred phase. Using this principle, a 2D phase diagram is approximated in Fig. 3C. The interplay between the particle shape and the size ratio q suggested by this diagram is qualitatively apparent in the experimentally realized structures (Fig. 4).Open in a separate windowFig. 3.Two-dimensional predicted diagram for depletion-stabilized superball phases. The favorability of each lattice type is determined by calculating the bound state energy of a particle. (A) Operationally, the bound state energy is found by computing the difference in the excluded volume for a particular lattice (A, i) and the excluded volume of that lattice when a particle is removed from the interior (A, ii). (B) Change in excluded volume for each lattice type with varying m but fixed q = 2R_g/L, where R_g is the radius of gyration of the depletant and L is the diameter of the superball. To illustrate the behavior of ΔV_ex, the range of m used in this plot is larger than the experimentally investigated range. Background color indicates the preferred phase. (C) Two-dimensional phase diagram for experimental range of q and m. (D) Difference in ΔV_ex between two most favorable lattice types. Near phase boundaries, the phases become degenerate. In addition, for large depletants, the benefit of choosing a particular phase is small (see also Fig. S3). Recent molecular dynamics simulations (12) of convex superdisks have shown that the critical value of m when the densest packings change from Λ₁ to Λ₀ is at m ≈ 2.572.Open in a separate windowFig. 4.Comparison between experimental observations, bulk crystal simulations, and calculated phase diagram for superballs at different m and q values. Circles indicate the experimental results, open circles indicate simulation results, and the background color indicates the predicted phase. The approximated phase diagram qualitatively agrees with our experimental and simulation results.Indeed, the calculations agree with the experimental result that for sufficiently small depletants and sufficiently large m, square lattices, although they are not the densest packings for any finite value of m, are preferred. Square lattices occur when m is large enough such that the overlap in exclusion zones resulting from face-to-face contact is considerable and for q small enough such that depletants are able to fit into the interparticle pores made where the rounded edges of the superballs meet. When the osmotic pressure exerted by a depletant within an interparticle pore is substantial, the cubic phase is stabilized. However, when intermediate-sized depletants, which can no longer fit into the spaces within the lattice, are dispersed with superballs possessing these larger values of m (3.5 and 3.9), the densely packed Λ₁ phase emerges. As the size ratio gets larger, we note the distribution of bond angles within a crystallite begins to broaden. In the case of m? = ?3.9, for the highest size ratio q we tested, the distribution was too broad to identify the experimental structure with one of the three lattices, leaving the structure undetermined. Whereas our calculations suggest that the Λ₁ phase is energetically favorable, Fig. 3D shows that the difference in ΔV_ex between the lattices with the two highest values becomes negligible for large q. This suggests that the energetic benefit of choosing a particular phase decreases, which is consistent with our observation that the variance in experimental bond angle distributions increases for high q.As mentioned previously, spherical superballs form triangular lattices, which are equivalent to the Λ₀ lattice for m = 2. For small deviations from spheres, our calculations suggest that the Λ₀ lattice also tends to maximize ΔV_ex. As the deformation parameter is increased, however, the value of ΔV_ex for a different lattice, depending on q, surpasses that of the Λ₀ lattice. This can be seen, for example, in Fig. 3B where the curve representing the square lattice intersects the curve representing the Λ₀ lattice. At this intersection point, the lowest energy state becomes degenerate. Near these regions, the difference in energy between the most favorable lattices is small (Fig. 3D). As a result, we find experimental structures near phase boundaries fail to conform to a single coherent crystal type. Again, here we find some experimental structures are characterized by broad variances in bond angle distributions, which disallow the identification of a particular crystal type. Often, however, although there are insufficient statistical data to make a precise classification, we find the appearance of mixed assortments of crystallites (SI Text) of both cubic structures and undetermined, noncubic structures within a single sample cell.To more carefully probe the stability of our observed lattices, we perform idealized simulations of superballs and depletants (SI Text and Fig. S4). We first simulate finite crystallites and find the results qualitatively agree with experiments (Figs. S5 and S6). A particular choice of initial conditions, however, may influence the vulnerability of the resulting assembly to fall into kinetic traps. To probe the true stability of our candidate lattices, and to remove surface effects that exist in finite crystallites, we perform bulk crystal simulations, using periodic boundary conditions of each candidate lattice (Fig. S7). Fig. 4 shows the resulting stable lattices determined from these simulations. The results