Background: SecA targets preproteins to the protein-conducting channel in bacteria. Results: Both the single and double copies of SecA bind to the 70S ribosome. Conclusion: Two copies of SecA completely surround the polypeptide tunnel exit. Significance: The structures suggest a function of the dimeric form of SecA on the ribosome.

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein implicated in the pathogenesis of both familial and sporadic Parkinson’s disease (PD), and currently one of the most promising therapeutic targets for drug design in Parkinson’s disease. In contrast, LRRK1, the closest homologue to LRRK2, does not play any role in PD. Here, we use cryo-electron microscopy (cryo-EM) and single particle analysis to gain structural insight into the full-length dimeric structures of LRRK2 and LRRK1. Differential scanning fluorimetry-based screening of purification buffers showed that elution of the purified LRRK2 protein in a high pH buffer is beneficial in obtaining high quality cryo-EM images. Next, analysis of the 3D maps generated from the cryo-EM data show 16 and 25 Å resolution structures of full length LRRK2 and LRRK1, respectively, revealing the overall shape of the dimers with two-fold symmetric orientations of the protomers that is closely similar between the two proteins. These results suggest that dimerization mechanisms of both LRRKs are closely related and hence that specificities in functions of each LRRK are likely derived from LRRK2 and LRRK1’s other biochemical functions. To our knowledge, this study is the first to provide 3D structural insights in LRRK2 and LRRK1 dimers in parallel.

Membrane proteins are vital to life and major therapeutic targets. Yet, understanding how they function is limited by a lack of structural information. In biological cells, membrane proteins reside in lipidic membranes and typically experience different buffer conditions on both sides of the membrane or even electric potentials and transmembrane gradients across the membranes. Proteoliposomes, which are lipidic vesicles filled with reconstituted membrane proteins, provide an ideal model system for structural and functional studies of membrane proteins under conditions that mimic nature to a certain degree. We discuss methods for the formation of liposomes and proteoliposomes, their imaging by cryo-electron microscopy, and the structural analysis of proteins present in their bilayer. We suggest the formation of ordered arrays akin to weakly ordered two-dimensional (2D) crystals in the bilayer of liposomes as a means to achieve high-resolution, and subsequent buffer modification as a method to capture snapshots of membrane proteins in action.

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Background: Microtubules are dynamic protein filaments that are crucial for cell division and constitute key elements of the cytoskeleton. They are assembled from αβ-tubulin heterodimers that form hollow cylindrical structures. There is a large number of naturally occurring compounds that are known to interact with tubulin, including alkaloids, macrolides and peptides, which are collectively called microtubule-targeting agents (MTAs). Based on their activities, MTAs can be classified as microtubulestabilizing agents (MSAs) that enhance MT assembly, and microtubule-destabilizing agents (MDAs) that suppress MT assembly. The chemical structure of these drugs and their binding mode to microtubules varies greatly amongst each other and confer the ability to act either synergistically or competitively on microtubules. Owing to their effects on microtubule dynamics, MTAs are of great interest and widely used in a variety of medical applications as antiparasitic agents, herbicides and, most importantly, as chemotherapeutic drugs used for the treatment of cancer. In the past years, we and others solved the structures of a large number of different MTAs bound to tubulin to high resolution using X-ray crystallography. Very recently, however, with the advent of the "Resolution Revolution" in cryo-electron microscopy (cryo-EM), atomic structures of known MSAs bound to microtubules have also been obtained. These cryo-EM structures confirmed that the sites and modes of binding described in the previous X-ray crystallographic studies are similar in the context of the assembled microtubule but additionally explain the effects of MTAs on lattice parameters in microtubules and the lateral contacts between protofilaments, especially at the microtubule seam. Methods: Cryo-EM and model building. Results: We solved several high-resolution structures of microtubules bound to novel MSAs and identified differential binding modes in comparison to that revealed by X-ray crystallography. Conclusion: Cryo-EM and X-ray crystallography can be used in a complementary manner to investigate the molecular mechanism of action of MSAs in detail.

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