Baseplate Structure Research Articles

Second site reversion of HIV-1 envelope protein baseplate mutations maps to the matrix protein.

The HIV-1 Envelope (Env) protein cytoplasmic tail (CT) recently has been shown to assemble an unusual trimeric baseplate structure that locates beneath Env ectodomain trimers. Mutations at linchpin residues that help organize the baseplate impair virus replication in restrictive T cell lines but not in permissive cell lines. We have identified and characterized a second site suppressor of these baseplate mutations, located at residue 34 in the viral matrix (MA) protein, that rescues viral replication in restrictive cells. The suppressor mutation was dependent on the CT to exert its activity and did not appear to affect Env protein traffic or fusion functions in restrictive cells. Instead, the suppressor mutation increased Env incorporation into virions 3-fold and virus infectivity in single-round infections 10-fold. We also found that a previously described suppressor of Env-incorporation defects that stabilizes the formation of MA trimers was ineffective at rescuing Env baseplate mutations. Our results support an interpretation in which changes at MA residue 34 induce conformational changes that stabilize MA lattice trimer-trimer interactions and/or direct MA-CT associations.IMPORTANCEHow HIV-1 Env trimers assemble into virus particles remains incompletely understood. In restrictive cells, viral incorporation of Env is dependent on the Env CT and on the MA protein, which assembles lattices composed of hexamers of trimers in immature and mature viruses. Recent evidence indicates that CT assembles trimeric baseplate structures that require membrane-proximal residues to interface with trimeric transmembrane domains and C-terminal helices in the CT. We found that mutations of these membrane-proximal residues impaired replication in restrictive cells. This defect was countered by a MA mutation that does not localize to any obvious interprotein regions but was only inefficiently suppressed by a MA mutation that stabilizes MA trimers and has been shown to suppress other CT-dependent Env defects. Our results suggest that efficient suppression of baseplate mutations involves stabilization of MA inter-trimer contacts and/or direct MA-CT associations. These observations shed new light on how Env assembles into virions.

Analysis of HIV-1 envelope cytoplasmic tail effects on viral replication

Trimers of the HIV-1 envelope (Env) protein perform receptor binding and virus-cell fusion functions during the virus life cycle. The cytoplasmic tail (CT) of Env forms an unusual baseplate structure, and is palmitoylated, rich in arginines, carries trafficking motifs, binds cholesterol, and interacts with host proteins. To dissect CT activities, we examined a panel of Env variants, including CT truncations, mutations, and an extension. We found that whereas all variants could replicate in permissive cells, viruses with CT truncations or baseplate mutations were defective in restrictive cells. We also identified a determinant in HIV-1 amphotericin sensitivity, and characterized variants that escape amphotericin inhibition via viral protease-mediated CT cleavage. Results additionally showed that full-length, his tagged Env can oligomerize and be co-assembled with CT truncations that delete portions of the baseplate, host protein binding sites, and trafficking signals. Our observations illuminate novel aspects of HIV-1 CT structure, interactions, and functions.

Multi-Mode Shape Control of Active Compliant Aerospace Structures Using Anisotropic Piezocomposite Materials in Antisymmetric Bimorph Configuration

The mission performance of future advanced aerospace structures can be synthetically improved via active shape control utilizing piezoelectric materials. Multiple work modes are required. Bending/twisting mode control receives special attention for many classic aerospace structures, such as active reflector systems, active blades, and compliant morphing wings. Piezoelectric fiber composite (Piezocomposite) material features in-plane anisotropic actuation, which is very suitable for multiple work modes. In this study, two identical macro-fiber composite (MFC) actuators of the F1 type were bonded to the base plate structure in an “antisymmetric angle-ply bimorph configuration” in order to achieve independent bending/twisting shape control. In terms of the finite element model and homogenization strategy, the locations of bimorph MFCs were determined by considering the effect of trade-off control capabilities on the bending and twisting shapes. The modal characteristics were investigated via both experimental and theoretical approaches. The experimental tests implied that the shape control accuracy was heavily reduced due to various uncertainties and nonlinearities, including hysteresis and the creep effect of the actuators, model errors, and external disturbances. A multi-mode feedback control law was designed and the experimental tests indicated that synthetic (independent and coupled) bending/twisting deformations were achieved with improved shape accuracy. This study provides a feasible multi-mode shape control approach with high surface accuracy, especially by employing piezocomposite materials.

