Feasibility and Accuracy of Thoracolumbar Pedicle Screw Placement Using an Augmented Reality Head Mounted Device.

BACKGROUND: To investigate the accuracy of augmented reality (AR) navigation using the Magic Leap head mounted device (HMD), pedicle screws were minimally invasively placed in four spine phantoms. METHODS: AR navigation provided by a combination of a conventional navigation system integrated with the Magic Leap head mounted device (AR-HMD) was used. Forty-eight screws were planned and inserted into Th11-L4 of the phantoms using the AR-HMD and navigated instruments. Postprocedural CT scans were used to grade the technical (deviation from the plan) and clinical (Gertzbein grade) accuracy of the screws. The time for each screw placement was recorded. RESULTS: The mean deviation between navigation plan and screw position was 1.9 ± 0.7 mm (1.9 [0.3-4.1] mm) at the entry point and 1.4 ± 0.8 mm (1.2 [0.1-3.9] mm) at the screw tip. The angular deviation was 3.0 ± 1.4° (2.7 [0.4-6.2]°) and the mean time for screw placement was 130 ± 55 s (108 [58-437] s). The clinical accuracy was 94% according to the Gertzbein grading scale. CONCLUSION: The combination of an AR-HMD with a conventional navigation system for accurate minimally invasive screw placement is feasible and can exploit the benefits of AR in the perspective of the surgeon with the reliability of a conventional navigation system.

Force sensing by the vascular protein von Willebrand factor is tuned by a strong intermonomer interaction.

The large plasma glycoprotein von Willebrand factor (VWF) senses hydrodynamic forces in the bloodstream and responds to elevated forces with abrupt elongation, thereby increasing its adhesiveness to platelets and collagen. Remarkably, forces on VWF are elevated at sites of vascular injury, where VWF's hemostatic potential is important to mediate platelet aggregation and to recruit platelets to the subendothelial layer. Adversely, elevated forces in stenosed vessels lead to an increased risk of VWF-mediated thrombosis. To dissect the remarkable force-sensing ability of VWF, we have performed atomic force microscopy (AFM)-based single-molecule force measurements on dimers, the smallest repeating subunits of VWF multimers. We have identified a strong intermonomer interaction that involves the D4 domain and critically depends on the presence of divalent ions, consistent with results from small-angle X-ray scattering (SAXS). Dissociation of this strong interaction occurred at forces above [Formula: see text]50 pN and provided [Formula: see text]80 nm of additional length to the elongation of dimers. Corroborated by the static conformation of VWF, visualized by AFM imaging, we estimate that in VWF multimers approximately one-half of the constituent dimers are firmly closed via the strong intermonomer interaction. As firmly closed dimers markedly shorten VWF's effective length contributing to force sensing, they can be expected to tune VWF's sensitivity to hydrodynamic flow in the blood and to thereby significantly affect VWF's function in hemostasis and thrombosis.

Time-Resolved Small-Angle X-ray Scattering Reveals Millisecond Transitions of a DNA Origami Switch.

Self-assembled DNA structures enable creation of specific shapes at the nanometer-micrometer scale with molecular resolution. The construction of functional DNA assemblies will likely require dynamic structures that can undergo controllable conformational changes. DNA devices based on shape complementary stacking interactions have been demonstrated to undergo reversible conformational changes triggered by changes in ionic environment or temperature. An experimentally unexplored aspect is how quickly conformational transitions of large synthetic DNA origami structures can actually occur. Here, we use time-resolved small-angle X-ray scattering to monitor large-scale conformational transitions of a two-state DNA origami switch in free solution. We show that the DNA device switches from its open to its closed conformation upon addition of MgCl2 in milliseconds, which is close to the theoretical diffusive speed limit. In contrast, measurements of the dimerization of DNA origami bricks reveal much slower and concentration-dependent assembly kinetics. DNA brick dimerization occurs on a time scale of minutes to hours suggesting that the kinetics depend on local concentration and molecular alignment.

Quantifying the influence of the ion cloud on SAXS profiles of charged proteins.