qualitatively agree with our excluded volume calculations. It is interesting to note that near phase boundaries both Λ₁ and square lattices often can be stable for the same parameters, as suggested by the appearance of mixed crystallites in experimental structures. In addition, we find that, for m values between 2 and 3, particles often assemble with irregular orientations with respect to their neighbors, consistent with the observation of indeterminate experimental structures.It is particularly interesting to note that for both experiment and simulation, we identify different crystalline structures as q is varied for m  ≥  3.5. In principle, it is thus experimentally possible to use size-variable depletants to reversibly switch the lattice structure within a single sample. To explore this possibility, we use thermosensitive poly(N-isopropylacrylamide) (pNIPAM) microgel spheres as depletants. Although we find inducing a structural transition with pNIPAM depletants alone is difficult (SI Text), we find that, using a bidepletant mixture, we are able to entropically drive a solid–solid transition. This transition demonstrates a powerful mechanism in which leveraging different geometric features of individual particles enables one to controllably and reversibly tune their assembly.To drive this transition, we use superballs with shape parameter m = 3.9 and a mixture of polyethylene oxide (PEO) and pNIPAM as depletant. Using the pNIPAM alone, the superballs form square lattices at 25 °C. When they are heated to 29 °C, we find the overall superball interactions induced by pNIPAM decrease sufficiently to melt this square lattice (Movie S1). Moreover, when the superballs are dispersed with PEO (molecular weight of 8 M) alone as depletant, we find we are able to stabilize a Λ₁ lattice of superballs (Movie S2).Using a mixture of the two depletants, however, allows us to reversibly switch between the two lattice types by varying the temperature. At room temperature, the interactions induced by the pNIPAM are activated, and the superballs once again favor a square lattice. As the temperature is increased, the relative energetic contribution of the pNIPAM depletant decreases, while the contribution of the PEO remains the same. Because the PEO dominates the overall energy at high temperatures, the Λ₁ lattice emerges. Fig. 5 and Movie S3 demonstrate this reversible solid–solid phase transition.Open in a separate windowFig. 5.Demonstration of reversible solid–solid phase transition of superballs. (A) Colloidal superballs with shape parameter m = 3.9 dispersed in depletant mixture of PEO and pNIPAM. At 27.5 °C, superballs assemble into a square lattice. At 31 °C, energetic contribution of pNIPAM becomes negligible, while that of PEO stays fixed, resulting in the transition into a Λ₁ lattice. (B) Simulated phase transition in a bulk crystal of superballs and depletants. A periodic lattice of superballs is simulated along with a mixture of two species of depletants, one with fixed size ratio of q₁ = 0.35 (which favors a Λ₁ lattice) and a smaller depletant (which favors a square lattice) of size ratio varying from q₂ = 0.04 to 0.032. As the smaller depletant is reduced in size, its overall energetic contribution decreases and the lattice transitions to a Λ₁ structure. When the size of the smaller depletant is once again increased, the square lattice once again emerges.By performing simulations of bidepletant superball dispersions we provide further evidence of the simple entropic nature of the geometric mechanism that induces this solid–solid transition. Again we perform periodic simulations of a bulk crystal as well as simulations of finite crystallites. Superballs are dispersed with two species of depletant, one with fixed size ratio q₁ = 0.35, which is found to stabilize a Λ₁ lattice, and a variable-size depletant with initial size ratio q₂ = 0.04, which is found to stabilize a square lattice. When dispersed in a mixture with number densities n₁ = 24.9L^?3 and n₂ = 596.8L^?3 for the large and small depletant, respectively, superballs with shape parameter m = 4 arrange into a square lattice. Here L is the diameter of the superball. As the smaller depletant is shrunk by 20% at fixed number density, its induced pressure remains fixed while its overall energetic contribution is lowered. We find, consistent with our experimental observations, that the lattice becomes canted. Upon increasing q₂ once again, we find the square lattice is restored (Figs. S8 and S9 and Movie S4).In this article we have demonstrated the reversible assembly of the same superball-shaped colloidal particles into both a square phase and the recently predicted Λ₁ phase. We show depletant size can be used to tune interparticle interactions. As a result, both particle shape and depletant size are used to determine the resulting phases. By mixing large depletants and small thermosensitive depletants we demonstrate a fully reversible solid-to-solid transition between square and Λ₁ superball phases. The sensitivity of the assembled phase to a fine feature of the particle shape, combined with a mechanism to reversibly activate a depletant on that scale, demonstrates that depletants can be used to tune interactions. These results create previously unidentified opportunities for controlling the reversible self-assembly of colloidal particles and controlling phases, for example through solid-to-solid phase transitions.     

