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Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting “preferred orientations” on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

 

Abstract We present a passive (unpowered) exoskeleton that assists the back during lifting. Our exoskeleton uses carbon fiber beams as the sole means to store energy and return it to the wearer. To motivate the design, we present general requirements for the design of a lifting exoskeleton, including calculating the required torque to support the torso for people of different weights and heights. We compare a number of methods of energy storage for exoskeletons in terms of mass, volume, hysteresis, and cycle life. We then discuss the design of our exoskeleton, and show how the torso assembly leads to balanced forces. We characterize the energy storage in the exoskeleton and the torque it provides during testing with human subjects. Ten participants performed freestyle, stoop, and squat lifts. Custom image processing software was used to extract the curvature of the carbon fiber beams in the exoskeleton to determine the stored energy. During freestyle lifting, it stores an average of 59.3 J and provides a peak torque of 71.7 Nm.