Person:

Leschziner, Andres E

Electron microscopy of frozen-hydrated samples (cryo-EM) can yield high resolution structures of macromolecular complexes by accurately determining the orientation of large numbers of experimental views of the sample relative to an existing 3D model. The “initial model problem”, the challenge of obtaining these orientations ab initio, remains a major bottleneck in determining the structure of novel macromolecules, chiefly those lacking internal symmetry. We previously proposed a method for the generation of initial models – orthogonal tilt reconstruction (OTR) – that bypasses limitations inherent to the other two existing methods, random conical tilt (RCT) and angular reconstitution (AR). Here we present a validation of OTR with a biological test sample whose structure was previously solved by RCT: the complex between the yeast exosome and the subunit Rrp44. We show that, as originally demonstrated with synthetic data, OTR generates initial models that do not exhibit the “missing cone” artifacts associated with RCT and show an isotropic distribution of information when compared with the known structure. This eliminates the need for further user intervention to solve these artifacts and makes OTR ideal for automation and the analysis of heterogeneous samples. With the former in mind, we propose a set of simple quantitative criteria that can be used, in combination, to select from a large set of initial reconstructions a subset that can be used as reliable references for refinement to higher resolution.

The advent of a new generation of electron microscopes and direct electron detectors has realized the potential of single particle cryo-electron microscopy (cryo-EM) as a technique to generate high-resolution structures. Calculating these structures requires high performance computing clusters, a resource that may be limiting to many likely cryo-EM users. To address this limitation and facilitate the spread of cryo-EM, we developed a publicly available ‘off-the-shelf’ computing environment on Amazon's elastic cloud computing infrastructure. This environment provides users with single particle cryo-EM software packages and the ability to create computing clusters with 16–480+ CPUs. We tested our computing environment using a publicly available 80S yeast ribosome dataset and estimate that laboratories could determine high-resolution cryo-EM structures for $50 to $1500 per structure within a timeframe comparable to local clusters. Our analysis shows that Amazon's cloud computing environment may offer a viable computing environment for cryo-EM. DOI: http://dx.doi.org/10.7554/eLife.06664.001

Regulation of cytoplasmic dynein's motor activity is essential for diverse eukaryotic functions, including cell division, intracellular transport, and brain development. The dynein regulator Lis1 is known to keep dynein bound to microtubules; however, how this is accomplished mechanistically remains unknown. We have used three-dimensional electron microscopy, single-molecule imaging, biochemistry, and in vivo assays to help establish this mechanism. The three-dimensional structure of the dynein–Lis1 complex shows that binding of Lis1 to dynein's AAA+ ring sterically prevents dynein's main mechanical element, the ‘linker’, from completing its normal conformational cycle. Single-molecule experiments show that eliminating this block by shortening the linker to a point where it can physically bypass Lis1 renders single dynein motors insensitive to regulation by Lis1. Our data reveal that Lis1 keeps dynein in a persistent microtubule-bound state by directly blocking the progression of its mechanochemical cycle. DOI: http://dx.doi.org/10.7554/eLife.03372.001