Tryptophan Residues Are Critical for Portal Protein Assembly and Incorporation in Bacteriophage P22

The oligomerization and incorporation of the bacteriophage P22 portal protein complex into procapsids (PCs) depends upon an interaction with scaffolding protein, but the region of the portal protein that interacts with scaffolding protein has not been defined. In herpes simplex virus 1 (HSV-1), conserved tryptophan residues located in the wing domain are required for portal-scaffolding protein interactions. In this study, tryptophan residues (W) present at positions 41, 44, 207 and 211 within the wing domain of the bacteriophage P22 portal protein were mutated to both conserved and non-conserved amino acids. Substitutions at each of these positions were shown to impair portal function in vivo, resulting in a lethal phenotype by complementation. The alanine substitutions caused the most severe defects and were thus further characterized. An analysis of infected cell lysates for the W to A mutants revealed that all the portal protein variants except W211A, which has a temperature-sensitive incorporation defect, were successfully recruited into procapsids. By charge detection mass spectrometry, all W to A mutant portal proteins were shown to form stable dodecameric rings except the variant W41A, which dissociated readily to monomers. Together, these results suggest that for P22 conserved tryptophan, residues in the wing domain of the portal protein play key roles in portal protein oligomerization and incorporation into procapsids, ultimately affecting the functionality of the portal protein at specific stages of virus assembly.     

Assembly and release of infectious hepatitis C virus involving unusual organization of the secretory pathway

AIM: To determine if calnexin(CANX), RAB1 and alphatubulin were involved in the production of hepatitis C virus(HCV) particles by baby hamster kidney-West Nile virus(BHK-WNV) cells. METHODS: Using a si RNA-based approach complemented with immuno-fluorescence confocal microscope and Western blot studies, we examined the roles of CANX, RAB1 and alpha-tubulin in the production of HCV particles by permissive BHK-WNV cells expressing HCV structural proteins or the full-length genome of HCV genotype 1a. Immuno-fluorescence studies in producer cells were performed with monoclonal antibodies against HCV structural proteins, as well as immunoglobulin from the serum of a patient recently cured from an HCV infection of same genotype. The cellular compartment stained by the serum immunoglobulin was also observedin thin section transmission electron microscopy. These findings were compared with the JFH-1 strain/Huh-7.5 cell model.RESULTS: We found that CANX was necessary for the production of HCV particles by BHK-WNV cells. This process involved the recruitment of a subset of HCV proteins, detected by immunoglobulin of an HCV-cured patient, in a compartment of rearranged membranes bypassing the endoplasmic reticulum-Golgi intermediary compartment and surrounded by mitochondria. It also involved the maturation of N-linked glycans on HCV envelope proteins, which was required for assembly and/or secretion of HCV particles. The formation of this specialized compartment required RAB1; upon expression of HCV structural genes, this compartment developed large vesicles with viral particles. RAB1 and alpha-tubulin were required for the release of HCV particles. These cellular factors were also involved in the production of HCVcc in the JFH-1 strain/Huh-7.5 cell system, which involves HCV RNA replication. The secretion of HCV particles by BHK-WNV cells presents similarities with a pathway involving caspase-1; a caspase-1 inhibitor was found to suppress the production of HCV particles from a full-length genome.CONCLUSION: Prior activity of the WNV subgenomic replicon in BHK-21 cells promoted re-wiring of host factors for the assembly and release of infectious HCV in a caspase-1-dependent mechanism.     