Understanding Uncertainty in Microstructure Evolution and Constitutive Properties in Additive Process Modeling

Coupled process–microstructure–property modeling, and understanding the sources of uncertainty and their propagation toward error in part property prediction, are key steps toward full utilization of additive manufacturing (AM) for predictable quality part development. The OpenFOAM model for process conditions, the ExaCA model for as-solidified grain structure, and the ExaConstit model for constitutive mechanical properties are used as part of the ExaAM modeling framework to examine a few of the various sources of uncertainty in the modeling workflow. In addition to “random” uncertainty (due to random number generation in the orientations and locations of grains present), the heterogeneous nucleation density N0 and the mean substrate grain spacing S0 are varied to examine their impact of grain area development as a function of build height in the simulated microstructure. While mean grain area after 1 mm of build is found to be sensitive to N0 and S0, particularly at small N0 and large S0 (despite some convergence toward similar values), the resulting grain shapes and overall textures develop in a reasonably similar manner. As a result of these similar textures, ExaConstit simulation using ExaCA representative volume elements (RVEs) from various permutations of N0, S0, and location within the build resulted in similar yield stress, stress–strain curve shape, and stress triaxiality distributions. It is concluded that for this particular material and scan pattern, 15 layers is sufficient for ExaCA texture and ExaConstit predicted properties to become relatively independent of additional layer simulation, provided that reasonable estimates for N0 and S0 are used. However, additional layers of ExaCA will need to be run to obtain mean grain areas independent of build height and baseplate structure.

Design of experiment for optimization of 3D printing parameters of base plate structures in colostomy bag for newborns

ABSTRACT This study introduces a low-cost ostomy bag with sufficient softness for a newborn’s body to improve newborn medical care. Fused-deposition modeling was performed with a thermoplastic elastomer as the 3D printing material. The experimental design of the Taguchi L9(34) orthogonal arrays was used for the parameter design. To assess quality, the hardness was measured, with a focus on the smaller-the-better characteristic of the average hardness. The experimental results showed that the optimal combination of the structural hardness parameters is A1B3C1D1. As per the A scale, the average Shore hardness of the experimental model was 41.9. The signal-to-noise ratio of the experimental sample was −32.4526 dB, which is within the confidence interval, indicating that the factor effect has a reproduction characteristic that represents the reliability of this experimental result. The original material’s Shore hardness was reduced by 44.95%, from 85 A to 46.79 A, which represents an effective improvement in wearability.

Abstract OR-6: Baseplate Structure of Bacteriophage Phi812 and Mechanism of Cell Wall Binding and Penetration

Background: Antibiotic-resistant strains of Staphylococcus aureus cause human infections that are difficult to treat and can lead to death. Bacteriophage (phage) phi812K1/420 from the family Myoviridae infects 95% of clinical isolates of S. aureus and therefore is a promising candidate for a phage therapy agent. As the native phage particle approaches its host cell, phage receptor-binding proteins make a contact with the host cell wall. This interaction triggers a cascade of structural changes in the baseplate resulting in phage tail contraction and genome ejection. Mechanistic description of the baseplate re-organization, however, remains unknown. Methods: Using cryo-electron microscopy (cryo-EM), we studied the baseplate of the phage phi812K1/420. Also, selected proteins involved in the host cell wall binding and penetration were produced in recombinant form and their structures were solved using X-ray crystallography and cryo-EM single-particle reconstruction. Results: We reconstructed the phage baseplate in native and contracted states. The reconstruction of the native baseplate reaches a resolution of 4 Å, which enables us to discern individual protein structures. Solved protein structures will be fitted into the reconstruction of the contracted baseplate. Conclusion: Our results provide the first structural characterization of contractile phage infecting a Gram-positive bacterium. Comparison of the two distinct baseplate states will allow us to describe the molecular mechanism of the initial stage of phage infection in detail.

Action of a minimal contractile bactericidal nanomachine.

R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics1–4. Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold1,2. Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of (DNA-containing) T4 bacteriophage5. Here we report the atomic model of the complete R2 pyocin in its pre- and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the sequence of events during pyocin contraction: tail fibers trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.

