Author response: Molecular dynamics-based refinement and validation for sub-5 Å cryo-electron microscopy maps

Advances in the molecular dynamics flexible fitting method for cryo-EM modeling

Advances in cryoelectron microscopy (cryo-EM) have revolutionized the structural investigation of large macromolecular assemblies. In this review, we first provide a broad overview of modeling methods used for flexible fitting of molecular models into cryo-EM density maps. We give special attention to approaches rooted in molecular simulations-atomistic molecular dynamics and Monte Carlo. Concise descriptions of the methods are given along with discussion of their advantages, limitations, and most popular alternatives. We also describe recent extensions of the widely used molecular dynamics flexible fitting (MDFF) method and discuss how different model-building techniques could be incorporated into new hybrid modeling schemes and simulation workflows. Finally, we provide two illustrative examples of model-building and refinement strategies employing MDFF, cascade MDFF, and RosettaCM. These examples come from recent cryo-EM studies that elucidated transcription preinitiation complexes and shed light on the functional roles of these assemblies in gene expression and gene regulation.

Hybrid structure fitting methods combine data from cryo-electron microscopy and X-ray crystallography with molecular dynamics simulations for the determination of all-atom structures of large biomolecular complexes. Evaluating the quality-of-fit obtained from hybrid fitting is computationally demanding, particularly in the context of a multiplicity of structural conformations that must be evaluated. Existing tools for quality-of-fit analysis and visualization have previously targeted small structures and are too slow to be used interactively for large biomolecular complexes of particular interest today such as viruses or for long molecular dynamics trajectories as they arise in protein folding. We present new data-parallel and GPU-accelerated algorithms for rapid interactive computation of quality-of-fit metrics linking all-atom structures and molecular dynamics trajectories to experimentally-determined density maps obtained from cryo-electron microscopy or X-ray crystallography. We evaluate the performance and accuracy of the new quality-of-fit analysis algorithms vis-à-vis existing tools, examine algorithm performance on GPU-accelerated desktop workstations and supercomputers, and describe new visualization techniques for results of hybrid structure fitting methods.

Striated muscle contraction is regulated in a calcium-dependent manner through dynamic motions of the tropomyosin/troponin polymer, a multicomponent complex wrapped around actin-containing thin filaments. Tropomyosin/troponin sterically blocks myosin-binding at low-calcium concentrations but moves to expose myosin-binding sites at high-calcium concentrations leading to force development. Understanding the key intermolecular interactions that define these dynamic motions will promote our understanding of mutation-induced contractile dysfunction that eventually leads to hypertrophic cardiomyopathy, dilated cardiomyopathy, and skeletal myopathies. Advancements in cryoelectron microscopy (cryoEM) have resulted in a partial elucidation of structures of the thin filament, revealing many atomic-level interactions between the component proteins and critical calcium-dependent conformational alterations. However, building models at the resolutions achieved can be challenging since landmarks in the maps are often missing or ambiguous. Therefore, current computational analyses including de novo structure prediction, protein-protein docking, molecular dynamics flexible fitting, and molecular dynamics simulations are needed to ensure good quality models. We review here our efforts to model the troponin T domain spanning the head-to-tail overlap domain of tropomyosin, improving previous models. Next, we refined the published cryoEM modeled structures, which had mistakenly compressed alpha helices, with a model that has expected helical parameters while matching densities in the cryoEM volume. Lastly, we used this model to reinterpret the interactions between tropomyosin and troponin I showing key features that hold the tropomyosin cable in its low-calcium, sterically blocking position. These revised thin filament models show improved intermolecular interactions in the key low- and high-calcium regulatory states, providing novel insights into function.

Human α-defensins are proteins of the innate immune system that suppress viral and bacterial infections by multiple mechanisms including membrane disruption. For viruses that lack envelopes, such as human adenovirus (HAdV), other, less well defined, mechanisms must be involved. A previous structural study on the interaction of an α-defensin, human α-defensin 5 (HD5), with HAdV led to a proposed mechanism in which HD5 stabilizes the vertex region of the capsid and blocks uncoating steps required for infectivity. Studies with virus chimeras comprised of capsid proteins from sensitive and resistant serotypes supported this model. To further characterize the critical binding site, we determined subnanometer resolution cryo-electron microscopy (cryoEM) structures of HD5 complexed with both neutralization-sensitive and -resistant HAdV chimeras. Models were built for the vertex regions of these chimeras with monomeric and dimeric forms of HD5 in various initial orientations. CryoEM guided molecular dynamics flexible fitting (MDFF) was used to restrain the majority of the vertex model in well-defined cryoEM density. The RGD-containing penton base loops of both the sensitive and resistant virus chimeras are predicted to be intrinsically disordered, and little cryoEM density is observed for them. In simulations these loops from the sensitive virus chimera, interact with HD5, bridge the penton base and fiber proteins, and provides significant stabilization with a three-fold increase in the intermolecular nonbonded interactions of the vertex complex. In the case of the resistant virus chimera, simulations revealed fewer bridging interactions and reduced stabilization by HD5. This study implicates a key dynamic region in mediating a stabilizing interaction between a viral capsid and a protein of the innate immune system with potent anti-viral activity.