Molecular Mechanism of Arenavirus Assembly and Budding

Arenaviruses have a bisegmented negative-strand RNA genome, which encodes four viral proteins: GP and NP by the S segment and L and Z by the L segment. These four viral proteins possess multiple functions in infection, replication and release of progeny viruses from infected cells. The small RING finger protein, Z protein is a matrix protein that plays a central role in viral assembly and budding. Although all arenaviruses encode Z protein, amino acid sequence alignment showed a huge variety among the species, especially at the C-terminus where the L-domain is located. Recent publications have demonstrated the interactions between viral protein and viral protein, and viral protein and host cellular protein, which facilitate transportation and assembly of viral components to sites of virus egress. This review presents a summary of current knowledge regarding arenavirus assembly and budding, in comparison with other enveloped viruses. We also refer to the restriction of arenavirus production by the antiviral cellular factor, Tetherin/BST-2.     

Assembly and subunit stoichiometry of the functional helicase-primase (primosome) complex of bacteriophage T4

Physical biochemical techniques are used to establish the structure, subunit stoichiometry, and assembly pathway of the primosome complex of the bacteriophage T4 DNA replication system. Analytical ultracentrifugation and fluorescence anisotropy methods show that the functional T4 primosome consists of six gp41 helicase subunits that assemble into a hexagon, driven by the binding of six NTPs (or six nonhydrolyzable GTPγS analogues) that are located at and stabilize the intersubunit interfaces, together with a single tightly bound gp61 primase subunit. Assembling the components of the primosome onto a model DNA replication fork is a multistep process, but equilibrium cannot be reached along all mixing pathways. Producing a functional complex requires that the helicase hexamer be assembled in the presence of the DNA replication fork construct prior to the addition of the primase to avoid the formation of metastable DNA-protein aggregates. The gp41 helicase hexamer binds weakly to fork DNA in the absence of primase, but forms a much more stable primosome complex that expresses full and functional helicase (and primase) activities when bound to a gp61 primase subunit at a helicase:primase subunit ratio of 61. The presence of additional primase subunits does not change the molecular mass or helicase activity of the primosome, but significantly inhibits its primase activity. We develop both an assembly pathway and a minimal mechanistic model for the structure and function of the T4 primosome that are likely to be relevant to the assembly and function of the replication primosome subassemblies of higher organisms as well.     

The Beauty of Bacteriophage T4 Research: Lindsay W. Black and the T4 Head Assembly

Viruses are biochemically complex structures and mainly consist of folded proteins that contain nucleic acids. Bacteriophage T4 is one of most prominent examples, having a tail structure that contracts during the infection process. Intracellular phage multiplication leads to separate self-directed assembly reactions of proheads, tails and tail fibers. The proheads are packaged with concatemeric DNA produced by tandem replication reactions of the parental DNA molecule. Once DNA packaging is completed, the head is joined with the tail and six long fibers are attached. The mature particles are then released from the cell via lysis, another tightly regulated process. These processes have been studied in molecular detail leading to a fascinating view of the protein-folding dynamics that direct the structural interplay of assembled complexes. Lindsay W. Black dedicated his career to identifying and defining the molecular events required to form the T4 virion. He leaves us with rich insights into the astonishingly precise molecular clockwork that co-ordinates all of the players in T4 assembly, both viral and cellular. Here, we summarize Lindsay’s key research contributions that are certain to stimulate our future science for many years to come.     