Integrated topology optimization for vibration suppression in a vertical pump

This article presents a new approach aiming to reducing pump vibration by modifying its baseplate structure. The finite element models of the vertical pump were established and validated by the experimental impact test. The natural frequencies of pump were mapped in both experimental and numerical methods. The weak stiffness of the baseplate was identified as the root cause for the pump vibration. A topology optimization was used for enhancing the stiffness of baseplate and controlling its weight. The new baseplate was designed according to the inputs from optimization results and manufactured by the casting method. Both the vibration tests and the numerical simulations were carried out to investigate the vibration behaviors of the optimized pump model. The differences of vibration characteristics between original and optimized pumps were evaluated using 1/3 octave-band filter technique. Results show that the vibration was suppressed, and the resonance at 31.5 Hz was eliminated using the optimized baseplate. In particular, the maximum vibration amplitude of the vertical pump was reduced from 4.05 to 1.75 mm/s at the low flow rate condition. It was experimentally confirmed that the vibration amplitude of the optimized model complies with the requirements of the International Organization for Standardization standard and ensures the pump can operate stable for a long time.

Identification of Dual Receptor Binding Protein Systems in Lactococcal 936 Group Phages.

Siphoviridae of the lactococcal 936 group are the most commonly encountered bacteriophages in the dairy processing environment. The 936 group phages possess a discrete baseplate at the tip of their tail—a complex harbouring the Receptor Binding Protein (RBP) which is responsible for host recognition and attachment. The baseplate-encoding region is highly conserved amongst 936 phages, with 112 of 115 publicly available phages exhibiting complete synteny. Here, we detail the three exceptions (Phi4.2, Phi4R15L, and Phi4R16L), which differ from this genomic architecture in possessing an apparent second RBP-encoding gene upstream of the “classical” rbp gene. The newly identified RBP possesses an elongated neck region relative to currently defined 936 phage RBPs and is genetically distinct from defined 936 group RBPs. Through detailed characterisation of the representative phage Phi4.2 using a wide range of complementary techniques, we demonstrated that the above-mentioned three phages possess a complex and atypical baseplate structure. Furthermore, the presence of both RBPs in the tail tip of the mature virion was confirmed, while the anticipated host-binding capabilities of both proteins were also verified.

Structure and Analysis of R1 and R2 Pyocin Receptor-Binding Fibers.

The R-type pyocins are high-molecular weight bacteriocins produced by some strains of Pseudomonas aeruginosa to specifically kill other strains of the same species. Structurally, the R-type pyocins are similar to “simple” contractile tails, such as those of phage P2 and Mu. The pyocin recognizes and binds to its target with the help of fibers that emanate from the baseplate structure at one end of the particle. Subsequently, the pyocin contracts its sheath and drives the rigid tube through the host cell envelope. This causes depolarization of the cytoplasmic membrane and cell death. The host cell surface-binding fiber is ~340 Å-long and is attached to the baseplate with its N-terminal domain. Here, we report the crystal structures of C-terminal fragments of the R1 and R2 pyocin fibers that comprise the distal, receptor-binding part of the protein. Both proteins are ~240 Å-long homotrimers in which slender rod-like domains are interspersed with more globular domains—two tandem knob domains in the N-terminal part of the fragment and a lectin-like domain at its C-terminus. The putative substrate binding sites are separated by about 100 Å, suggesting that binding of the fiber to the cell surface causes the fiber to adopt a certain orientation relative to the baseplate and this then triggers sheath contraction.

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species

ABSTRACTBacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCE Streptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.

Energetics of Sheath Contraction in Contractile Injection Systems

The process of protein and DNA translocation across lipid membranes is central to the function of any organism. A large class of large multicomponent organelles, such as bacteriophage tails, Type VI Secretion System, R-type pyocins, Serratia antifeeding prophage, and others, translocate their substrates using a rigid tube/contractile sheath mechanism. Functionally and structurally, these ‘contractile injection systems’ resemble a stretched spring (or sheath) wound around a non-contractile tube. The system is locked in a high-energy metastable state by a baseplate structure that plays an important role in sheath assembly and contraction triggering. Upon interaction of the baseplate with a target cell membrane, the sheath contracts and drives the tube through the cell envelope. A full atomic model of the sheath and tube in the extended and contracted state is available for R-type pyocin, one of the simplest representatives of contractile injection systems. The structure suggests that the contraction is accomplished by rigid body rearrangement of sheath subunits and that the transformation is driven by the energy stored in the extended conformation of the sheath during assembly. Other data show that the contraction starts at the baseplate and propagates through the sheath as a wave. The individual subunits’ trajectories are however unknown. We will discuss how we can derive these trajectories from energetics considerations. These finding are important for understanding the substrates that can be translocated by contractile injection systems.