Several bacterial solute transport mechanisms involve members of the periplasmic binding protein (PBP) superfamily that bind and deliver ligand to integral membrane transport proteins in the ATP-binding cassette, tripartite tricarboxylate transporter, or tripartite ATP-independent (TRAP) families. PBPs involved in ATP-binding cassette transport systems have been well characterized, but only a few PBPs involved in TRAP transport have been studied. We have measured the thermal stability, determined the oligomerization state by small angle x-ray scattering, and solved the x-ray crystal structure to 1.9 A resolution of a TRAP-PBP (open reading frame tm0322) from the hyperthermophilic bacterium Thermotoga maritima (TM0322). The overall fold of TM0322 is similar to other TRAP transport related PBPs, although the structural similarity of backbone atoms (2.5-3.1 A root mean square deviation) is unusually low for PBPs within the same group. Individual monomers within the tetrameric asymmetric unit of TM0322 exhibit high root mean square deviation (0.9 A) to each other as a consequence of conformational heterogeneity in their binding pockets. The gel filtration elution profile and the small angle x-ray scattering analysis indicate that TM0322 assembles as dimers in solution that in turn assemble into a dimer of dimers in the crystallographic asymmetric unit. Tetramerization has been previously observed in another TRAP-PBP (the Rhodobacter sphaeroides alpha-keto acid-binding protein) where quaternary structure formation is postulated to be an important requisite for the transmembrane transport process.

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

Three-Dimensional Quantitative Comparative Analysis of Trapezial-Metacarpal Joint Surface Curvatures in Human Populations

Force and vibration generated in apical direction by three endodontic files of different kinematics during simulated canal preparation: An in vitro analytical study.

The aim of the present research is to calculate the Relative Accuracy (R.A) and Root Mean Square Error (RMSE) for the location of Technical Institute, Mosul in Nineveh Governorate, Iraq. at the northern part of Mosul city by using surveying Instruments (GPS 1230 , Total Station 06 & Level NAK2), to calculate the coordinates and elevations of (16) Ground Control Points which covered the study area. The field data, remote sensing techniques and GIS software (Arc Map GIS10.3) are used to check the digital map of study area that helped the administrator to take the correct decision, determining positions of the future expansionist for buildings, roads, measuring lengths, areas and there coordinates, directions as well as the total costs estimation needed for any development from the final map. The gradient percentage between the highest and lowest points is calculated to be (9.954%). Relative accuracy for the based measured points (R.A) = 1:60000), and Root Mean Square error in Easting coordinates (RMSE) = ± 0.102m, in Northing coordinates (RMSN) = ± 0.096m, the Root Mean Square for the resultant (RMSR) = ± 0.099m, and Root Mean Square for Elevations ( RMSRL)= ± 0.005m. We Matched and correlated (10) measured points from study area with their graphic data positions on the aerial photo, determining the offsets for position and elevations as follows: (RMSE = ± 0.076m), (RMSN = ± 0.125m), (RMSR = ± 0.088m), and (Root Mean Square for Elevations = RMSRL= ± 0.045m). This research enable us after checking the results to produce new maps from Aerial photograph or Digital Image to use it for land uses and future extensions in whole study area.

Soil Roughness Measuring System Combined With Image Processing

Radar antennas in the open air are affected by dynamic wind loading during azimuthal scanning. In this state, the acquisition and prediction of the aerodynamic load is the focus of the structure design for the antenna. We performed wind tunnel force tests on flat plate antennas with different aspect ratios. The changes in mean, maximum, and root mean square values of the aerodynamic coefficients relative to the antenna aspect ratio and reduced frequency are given for the first time. The effects of the antenna aspect ratio and reduced frequency on aerodynamic characteristics are analyzed. The experimental results indicate that the magnitude of the azimuthal moment is related to the antenna aspect ratio and the reduced frequency. It is worth noting that the antenna aspect ratio has a significant impact on the mean, maximum, and root mean square values of dynamic aerodynamic coefficients. The mean, maximum, and root mean square values of the rolling moment coefficient, azimuth moment coefficient, and pitching moment coefficient monotonously decrease with the increase in aspect ratio. In addition, the influence of reduced frequency on the dynamic aerodynamic coefficient is closely related to the antenna aspect ratio. Under the same antenna aspect ratio, increasing the reduced frequency will increase the mean, maximum, and root mean square values of the aerodynamic coefficient. For flat plate antennas with different aspect ratios, at the condition of increasing the aspect ratios, the mean, maximum, and root mean square values of aerodynamic coefficients do not change significantly with the change of reduced frequency.