Role of Human Cytomegalovirus Tegument Proteins in Virion Assembly

Like other herpesviruses, human cytomegalovirus (HCMV) contains a unique proteinaceous layer between the virion envelope and capsid, termed the tegument. Upon infection, the contents of the tegument layer are delivered to the host cell, along with the capsid and the viral genome, where they facilitate the initial stages of virus replication. The tegument proteins also play important roles in virion assembly and this dual nature makes them attractive potential targets for antiviral therapies. While our knowledge regarding tegument protein function during the initiation of infection has been the subject of intense study, their roles in assembly are much less well understood. In this review, we will focus on recent studies that highlight the functions of HCMV tegument proteins during assembly, and pose key questions for further investigation.     

Assembly of vorticity-aligned hard-sphere colloidal strings in a simple shear flow

Colloidal suspensions self-assemble into equilibrium structures ranging from face- and body-centered cubic crystals to binary ionic crystals, and even kagome lattices. When driven out of equilibrium by hydrodynamic interactions, even more diverse structures can be accessed. However, mechanisms underlying out-of-equilibrium assembly are much less understood, though such processes are clearly relevant in many natural and industrial systems. Even in the simple case of hard-sphere colloidal particles under shear, there are conflicting predictions about whether particles link up into string-like structures along the shear flow direction. Here, using confocal microscopy, we measure the shear-induced suspension structure. Surprisingly, rather than flow-aligned strings, we observe log-rolling strings of particles normal to the plane of shear. By employing Stokesian dynamics simulations, we address the mechanism leading to this out-of-equilibrium structure and show that it emerges from a delicate balance between hydrodynamic and interparticle interactions. These results demonstrate a method for assembling large-scale particle structures using shear flows.     

Acoustically trapped colloidal crystals that are reconfigurable in real time

Photonic and phononic crystals are metamaterials with repeating unit cells that result in internal resonances leading to a range of wave guiding and filtering properties and are opening up new applications such as hyperlenses and superabsorbers. Here we show the first, to our knowledge, 3D colloidal phononic crystal that is reconfigurable in real time and demonstrate its ability to rapidly alter its frequency filtering characteristics. Our reconfigurable material is assembled from microspheres in aqueous solution, trapped with acoustic radiation forces. The acoustic radiation force is governed by an energy landscape, determined by an applied high-amplitude acoustic standing wave field, in which particles move swiftly to energy minima. This creates a colloidal crystal of several milliliters in volume with spheres arranged in an orthorhombic lattice in which the acoustic wavelength is used to control the lattice spacing. Transmission acoustic spectroscopy shows that the new colloidal crystal behaves as a phononic metamaterial and exhibits clear band-pass and band-stop frequencies which are adjusted in real time.Artificially engineered metamaterials have attracted significant research interest due to their useful wave guiding and transmission properties. The interest in these materials stems from the possibility of gaining previously unheralded control over wave phenomena, for example, controlling the path of waves leads to incredibly efficient lenses (1) or invisibility cloaking (2, 3) and controlling their transmission and reflection leads to highly efficient filters (4), diodes (5–7), or superabsorbers (8).Photonic and phononic crystals are periodic metamaterials typically created from multiple elementary repeating cells. They exhibit a well-known series of band-pass and band-stop frequencies that have attracted significant attention for use as filters and absorbers. Here we demonstrate a phononic crystal reconfigurable in real time, although the same principles could be used to fabricate other metamaterials including photonic crystals, as well as the more elaborate configurations required for applications such as cloaking.The frequencies of the band gaps in phononic crystals can be tuned by changing the lattice geometry (9), and the width of the gaps depends on the contrast between the densities and sound velocities of the component materials. Most of the work to date has not involved any reconfigurability, for example, 2D phononic crystals with a periodicity millimeter order and above have been assembled manually and used to explore their basic properties (10, 11). A reconfigurable phononic crystal has been created in 2D using optical tweezers but this is limited to a relatively small scale ( area) and to transparent or dielectric particles (12). Three-dimensional experimental realizations of phononic crystals have either been manufactured with millimeter periodicity or as colloids (13, 14). Colloids represent a particularly attractive option as they are known to self-assemble into simple 3D crystalline structures. Recent work on 3D hypersonic (GHz) colloidal crystals has led to experimental observation of the hypersonic Bragg gaps (15). However, whereas the choice of particle shape, size, volume fraction, etc. allows the colloidal crystal to be tuned, they do not facilitate real-time reconfigurability and so cannot be used in an active mode.In this article we report for the first time, to our knowledge, the experimental realization of a 3D colloidal metamaterial that is reconfigurable in real time and demonstrate its ability to rapidly alter its acoustic filtering characteristics. The reconfigurable metamaterial is assembled from a low-density aqueous suspension of microspheres. Our metadevice generates a high-amplitude megahertz-frequency (MHz) acoustic standing wave which results in acoustic radiation forces that lead to particles moving to, and becoming trapped in, a simple orthorhombic lattice (although here we explore only simple tetragonal forms). By varying the frequency of the acoustic standing wave in the metadevice we are able to control the lattice geometry. We use transmission acoustic spectroscopy to explore the reconfigurability of the resulting metamaterial. These measurements reveal a complex and tunable distribution of band-gap and band-stop phenomena over a wide range of frequencies.     

