Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ≅ 15 μs relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants.

The past three decades of hybrid modeling has seen molecular dynamics (MD) simulations leverage NMR, X-ray crystallography and cryo EM data for the determination of medium-to high-resolution structures atomic structures. However, all these approaches, including our popular Molecular Dynamics Flexible Fitting (MDFF), and its various extensions work under the conventional molecular replacement paradigm, whereby any initial search model is morphed to satiate the data-imposed constraints. As a natural consequence, quality of the determined model remains heavily biased by choices of the initial model. Here, we deliver a novel modeling pipeline that iteratively combines minimum spanning tree-based backbone tracing tool (MAIN-MAST), Bayesian-likelihood based protein-folding methodology (MELD), and a resolution exchange-based fitting protocol (ReMDFF). Starting from only sequence information, the algorithm places C-alpha atoms into the density, fits a random coil to this C-alpha trace, generates protein secondary structures on-the-fly, and exhaustively samples the backbone and side chain geometries to deliver a refined model. Overcoming limitations of traditional approaches, the need for an initial model or homology information is completely subsided, and de novo modeling is now made feasible even at low resolutions. Available on cloud computing resources, the method is equally applicable for determining globular and transmembrane protein structures, as demonstrated for ubiquitin, TRPV1 and Magnesium-channel CorA (all in the 3-5 Å resolution range), and the de novo atomic structure of a lipoprotein flp3. Automating the segmentation of biological structures at the cell and organelle level is essential for making efficient use of 3-D scanning electron microscopy (SEM) technologies, such as serial block-face (SBF)-SEM and focused ion beam (FIB)-SEM. Currently, automated segmentation using convolutional neural networks (CNNs) typically addresses a specific biological system, and it is necessary to train a CNN for each new problem. However, addressing each problem individually imposes impractical labor requirements for machine learning developers, and requires large amounts of annotated training data, which are costly to produce. Here, we present our work on reducing this burden by applying transfer learning techniques to neural network architectures to solve instance segmentation and semantic segmentation problems for multiple biological datasets imaged via 3-D SEM. We are able to train instance segmentation networks to detect and localize objects across different cells and tissues. By sharing semantic segmentation network pathways between biological systems, we are able to reduce the amount of training data required for effective segmentation of new EM datasets. Cryo-electron microscopy (CryoEM) has become a routine tool to determine the structure of protein complexes, alongside X-ray crystallography (XRD) and Nuclear magnetic resonance spectroscopy (NMR). However, several structures determined employing CryoEM and deposite...

The Escherichia coli UvrD protein is a 3 0 to 5 0 superfamily 1 DNA helicase that functions in nucleotide excision repair and methyl-directed mismatch repair of DNA, as well as DNA replication of certain plasmids. UvrD uses the energy of ATP binding and hydrolysis to unwind double-stranded DNA (dsDNA) and translocate along single-stranded DNA (ssDNA) with biased 3 0 to 5 0 directionality. Single turnover pre-steady state DNA unwinding kinetics experiments have shown that the UvrD dimer is the active form of the helicase in vitro, although a UvrD monomer can translocate along ssDNA with the same directionality as used in unwinding. Crystal structures show that UvrD can exist in two dramatically different conformations, ''open'' in the apo state and ''closed'' when forming a complex with a 3 0 -ssDNA-dsDNA junction. The rotational orientations of the 2B domain differ in these two states by about 100 degrees. To study the conformational changes of the 2B domain, double cysteine mutants with one pair on 1B and 2B domains and another pair on 2A and 2B domains were constructed and labeled with a mixture of donor-acceptor fluorophores such that the movement of 2B domain results in either an increase or a decrease in FRET, depending on the positions of the labeled fluorophores. Our ensemble studies show that the 2B domain is in the closed conformation at low salt and swivels open at high salt in the absence of DNA. The open and closed conformations are in dynamic equilibrium. The binding of UvrD to ssDNA induces the open conformation of the 2B domain. The swiveling of the 2B domain is also coordinated with ATP binding and hydrolysis.

Voltage-gated sodium channels are transmembrane proteins involved in generating action potentials for nerve signalling and muscle contractions. They are of a great deal of interest, as mutations in voltage-gated sodium channels are responsible for a variety of disorders, including epilepsy and chronic pain. Designing better inhibitors and treatments, including ones which are