Dynamic Characteristics Analysis of a Seismic Vibrator-Ground Coupling System

A mathematical model to describe the seismic vibrator and ground coupling is proposed based on dynamic analyses of the vibrator baseplate structure and properties of soil. This mathematical model is solved using Newmark’s method. It produces reasonable results and the trend of amplitude envelope follows the experimental data. With this model, characterization of the vibrator-ground coupling is performed. Modal analysis study shows that the motions between the vibrator reaction mass and baseplate experience a combination of a first-order vibration mode and a second-order vibration mode. The corresponding frequencies of two vibration modes are located at 2.77 Hz and 488.7 Hz, respectively. As the frequency goes up, the motion behavior of the reaction mass and baseplate is dominated by the second-order vibration mode. It is realized that the second-order vibration mode is responsible for the phase difference between the input sweep signal and the output force signal. Furthermore, the impact of the coupling system parameters on the vibrator output force is also investigated. It is observed that the vibrator output force decreases as the sweep frequency increases. The weight ratio of the reaction mass and the baseplate has an impact on the vibrator output force.

Molecular assembly and structure of the bacteriophage T4 tail.

The tail of bacteriophage T4 undergoes large structural changes upon infection while delivering the phage genome into the host cell. The baseplate is located at the distal end of the contractile tail and plays a central role in transmitting the signal to the tail sheath that the tailfibers have been adsorbed by a host bacterium. This then triggers the sheath contraction. In order to understand the mechanism of assembly and conformational changes of the baseplate upon infection, we have determined the structure of an in vitro assembled baseplate through the three-dimensional reconstruction of cryo-electron microscopy images to a resolution of 3.8Å from electron micrographs. The atomic structure was fitted to the baseplate structure before and after sheath contraction in order to elucidate the conformational changes that occur after bacteriophage T4 has attached itself to a cell surface. The structure was also used to investigate the protease digestion of the assembly intermediates and the mutation sites of the tail genes, resulting in a number of phenotypes.

The Baseplate of Lactobacillus delbrueckii Bacteriophage Ld17 Harbors a Glycerophosphodiesterase

Glycerophosphodiester phosphodiesterases (GDPDs; EC 3.1.4.46) typically hydrolyze glycerophosphodiesters to sn-glycerol 3-phosphate (Gro3P) and their corresponding alcohol during patho/physiological processes in bacteria and eukaryotes. GDPD(-like) domains were identified in the structural particle of bacterial viruses (bacteriophages) specifically infecting Gram-positive bacteria. The GDPD of phage 17 (Ld17; GDPDLd17), representative of the group b Lactobacillus delbrueckii subsp. bulgaricus (Ldb)-infecting bacteriophages, was shown to hydrolyze, besides the simple glycerophosphodiester, two complex surface-associated carbohydrates of the Ldb17 cell envelope: the Gro3P decoration of the major surface polysaccharide d-galactan and the oligo(glycerol phosphate) backbone of the partially glycosylated cell wall teichoic acid, a minor Ldb17 cell envelope component. Degradation of cell wall teichoic acid occurs according to an exolytic mechanism, and Gro3P substitution is presumed to be inhibitory for GDPDLd17 activity. The presence of the GDPDLd17 homotrimer in the viral baseplate structure involved in phage-host interaction together with the dependence of native GDPD activity, adsorption, and efficiency of plating of Ca(2+) ions supports a role for GDPDLd17 activity during phage adsorption and/or phage genome injection. In contrast to GDPDLd17, we could not identify any enzymatic activity for the GDPD-like domain in the neck passage structure of phage 340, a 936-type Lactococcus lactis subsp. lactis bacteriophage.

Structure of the host-recognition device of Staphylococcus aureus phage ϕ11.

