Synthetic antibodies against BRIL as universal fiducial marks for single−particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins

Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM

“Universal” synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric G_i and G_s, as well as mini-G_s, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ) protein. Several Gβγ-specific sABs, cross-reactive between trimeric G_i and G_s, were identified suggesting they could be used across all subclasses in a “plug and play” fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes

Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM

“Universal” synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric G_i and G_s, as well as mini-G_s, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ) protein. Several Gβγ-specific sABs, cross-reactive between trimeric G_i and G_s, were identified suggesting they could be used across all subclasses in a “plug and play” fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes

Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Thermoelastic analysis of a geological repository with distributed decay heat sources by the image method in combination with a numerical integration scheme

The disposal of nuclear waste involves thermo-mechanical reaction of the host rock to the buried waste – a distributed heat source that decays. To solve the problem within the half infinite space confined by the ground surface, an image method is developed. Specifically, a negative image of the heat source with the ground surface as the mirror, i.e. a mirrored heat sink is utilized so that the normal traction generated by the heat source can be counterbalanced, and a numerical scheme of integration of the classical Cerruti solution is developed to include the effect of tangential shear traction on the ground surface caused by the heat sources and their mirrored sinks. For a conceptual repository model, large thermal shear stress, tensile stress, and deformation occur at the corner, between adjacent drifts, and at the boundary of the repository area, respectively. For a prescribed thermal loading, it is more efficient to mitigate the thermo-mechanical effects through enlarging the pit spacing than increasing the drift spacing

An Analytical Continuous Upper Bound Limit Analysis of Pore Water Effect on the Tail Stability of Underwater Shield Tunnels during Construction

An analytical continuous upper bound limit analysis is developed to analyse the effects of seepage on the transverse stability of underwater shield tunnels. The approach is based on an analytical continuous upper bound limit analysis method for cohesive-frictional soils. It employs the complex variables solution of the displacement field due to tunnel deformation and movement, and the analytical solution of the pore water pressure field for steady state seepage due to pore water influx at the tunnel perimeter. The most critical slip line position and the minimum required tunnel support pressure are determined by using a particle swarm optimization scheme for various generic situations. The method is verified via finite element simulation and comparison with the solution from using rigid block upper bound limit analysis. The parametric analysis revealed among other things that both the infimum of the necessary tunnel support pressure and the most critical plastic zone increase when the hydraulic head at the ground surface increases, but decrease when the tunnel influx increases due to the fact that pore water pressure at the tunnel perimeter decreases with the tunnel influx

A Semi-Analytical Method for Three-Dimensional Heat Transfer in Multi-Fracture Enhanced Geothermal Systems

Multiple fractures have been proposed for improving the heat extracted from an enhanced geothermal system (EGS). For calculating the production temperature of a multi-fracture EGS, previous analytical or semi-analytical methods have all been based on an infinite scale of fractures and one-dimensional conduction in the rock matrix. Here, a temporal semi-analytical method is presented in which finite-scale fractures and three-dimensional conduction in the rock matrix are both considered. Firstly, the developed model was validated by comparing it with the analytical solution, which only considers one-dimensional conduction in the rock matrix. Then, the temporal semi-analytical method was used to predict the production temperature in order to investigate the effects of fracture spacing and fracture number on the response of an EGS with a constant total injection rate. The results demonstrate that enlarging the spacing between fractures and increasing the number of fractures can both improve the heat extraction; however, the latter approach is much more effective than the former. In addition, the temporal semi-analytical method is applicable for optimizing the design of an EGS with multiple fractures located equidistantly or non-equidistantly

A Semi-Analytical Model and Parameter Analysis of a Collaborative Drainage Scheme for a Deeply Buried Tunnel and Parallel Adit in Water-Rich Ground

For a railway or highway tunnel under high water pressure during operation, various factors such as the design of the drainage system, material aging, and pipeline blockage must be considered for the tunnels to work with the parallel adit to drain and control the external water pressure on the tunnel lining. A simplified steady-state seepage model in a semi-infinite multi-connected domain for the tunnel and parallel adit was established and was solved iteratively using the complex variable method and the Schwartz alternating method. After verifying the numerical simulation, parametric analysis, orthogonal tests, and multivariate nonlinear regression were also carried out. Results show that the simplified theoretical model and its semi-analytical algorithm have a fast convergence speed, and the obtained regression formula is simple, which is suitable for calculation and parameter analysis. A scheme that primarily relies on the parallel adit for drainage would make the external water pressure of the lining facing the parallel adit side less than that of the opposite side. Therefore, to reduce pressure uniformly and meet the requirements of surrounding rock stability, the horizontal net distance between the parallel adit and the tunnel should be no less than the tunnel diameter. Drainage volume of the parallel adit is linearly negatively correlated with tunnel water pressure on the lining and has the most significant effect on pressure reduction. The influence of the vertical distance between the parallel adit and the tunnel on water pressure is small