Proteins are the molecules carry out the vital functions and make more than the half of dry weight in every cell. Protein in nature folds into a unique and energetically favorable 3-Dimensional (3-D) structure which is critical and unique to its biological function. In contrast to other methods for protein structure determination, Electron Cryo-rricroscopy (CryoEM) is able to produce volumetric maps of proteins that are poorly soluble, large and hard to crystallize. Furthermore, it studies the proteins in their native environment. Unfortunately, the volumetric maps generated by current advances in CryoEM technique produces protein maps at medium resolution about (~5 to 10A) in which it is hard to determine the atomic-structure of the protein. However, the resolution of the volumetric maps is improving steadily, and recent works could obtain atomic models at higher resolutions (~3A). De novo protein modeling is the process of building the structure of the protein using its CryoEM volumetric map. Thereupon, the volumetric maps at medium resolution generated by CryoEM technique proposed a new challenge. At the medium resolution, the location and orientation of secondary structure elements (SSE) can be visually and computationally identified. However, the order and direction (called protein topology) of the SSEs detected from the CryoEM volumetric map are not visible. In order to determine the protein structure, the topology of the SSEs has to be figured out and then the backbone can be built. Consequently, the topology problem has become a bottle neck for protein modeling using CryoEM In this dissertation, we focus to establish an effective computational framework to derive the atomic structure of a protein from the medium resolution CryoEM volumetric maps. This framework includes a topology graph component to rank effectively the topologies of the SSEs and a model building component. In order to generate the small subset of candidate topologies, the problem is translated into a layered graph representation. We developed a dynamic programming algorithm (TopoDP) for the new representation to overcome the problem of large search space. Our approach shows the improved accuracy, speed and memory use when compared with existing methods. However, the generating of such set was infeasible using a brute force method. Therefore, the topology graph component effectively reduces the topological space using the geometrical features of the secondary structures through a constrained K-shortest paths method in our layered graph. The model building component involves the bending of a helix and the loop construction using skeleton of the volumetric map. The forward-backward CCD is applied to bend the helices and model the loops.

Fexofenadine is a recommended in vivo probe drug for phenotyping P-glycoprotein (P-gp) and organic anion transporting polypeptide (OATP) 1B1/3 transporter activities. This study evaluated a limited sampling strategy using a population pharmacokinetic approach to estimate plasma fexofenadine exposure as an index of P-gp and OATP activities. In a previous study, a single oral dose of fexofenadine (120 mg) was administered alone or in combination with grapefruit juice, Panax ginseng , or Echinacea purpurea to healthy adult participants. Serial plasma samples were collected up to 72 hours after administration and fexofenadine concentrations were measured. A population pharmacokinetic model was developed using nonlinear mixed-effects modeling. Limited sampling models (LSMs) using single and 2-timepoint fexofenadine concentrations were compared with full profiles from intense sampling using empirical Bayesian post hoc estimations of systemic exposure derived from the population pharmacokinetic model. Predefined criteria for LSM selection and validation included a coefficient of determination (R 2 ) ≥ 0.90, relative percent mean prediction error ≥ -5 to ≤5%, relative percent mean absolute error ≤ 10%, and relative percent root mean square error ≤ 15%. Fexofenadine concentrations (n = 1520) were well described using a 2-compartment model. Grapefruit juice decreased the relative oral bioavailability of fexofenadine by 25%, whereas P. ginseng and E. purpurea had no effect. All the evaluated single timepoint fexofenadine LSMs showed unacceptable percent mean prediction error, percent mean absolute error, and/or percent root mean square error. Although adding a second time point improved precision, the predefined criteria were not met. Identifying novel fexofenadine LSMs to estimate P-gp and OATP1B1/3 activities in healthy adults for future transporter-mediated drug-drug interaction studies remains elusive.

There is a lack of experimental structural information about folding intermediates of multidomain proteins. Tick carboxypeptidase inhibitor (TCI) is a small, disulfide-rich protein consisting of two domains that fold and unfold autonomously through the formation of two major intermediates, IIIa and IIIb. Each intermediate contains three native disulfide bonds in one domain and six free cysteines in the other domain. Here we have determined the NMR structures of these two intermediates trapped and isolated at acidic pH in which they are stable and compared their structures with that of the native protein analyzed under the same conditions. Both IIIa and IIIb were found to contain a folded region that corresponds to the N- and C-terminal domains of TCI, respectively, with structures very similar to the corresponding regions of the native protein. The remainder of the polypeptide chains of the intermediates was shown to be unfolded in a random coil conformation. Solvent exchange measurements further indicated that the two protein domains are not completely independent, but affect each other in terms of dynamics and stability, in agreement with reported inhibitory activity data. The derived results provide structural evidence for symmetric TCI folding and unfolding mechanisms that converge in IIIa and IIIb and reveal the structural basis that accounts for the strong and simultaneous accumulation of both intermediates. Altogether, this work has important implications for a better understanding of the folding mechanisms of multidomain, disulfide-rich proteins.