Cellulose biogenesis: Polymerization and crystallization are coupled processes in Acetobacter xylinum

Calcofluor White ST, stilbene derivative used commerically as an optical brightener for cellulose, increased the rate of glucose polymerization into cellulose by resting cells of the gram-negative bacterium Acetobacter xylinum. This bacterium normally produces a ribbon of cellulose that is a composite of crystalline microfibrils. In concentrations above 0.1 mM, Calcofluor disrupts the assembly of crystalline cellulose I microfibrils and their integration into a composite ribbon by stoichiometric binding to glucose residues of newly polymerized glucan chains. Under these conditions, the rate of glucose polymerization increases up to 4 times the control rate, whereas oxygen uptake increases only 10-15%. These observed effects are readily reversible. If free Calcofluor is washed away or depleted below the threshold value by binding to cellulose as polymerization continues, ribbon production and the normal rate of polymerization resume. It is concluded that polymerization and crystallization are cell-directed, coupled processes and that the rate of crystallization determines the rate of polymerization. It is suggested that coupling must be maintained for biogenesis of crystalline cellulose I.     

沙门菌Ⅲ型分泌系统组成与装配研究进展

沙门菌(Salmonella)是引发人和动物食物中毒、胃肠炎的主要食源性病原菌,该菌III型分泌系统(T3SS)对其入侵宿主细胞发挥着重要作用,近年来,有关沙门菌T3SS的组成、装配以及相关致病机理的研究取得了一定进展。本文对沙门菌T3SS的组成与装配等研究作一综述,为深入研究沙门菌的致病机制以及预防、治疗该菌引发的疾病提供新的策略和手段。     

Structural Analysis of Retrovirus Assembly and Maturation

Retroviruses have a very complex and tightly controlled life cycle which has been studied intensely for decades. After a virus enters the cell, it reverse-transcribes its genome, which is then integrated into the host genome, and subsequently all structural and regulatory proteins are transcribed and translated. The proteins, along with the viral genome, assemble into a new virion, which buds off the host cell and matures into a newly infectious virion. If any one of these steps are faulty, the virus cannot produce infectious viral progeny. Recent advances in structural and molecular techniques have made it possible to better understand this class of viruses, including details about how they regulate and coordinate the different steps of the virus life cycle. In this review we summarize the molecular analysis of the assembly and maturation steps of the life cycle by providing an overview on structural and biochemical studies to understand these processes. We also outline the differences between various retrovirus families with regards to these processes.     