Small-angle X-ray scattering (SAXS) is a popular experimental technique used to obtain structural information on biomolecules in solution. SAXS is sensitive to the overall electron density contrast between the biomolecule and the buffer, including contrast contributions from the hydration layer and the ion cloud. This property may be used advantageously to probe the properties of the ion cloud around charged biomolecules. However, in turn, contributions from the hydration layer and ion cloud may complicate the interpretation of the data, because these contributions must be modelled during structure validation and refinement. In this work, we quantified the influence of the ion cloud on SAXS curves of two charged proteins, bovine serum albumin (BSA) and glucose isomerase (GI), solvated in five different alkali chloride buffers of 100 mM or 500 mM concentrations. We compared three computational methods of varying physical detail, for deriving the ion cloud effect on the radius of gyration Rg of the proteins, namely (i) atomistic molecular dynamics simulations in conjunction with explicit-solvent SAXS calculations, (ii) non-linear Poisson-Boltzmann calculations, and (iii) a simple spherical model in conjunction with linearized Poisson-Boltzmann theory. The calculations for BSA are validated against experimental data. We find favorable agreement among the three computational methods and the experiment, suggesting that the influence of the ion cloud on Rg, as detected by SAXS, may be predicted with nearly analytic calculations. Our analysis further suggests that the ion cloud effect on Rg is dominated by the long-range distribution of the ions around the proteins, as described by Debye-Hückel theory, whereas the local salt structure near the protein surface plays a minor role.

Temperature-Dependent Atomic Models of Detergent Micelles Refined against Small-Angle X-Ray Scattering Data.

Surfactants have found a wide range of industrial and scientific applications. In particular, detergent micelles are used as lipid membrane mimics to solubilize membrane proteins for functional and structural characterization. However, an atomic-level understanding of surfactants remains limited because many experiments provide only low-resolution structural information on surfactant aggregates. In this work, small-angle X-ray scattering is combined with molecular dynamics simulations to derive fully atomic models of two maltoside micelles at temperatures between 10 °C and 70 °C. The micelles take the shape of general tri-axial ellipsoids and decrease in size and aggregation number with increasing temperature. Density profiles of hydrophobic groups and water along the three principal axes reveal that the minor micelle axis closely mimics lipid membranes. The results suggest that coupling atomic simulations with low-resolution data allows the structural characterization of surfactant aggregates.

Conformational Changes and Flexibility of DNA Devices Observed by Small-Angle X-ray Scattering.

Self-assembled DNA origami nanostructures enable the creation of precisely defined shapes at the molecular scale. Dynamic DNA devices that are capable of switching between defined conformations could afford completely novel functionalities for diagnostic, therapeutic, or engineering applications. Developing such objects benefits strongly from experimental feedback about conformational changes and 3D structures, ideally in solution, free of potential biases from surface attachment or labeling. Here, we demonstrate that small-angle X-ray scattering (SAXS) can quantitatively resolve the conformational changes of a DNA origami two-state switch device as a function of the ionic strength of the solution. In addition, we show how SAXS data allow for refinement of the predicted idealized three-dimensional structure of the DNA object using a normal mode approach based on an elastic network model. The results reveal deviations from the idealized design geometries that are otherwise difficult to resolve. Our results establish SAXS as a powerful tool to investigate conformational changes and solution structures of DNA origami and we anticipate our methodology to be broadly applicable to increasingly complex DNA and RNA devices.

pH-Dependent Interactions in Dimers Govern the Mechanics and Structure of von Willebrand Factor.

