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Biology subjects

Walter, P.

Publications and source records attributed to Walter, P..

3 recordsLinked to original sources

Electrospray sample injection for single-particle imaging with X-ray lasers

The possibility of imaging single proteins constitutes an exciting challenge for X-ray lasers. Despite encouraging results on large particles, imaging small particles has proven to be difficult for two reasons: not quite high enough pulse intensity from currently available X-ray lasers and, as we demonstrate here, contamination of the aerosolised molecules by non-volatile contaminants in the solution. The amount of contamination on the sample depends on the initial droplet-size during aerosolisation. Here we show that with our electrospray injector we can decrease the size of aerosol droplets and demonstrate virtually contaminant-free sample delivery of organelles, small virions, and proteins. The results presented here, together with the increased performance of next generation X-ray lasers, constitute an important stepping stone towards the ultimate goal of protein structure determination from imaging at room temperature and high temporal resolution.

biophysics

Olfactory and Vomeronasal Receptor Feedback Employ Divergent Mechanisms of PERK Activation

Mutually-exclusive chemoreceptor expression in olfactory and vomeronasal sensory neurons (OSNs and VSNs) enables odorant discrimination. This configuration involves chemoreceptor mediated activation of the endoplasmic reticulum (ER)-resident kinase PERK. PERK drives translation of the transcription factor ATF5 to preclude additional chemoreceptor expression. ATF5 translation is transient in OSNs but persistent in VSNs, suggesting chemoreceptor-specific modes of PERK activation. Herein, we showed that the ER-lumenal domain (LD) of PERK recognized vomeronasal receptor (VR)-derived peptides, suggesting direct PERK activation drives persistent ATF5 translation in VSNs. In contrast, PERK LD did not recognize olfactory receptor (OR)-derived peptides in vitro, and facilitating OR maturation in vivo prevented PERK activation, suggesting that ORs activate PERK indirectly through a failure to exit the ER. Importantly, impairing or prolonging ATF5 expression drove specific chemoreceptor repertoire biases. Together, these results demonstrate mechanistic divergence in chemoreceptor feedback and establish that differences in PERK activation promote qualitatively different gene regulatory results.

cell biology

Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule

Regulation by the integrated stress response (ISR) converges on the phosphorylation of translation initiation factor eIF2 in response to a variety of stresses. Phosphorylation converts eIF2 from substrate to competitive inhibitor of its dedicated guanine nucleotide exchange factor, eIF2B, inhibiting translation. ISRIB, a drug-like eIF2B activator, reverses the effects of eIF2 phosphorylation and, remarkably, in rodents enhances cognition and corrects cognitive deficits after brain injury. To determine its mechanism of action, we solved an atomic-resolution structure of ISRIB bound in a deep cleft within decameric human eIF2B by electron cryo-microscopy. Structural and biochemical analyses revealed that formation of fully active, decameric eIF2B holoenzyme depended on the assembly of two identical tetrameric subcomplexes, and that ISRIB promoted this step by cross-bridging a central symmetry interface. Regulation of eIF2B assembly emerges as a rheostat for eIF2B activity that tunes translation during the ISR and that can be further modulated by ISRIB.

biochemistry