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Automated assembly machines operate continuously to achieve high production rates. Continuous operation increases the potential for faults such as jams, missing parts, and electromechanical failures of subsystems. The goal of this research project was to develop and validate a machine vision inspection (MVI) system to detect and classify multiple faults using a single camera as a sensor. An industrial automated O-ring assembly machine that places O-rings on to continuously moving plastic carriers at a rate of over 100 assemblies per minute was modified to serve as the test apparatus. An industrial camera with LED panel lights for illumination was used to acquire videos of the machine’s operation. A programmable logic controller (PLC) with a human-machine interface (HMI) allowed for the generation of faults in a controlled fashion. Three MVI methods, based on computer vision techniques available in the literature, were developed for this application. The methods used features extracted from the videos to classify the machine’s condition. The first method was based on Gaussian mixture models (GMMs); the second method used an optical flow approach; and the third method was based on running average and morphological image processing operations. In order to provide a single metric to quantify relative performance, a machine vision performance index (MVPI) was developed with five measures of performance: accuracy, processing time, speed of response, robustness against noise, and ease of tuning. The MVPI for the three MVI methods is reported along with the significance of the results.

Utilizing large numbers of microrobots to heterogeneously integrate small devices to build advanced structures has long been a goal in the field of manufacturing automation. In this paper, we demonstrate a novel milli-scale robotic assembly machine with highly parallel capabilities and assisted with a programmable magnetic field. The prototype machine consists of a 16 × 16 array of electromagnets. Using this machine, we have successfully demonstrated the manipulation of up to nine milli-scale robots simultaneously. Moreover, two microrobots have been operated to demonstrate the proof of concept of two simultaneous pick-and-place light-emitting diodes (LEDs). The design and modeling of the microrobots is discussed.

Mitochondria, chloroplasts and Gram-negative bacteria are encased in a double layer of membranes. The outer membrane contains proteins with a β-barrel structure 1 , 2 . β-Barrels are sheets of β-strands wrapped into a cylinder, in which the first strand is hydrogen-bonded to the final strand. Conserved multi-subunit molecular machines fold and insert these proteins into the outer membrane 3 – 5 . One subunit of the machines is itself a β-barrel protein that has a central role in folding other β-barrels. In Gram-negative bacteria, the β-barrel assembly machine (BAM) consists of the β-barrel protein BamA, and four lipoproteins 5 – 8 . To understand how the BAM complex accelerates folding without using exogenous energy (for example, ATP) 9 , we trapped folding intermediates on this machine. Here we report the structure of the BAM complex of Escherichia coli folding BamA itself. The BamA catalyst forms an asymmetric hybrid β-barrel with the BamA substrate. The N-terminal edge of the BamA catalyst has an antiparallel hydrogen-bonded interface with the C-terminal edge of the BamA substrate, consistent with previous crosslinking studies 10 – 12 ; the other edges of the BamA catalyst and substrate are close to each other, but curl inward and do not pair. Six hydrogen bonds in a membrane environment make the interface between the two proteins very stable. This stability allows folding, but creates a high kinetic barrier to substrate release after folding has finished. Features at each end of the substrate overcome this barrier and promote release by stepwise exchange of hydrogen bonds. This mechanism of substrate-assisted product release explains how the BAM complex can stably associate with the substrate during folding and then turn over rapidly when folding is complete. Cryo-electron microscopy structures of a folding intermediate on the BAM complex of Escherichia coli reveal how interactions between the BamA catalyst and substrate permit stable association during folding, followed by rapid turnover.

