Tubulin isoform composition tunes microtubule dynamics

Microtubules polymerize and depolymerize stochastically, a behavior essential for cell division, motility and differentiation. While many studies advanced our understanding of how microtubule-associated proteins tune microtubule dynamics in trans, we have yet to understand how tubulin genetic diversity regulates microtubule functions. The majority of in vitro dynamics studies are performed with tubulin purified from brain tissue. This preparation is not representative of tubulin found in many cell types. Here we report the 4.2Å cryo-EM structure and in vitro dynamics parameters of α1B/βI+βIVb microtubules assembled from tubulin purified from a human embryonic kidney cell line with isoform composition characteristic of fibroblasts and many immortalized cell lines. We find that these microtubules grow faster and transition to depolymerization less frequently compared to brain microtubules. Cryo-EM reveals that the dynamic ends of α1B/βI+βIVb microtubules are less tapered and that these tubulin heterodimers display lower curvatures. Interestingly, analysis of EB1 distributions at dynamic ends suggests no differences in GTP cap sizes. Lastly, we show that the addition of recombinant α1A/βIII tubulin, a neuronal isotype overexpressed in many tumors, proportionally tunes the dynamics of α1B/βI+βIVb microtubules. Our study is an important step towards understanding how tubulin isoform composition tunes microtubule dynamics

Scipion web tools: Easy to use cryo-EM image processing over the web

Macromolecular structural determination by Electron Microscopy under cryogenic conditions is revolutionizing the field of structural biology, interesting a large community of potential users. Still, the path from raw images to density maps is complex, and sophisticated image processing suites are required in this process, often demanding the installation and understanding of different software packages. Here, we present Scipion Web Tools, a web-based set of tools/workflows derived from the Scipion image processing framework, specially tailored to nonexpert users in need of very precise answers at several key stages of the structural elucidation process

Collection, pre-processing and on-the-fly analysis of data for high-resolution, single-particle cryo-electron microscopy

The dramatic growth in the use of cryo-electron microscopy (cryo-EM) to generate high-resolution structures of macromolecular complexes has changed the landscape of structural biology. The majority of structures deposited in the Electron Microscopy Data Bank (EMDB) at higher than 4-Å resolution were collected on Titan Krios microscopes. Although the pipeline for single-particle data collection is becoming routine, there is much variation in how sessions are set up. Furthermore, when collection is under way, there are a range of approaches for efficiently moving and pre-processing these data. Here, we present a standard operating procedure for single-particle data collection with Thermo Fisher Scientific EPU software, using the two most common direct electron detectors (the Thermo Fisher Scientific Falcon 3 (F3EC) and the Gatan K2), as well as a strategy for structuring these data to enable efficient pre-processing and on-the-fly monitoring of data collection. This protocol takes 3–6 h to set up a typical automated data collection session