@article {48623, title = {Taking the measure of MicroED.}, journal = {Current Opinion in Structural Biology}, volume = {46}, year = {2017}, month = {2017 Jun 22}, pages = {79-86}, abstract = {

It is now possible to routinely determine atomic resolution structures by electron cryo-microscopy (cryoEM), facilitated in part by the method known as micro electron-diffraction (MicroED). Since its initial demonstration in 2013, MicroED has helped determine a variety of protein structures ranging in molecular weight from a few hundred Daltons to several hundred thousand Daltons. Some of these structures were novel while others were previously known. The resolutions of structures obtained thus far by MicroED range from 3.2{\r A} to 1.0{\r A}, with most better than 2.5{\r A}. Crystals of various sizes and shapes, with different space group symmetries, and with a range of solvent content have all been studied by MicroED. The wide range of crystals explored to date presents the community with a landscape of opportunity for structure determination from nano crystals. Here we summarize the lessons we have learned during the first few years of MicroED, and from our attempts at the first ab initio structure determined by the method. We re-evaluate theoretical considerations in choosing the appropriate crystals for MicroED and for extracting the most meaning out of measured data. With more laboratories worldwide adopting the technique, we speculate what the first decade might hold for MicroED.

}, issn = {1879-033X}, doi = {10.1016/j.sbi.2017.06.004}, author = {Rodriguez, Jose A and Eisenberg, David S and Gonen, Tamir} } @article {45372, title = {A type VI secretion-related pathway in bacteroidetes mediates interbacterial antagonism.}, journal = {Cell Host Microbe}, volume = {16}, year = {2014}, month = {2014 Aug 13}, pages = {227-36}, abstract = {

Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.

}, issn = {1934-6069}, doi = {10.1016/j.chom.2014.07.007}, author = {Russell, Alistair B and Wexler, Aaron G and Harding, Brittany N and Whitney, John C and Bohn, Alan J and Goo, Young Ah and Tran, Bao Q and Barry, Natasha A and Zheng, Hongjin and Peterson, S Brook and Chou, Seemay and Gonen, Tamir and Goodlett, David R and Goodman, Andrew L and Mougous, Joseph D} } @article {6633, title = {Three-dimensional electron crystallography of protein microcrystals.}, journal = {eLife}, volume = {2}, year = {2013}, month = {2013 Nov 19}, pages = {01345}, abstract = {

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 {\r A} resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1{\textendash}1{\textdegree} and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 {\r A} resolution. This proof of principle paves the way for the implementation of a new technique, which we name {\textquoteleft}MicroED{\textquoteright}, that may have wide applicability in structural biology.

}, doi = {10.7554/eLife.01345}, author = {Shi, Dan and Nannenga, Brent L and Iadanza, Matthew G and Gonen, Tamir} } @article {3631, title = {Tension directly stabilizes reconstituted kinetochore-microtubule attachments.}, journal = {Nature}, volume = {468}, year = {2010}, month = {2010 Nov 25}, pages = {576-9}, abstract = {

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct {\textquoteright}bi-oriented{\textquoteright} kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for \>30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

}, keywords = {Chromosomes, Fungal Proteins, Kinetochores, Microtubules, Saccharomyces cerevisiae}, issn = {1476-4687}, doi = {10.1038/nature09594}, author = {Akiyoshi, Bungo and Sarangapani, Krishna K and Powers, Andrew F and Nelson, Christian R and Reichow, Steve L and Arellano-Santoyo, Hugo and Gonen, Tamir and Ranish, Jeffrey A and Asbury, Charles L and Biggins, Sue} } @article {45961, title = {Tension applied through the Dam1 complex promotes microtubule elongation providing a direct mechanism for length control in mitosis.}, journal = {Nature Cell Biology}, volume = {9}, year = {2007}, month = {2007 Jul}, pages = {832-7}, abstract = {

In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule fibres and tension at the kinetochore-microtubule interface promotes fibre elongation. Tension-dependent microtubule fibre elongation is thought to be essential for coordinating chromosome alignment and separation, but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore-microtubule interface composed of the yeast Dam1 complex bound to individual dynamic microtubule tips. Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibres in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule-length control in mitosis.

}, keywords = {Biomechanical Phenomena, Cell Cycle Proteins, Fungal Proteins, Kinetochores, Microspheres, Microtubule-Associated Proteins, Microtubules, Mitosis, Optical Tweezers, Saccharomyces cerevisiae Proteins}, issn = {1465-7392}, doi = {10.1038/ncb1609}, author = {Franck, Andrew D and Powers, Andrew F and Gestaut, Daniel R and Gonen, Tamir and Davis, Trisha N and Asbury, Charles L} }