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the β-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K . BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K. Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

Type II DNA topoisomerases are ubiquitous enzymes with essential functions in DNA replication, recombination and transcription. They change DNA topology by forming a transient covalent cleavage complex with a gate-DNA duplex that allows transport of a second duplex though the gate. Despite its biological importance and targeting by anticancer and antibacterial drugs, cleavage complex formation and reversal is not understood for any type II enzyme. To address the mechanism, we have used X-ray crystallography to study sequential states in the formation and reversal of a DNA cleavage complex by topoisomerase IV from Streptococcus pneumoniae, the bacterial type II enzyme involved in chromosome segregation. A high resolution structure of the complex captured by a novel antibacterial dione reveals two drug molecules intercalated at a cleaved B-form DNA gate and anchored by drug-specific protein contacts. Dione release generated drug-free cleaved and resealed DNA complexes in which the DNA gate instead adopts an unusual A/B-form helical conformation with a Mg(2+) ion repositioned to coordinate each scissile phosphodiester group and promote reversible cleavage by active-site tyrosines. These structures, the first for putative reaction intermediates of a type II topoisomerase, suggest how a type II enzyme reseals DNA during its normal reaction cycle and illuminate aspects of drug arrest important for the development of new topoisomerase-targeting therapeutics.

The outer membrane (OM) of gram-negative bacteria confers innate resistance to toxins and antibiotics. Integral β-barrel outer membrane proteins (OMPs) function to establish and maintain the selective permeability of the OM. OMPs are assembled into the OM by the β-barrel assembly machine (BAM), which is composed of one OMP—BamA—and four lipoproteins—BamB, C, D, and E. BamB, C, and E can be removed individually with only minor effects on barrier function; however, depletion of either BamA or BamD causes a global defect in OMP assembly and results in cell death. We have identified a gain-of-function mutation, bamAE470K ss, that bypasses the requirement for BamD. Although bamD::kan bamAE470K cells exhibit growth and OM barrier defects, they assemble OMPs with surprising robustness. Our results demonstrate that BamD does not play a catalytic role in OMP assembly, but rather functions to regulate the activity of BamA.

Atomic-resolution X-ray crystallography, functional analyses, and molecular dynamics simulations suggest a novel mechanism for the regulation of water flux through the yeast Aqy1 water channel. Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 Å resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity. All living organisms must regulate precisely the flow of water into and out of cells in order to maintain cell shape and integrity. Proteins of one family, the aquaporins, are found in virtually every living organism and play a major role in maintaining water homeostasis by acting as regulated water channels. Here we describe the first crystal structure of a yeast aquaporin, Aqy1, at 1.15 Å resolution, which represents the highest resolution structural data obtained to date for a membrane protein. Using this structural information, we address an outstanding biological question surrounding yeast aquaporins: what is the functional role of the amino-terminal extension that is characteristic of yeast aquaporins? Our structural data show that the amino terminus of Aqy1 fulfills a novel gate-like function by folding to form a cytoplasmic helical bundle with a tyrosine residue entering the water channel and occluding the cytoplasmic entrance. Molecular dynamics simulations and functional studies in combination with site-directed mutagenesis suggest that water flow is regulated through a combination of mechanosensitive gating and post-translational modifications such as phosphorylation. Our study therefore provides insight into a unique mechanism for the regulation of water flux in yeast.

Intermediate filaments (IFs), in addition to microtubules and microfilaments, are one of the three major components of the cytoskeleton in eukaryotic cells, playing a vital role in mechanotransduction and in providing mechanical stability to cells. Despite the importance of IF mechanics for cell biology and cell mechanics, the structural basis for their mechanical properties remains unknown. Specifically, our understanding of fundamental filament properties, such as the basis for their great extensibility, stiffening properties, and their exceptional mechanical resilience remains limited. This has prevented us from answering fundamental structure-function relationship questions related to the biomechanical role of intermediate filaments, which is crucial to link structure and function in the protein material's biological context. Here we utilize an atomistic-level model of the human vimentin dimer and tetramer to study their response to mechanical tensile stress, and describe a detailed analysis of the mechanical properties and associated deformation mechanisms. We observe a transition from alpha-helices to beta-sheets with subsequent interdimer sliding under mechanical deformation, which has been inferred previously from experimental results. By upscaling our results we report, for the first time, a quantitative comparison to experimental results of IF nanomechanics, showing good agreement. Through the identification of links between structures and deformation mechanisms at distinct hierarchical levels, we show that the multi-scale structure of IFs is crucial for their characteristic mechanical properties, in particular their ability to undergo severe deformation of approximately 300% strain without breaking, facilitated by a cascaded activation of a distinct deformation mechanisms operating at different levels. This process enables IFs to combine disparate properties such as mechanosensitivity, strength and deformability. Our results enable a new paradigm in studying biological and mechanical properties of IFs from an atomistic perspective, and lay the foundation to understanding how properties of individual protein molecules can have profound effects at larger length-scales.