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is activated for hemostasis by increased hydrodynamic forces at sites of vascular injury. Here, we present data from atomic force microscopy-based single-molecule force measurements, atomic force microscopy imaging, and small-angle x-ray scattering to show that the structure and mechanics of VWF are governed by multiple pH-dependent interactions with opposite trends within dimeric subunits. In particular, the recently discovered strong intermonomer interaction, which induces a firmly closed conformation of dimers and crucially involves the D4 domain, was observed with highest frequency at pH 7.4, but was essentially absent at pH values below 6.8. However, below pH 6.8, the ratio of compact dimers increased with decreasing pH, in line with a previous transmission electron microscopy study. These findings indicated that the compactness of dimers at pH values below 6.8 is promoted by other interactions that possess low mechanical resistance compared with the strong intermonomer interaction. By investigating deletion constructs, we found that compactness under acidic conditions is primarily mediated by the D4 domain, i.e., remarkably by the same domain that also mediates the strong intermonomer interaction. As our data suggest that VWF has the highest mechanical resistance at physiological pH, local deviations from physiological pH (e.g., at sites of vascular injury) may represent a means to enhance VWF's hemostatic activity where needed.

Structural Architecture of the Nucleosome Remodeler ISWI Determined from Cross-Linking, Mass Spectrometry, SAXS, and Modeling.

Chromatin remodeling factors assume critical roles by regulating access to nucleosomal DNA. To determine the architecture of the Drosophila ISWI remodeling enzyme, we developed an integrative structural approach that combines protein cross-linking, mass spectrometry, small-angle X-ray scattering, and computational modeling. The resulting structural model shows the ATPase module in a resting state with both ATPase lobes twisted against each other, providing support for a conformation that was recently trapped by crystallography. The autoinhibiting NegC region does not protrude from the ATPase module as suggested previously. The regulatory NTR domain is located near both ATPase lobes. The full-length enzyme is flexible and can adopt a compact structure in solution with the C-terminal HSS domain packing against the ATPase module. Our data imply a series of conformational changes upon activation of the enzyme and illustrate how the NTR, NegC, and HSS domains contribute to regulation of the ATPase module.

Quantitative evaluation of statistical errors in small-angle X-ray scattering measurements.

A new model is proposed for the measurement errors incurred in typical small-angle X-ray scattering (SAXS) experiments, which takes into account the setup geometry and physics of the measurement process. The model accurately captures the experimentally determined errors from a large range of synchrotron and in-house anode-based measurements. Its most general formulation gives for the variance of the buffer-subtracted SAXS intensity σ2(q) = [I(q) + const.]/(kq), where I(q) is the scattering intensity as a function of the momentum transfer q; k and const. are fitting parameters that are characteristic of the experimental setup. The model gives a concrete procedure for calculating realistic measurement errors for simulated SAXS profiles. In addition, the results provide guidelines for optimizing SAXS measurements, which are in line with established procedures for SAXS experiments, and enable a quantitative evaluation of measurement errors.

A Mo-anode-based in-house source for small-angle X-ray scattering measurements of biological macromolecules.

We demonstrate the use of a molybdenum-anode-based in-house small-angle X-ray scattering (SAXS) setup to study biological macromolecules in solution. Our system consists of a microfocus X-ray tube delivering a highly collimated flux of 2.5 × 10(6) photons/s at a beam size of 1.2 × 1.2 mm(2) at the collimation path exit and a maximum beam divergence of 0.16 mrad. The resulting observable scattering vectors q are in the range of 0.38 Å(-1) down to 0.009 Å(-1) in SAXS configuration and of 0.26 Å(-1) up to 5.7 Å(-1) in wide-angle X-ray scattering (WAXS) mode. To determine the capabilities of the instrument, we collected SAXS data on weakly scattering biological macromolecules including proteins and a nucleic acid sample with molecular weights varying from ∼12 to 69 kDa and concentrations of 1.5-24 mg/ml. The measured scattering data display a high signal-to-noise ratio up to q-values of ∼0.2 Å(-1) allowing for an accurate structural characterization of the samples. Moreover, the in-house source data are of sufficient quality to perform ab initio 3D structure reconstructions that are in excellent agreement with the available crystallographic structures. In addition, measurements for the detergent decyl-maltoside show that the setup can be used to determine the size, shape, and interactions (as characterized by the second virial coefficient) of detergent micelles. This demonstrates that the use of a Mo-anode based in-house source is sufficient to determine basic geometric parameters and 3D shapes of biomolecules and presents a viable alternative to valuable beam time at third generation synchrotron sources.