Phages play key roles in the pathogenicity and adaptation of the human pathogen Staphylococcus aureus. However, little is known about the molecular recognition events that mediate phage adsorption to the surface of S. aureus. The lysogenic siphophage ϕ11 infects S. aureus SA113. It was shown previously that ϕ11 requires α- or β-N-acetylglucosamine (GlcNAc) moieties on cell wall teichoic acid (WTA) for adsorption. Gp45 was identified as the receptor binding protein (RBP) involved in this process and GlcNAc residues on WTA were found to be the key component of the ϕ11 receptor. Here we report the crystal structure of the RBP of ϕ11, which assembles into a large, multidomain homotrimer. Each monomer contains a five-bladed propeller domain with a cavity that could accommodate a GlcNAc moiety. An electron microscopy reconstruction of the ϕ11 host adhesion component, the baseplate, reveals that six RBP trimers are assembled around the baseplate core. The Gp45 and baseplate structures provide insights into the overall organization and molecular recognition process of the phage ϕ11 tail. This assembly is conserved among most glycan-recognizing Siphoviridae, and the RBP orientation would allow host adhesion and infection without an activation step.

Structure of the T4 baseplate and its function in triggering sheath contraction.

Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.

Alternative Excitonic Structure in the Baseplate (BChl a-CsmA Complex) of the Chlorosome from Chlorobaculum tepidum.

In the photosynthetic green sulfur bacterium Chlorobaculum tepidum, the baseplate mediates excitation energy transfer from the light-harvesting chlorosome to the Fenna-Matthews-Olson (FMO) complex and subsequently toward the reaction center (RC). Literature data suggest that the baseplate is a 2D lattice of BChl a-CsmA dimers. However, recently, it has been proposed, using 2D electronic spectroscopy (2DES) at 77 K, that at least four excitonically coupled BChl a are in close contact within the baseplate structure [ Dostál , J. ; et al., J. Phys. Chem. Lett. 2014 , 5 , 1743 ]. This finding is tested via hole burning (HB) spectroscopy (5 K). Our results indicate that the four excitonic states identified by 2DES likely correspond to contamination of the baseplate with the FMO antenna and possibly the RC. In contrast, HB reveals a different excitonic structure of the baseplate chromophores, where excitation is transferred to a localized trap state near 818 nm via exciton hopping, which leads to emission near 826 nm.

Novel Giant Siphovirus from Bacillus anthracis Features Unusual Genome Characteristics

Here we present vB_BanS-Tsamsa, a novel temperate phage isolated from Bacillus anthracis, the agent responsible for anthrax infections in wildlife, livestock and humans. Tsamsa phage is a giant siphovirus (order Caudovirales), featuring a long, flexible and non-contractile tail of 440 nm (not including baseplate structure) and an isometric head of 82 nm in diameter. We induced Tsamsa phage in samples from two different carcass sites in Etosha National Park, Namibia. The Tsamsa phage genome is the largest sequenced Bacillus siphovirus, containing 168,876 bp and 272 ORFs. The genome features an integrase/recombinase enzyme, indicative of a temperate lifestyle. Among bacterial strains tested, the phage infected only certain members of the Bacillus cereus sensu lato group (B. anthracis, B. cereus and B. thuringiensis) and exhibited moderate specificity for B. anthracis. Tsamsa lysed seven out of 25 B. cereus strains, two out of five B. thuringiensis strains and six out of seven B. anthracis strains tested. It did not lyse B. anthracis PAK-1, an atypical strain that is also resistant to both gamma phage and cherry phage. The Tsamsa endolysin features a broader lytic spectrum than the phage host range, indicating possible use of the enzyme in Bacillus biocontrol.

Dynamic behaviour and active sound radiation control of a two-stage vibration isolation system equipped on a flexible plate

This paper presents a general model of a two-stage vibration isolation system involving a flexible base plate with arbitrary boundary conditions. The dynamic behavior of the coupling system was obtained by using an impedance method in which the impedance matrix of the base plate was derived from a combined use of an improved Fourier series expansion and the Rayleigh-Ritz method. Subsequently, with the purpose of attenuation of sound radiation from the base plate structure, the optimal force information is formulated under various active control strategies including: 1) minimizing the total vibratory power of the coupled structure, 2) minimizing the sound power radiation from the base plate, 3) minimizing the vibratory power transmitted to the base plate, and 4) minimizing the mean square velocities at the isolator locations on the flexible plate. Numerical results were presented and discussed in detail. Finally, some concluding remarks are made.