The high-resolution structure of the rotor ring from alkaliphilic Bacillus pseudofirmus OF4 reveals a new type of ion binding in F1Fo-ATP synthases. We solved the crystal structure of a novel type of c-ring isolated from Bacillus pseudofirmus OF4 at 2.5 Å, revealing a cylinder with a tridecameric stoichiometry, a central pore, and an overall shape that is distinct from those reported thus far. Within the groove of two neighboring c-subunits, the conserved glutamate of the outer helix shares the proton with a bound water molecule which itself is coordinated by three other amino acids of outer helices. Although none of the inner helices contributes to ion binding and the glutamate has no other hydrogen bonding partner than the water oxygen, the site remains in a stable, ion-locked conformation that represents the functional state present at the c-ring/membrane interface during rotation. This structure reveals a new, third type of ion coordination in ATP synthases. It appears in the ion binding site of an alkaliphile in which it represents a finely tuned adaptation of the proton affinity during the reaction cycle. Like the wind turbines that generate electricity, the F1Fo-ATP synthases are natural “ion turbines” each made up of a stator and a rotor that turns, when driven by a flow of ions, to generate the cell's energy supply of ATP. The Fo motor rotates by reversible binding and release of coupling ions that flow down the electrochemical ion gradient across the cytoplasmic cell membrane (in the case of bacteria) or intracellular organelle membranes (in the case of eukaryotic cells). Here, we present the structure of a rotor (c-)ring from a Bacillus species (B. pseudofirmus OF4) determined at high-resolution by X-ray crystallography. This bacterium prefers alkaline environments where the concentration of protons (H+) is lower outside than inside the cell – the inverse of the situation usually found in organisms that prefer neutral or acidic environments. The amino acid sequence of the protein subunits in this rotor, nevertheless, has features common to an important group of ATP synthases in organisms from bacteria to man. The structure reveals a new type of ion binding in which a protonated glutamate residue in the protein associates with a water molecule. This finding raises the possibility considered by Nobel laureate Paul Boyer several decades ago that a hydronium ion (a protonated water molecule, H3O+), rather than a proton alone, might be the coupling species that energizes ATP synthesis. Also, it demonstrates the finely tuned adaptation of ATP synthase rotor rings and their ion-binding sites to the specific requirements of different organisms.

BamA is the central component of the essential β-barrel assembly machine (BAM), a conserved multi-subunit complex that dynamically inserts and folds β-barrel proteins into the outer membrane of Gram-negative bacteria. Despite recent advances in our mechanistic and structural understanding of BamA, there are few potent and selective tool molecules that can bind to and modulate BamA activity. Here, we explored in vitro selection methods and different BamA/BAM protein formulations to discover peptide macrocycles that kill Escherichia coli by targeting extreme conformational states of BamA. Our studies show that Peptide Targeting BamA-1 (PTB1) targets an extracellular divalent cation-dependent binding site and locks BamA into a closed lateral gate conformation. By contrast, PTB2 targets a luminal binding site and traps BamA into an open lateral gate conformation. Our results will inform future antibiotic discovery efforts targeting BamA and provide a template to prospectively discover modulators of other dynamic integral membrane proteins. BamA carries out the essential process of folding outer membrane β-barrels in Gram-negative bacteria and is a potential antibiotic target. Here, the authors discover macrocyclic peptide inhibitors that trap BamA in distinct structural conformations.