From the Cover: A geometric constraint, the head-to-tail exclusion rule, may be the basis for the isolated-pentagon rule in fullerenes with more than 60 vertices

Carbon atoms self-assemble into the famous soccer-ball shaped Buckminsterfullerene (C₆₀), the smallest fullerene cage that obeys the isolated-pentagon rule (IPR). Carbon atoms self-assemble into larger (n > 60 vertices) empty cages as well—but only the few that obey the IPR—and at least 1 small fullerene (n ≤ 60) with adjacent pentagons. Clathrin protein also self-assembles into small fullerene cages with adjacent pentagons, but just a few of those. We asked why carbon atoms and clathrin proteins self-assembled into just those IPR and small cage isomers. In answer, we described a geometric constraint—the head-to-tail exclusion rule—that permits self-assembly of just the following fullerene cages: among the 5,769 possible small cages (n ≤ 60 vertices) with adjacent pentagons, only 15; the soccer ball (n = 60); and among the 216,739 large cages with 60 < n ≤ 84 vertices, only the 50 IPR ones. The last finding was a complete surprise. Here, by showing that the largest permitted fullerene with adjacent pentagons is one with 60 vertices and a ring of interleaved hexagons and pentagon pairs, we prove that for all n > 60, the head-to-tail exclusion rule permits only (and all) fullerene cages and nanotubes that obey the IPR. We therefore suggest that self-assembly that obeys the IPR may be explained by the head-to-tail exclusion rule, a geometric constraint.     

Three-dimensional structure and organization of a receptor/signaling complex

Transmembrane signaling in bacterial chemotaxis has become an important model system for experimental and theoretical studies. These studies have provided a wealth of detailed molecular structures, including the structures of CheA, CheW, and the cytoplasmic domain of the serine receptor Tsr. How these three proteins interact to form the receptor/signaling complex remains unknown. By using EM and single-particle image analysis, we present a three-dimensional reconstruction of the receptor/signaling complex. The complex contains CheA, CheW, and the cytoplasmic portion of the aspartate receptor Tar. We observe density consistent with a structure containing 24 aspartate-receptor monomers and additional density sufficient to house the expected four CheA monomers and six CheW monomers. Within this bipolar structure are four groups of three receptor dimers that are not threefold symmetric and are therefore unlike the symmetric trimers observed in the x-ray crystal structure of the cytoplasmic domain of the serine receptor. In the latter, the interdimer contacts occur in the signaling domains near the hairpin loop. In our structure, the signaling domains within trimers appear spaced apart by the presence of CheA and CheW. This structure argues against models where one CheA and one CheW bind to the outer face of each of the dimers in the trimer. This structure of the receptor/signaling complex provides an additional basis for understanding the architecture of the large arrays of chemotaxis receptors, CheA, and CheW found at the cell poles in motile bacteria.     

From Crescent to Mature Virion: Vaccinia Virus Assembly and Maturation

Vaccinia virus (VACV) has achieved unprecedented success as a live viral vaccine for smallpox which mitigated eradication of the disease. Vaccinia virus has a complex virion morphology and recent advances have been made to answer some of the key outstanding questions, in particular, the origin and biogenesis of the virion membrane, the transformation from immature virion (IV) to mature virus (MV), and the role of several novel genes, which were previously uncharacterized, but have now been shown to be essential for VACV virion formation. This new knowledge will undoubtedly contribute to the rational design of safe, immunogenic vaccine candidates, or effective antivirals in the future. This review endeavors to provide an update on our current knowledge of the VACV maturation processes with a specific focus on the initiation of VACV replication through to the formation of mature virions.