Direct observation of the molecular mechanism underlying protein polymerization

Protein assembly is a main route to generating complexity in living systems. Revealing the relevant molecular details is challenging because of the intrinsic heterogeneity of species ranging from few to hundreds of molecules. Here, we use mass photometry to quantify and monitor the full range of actin oligomers during polymerization with single-molecule sensitivity. We find that traditional nucleation-based models cannot account for the observed distributions of actin oligomers. Instead, the key step of filament formation is a slow transition between distinct states of an actin filament mediated by cation exchange or ATP hydrolysis. The resulting model reproduces important aspects of actin polymerization, such as the critical concentration for filament formation and bulk growth behavior. Our results revise the mechanism of actin nucleation, shed light on the role and function of actin-associated proteins, and introduce a general and quantitative means to studying protein assembly at the molecular level

Femtosecond X-ray coherent diffraction of aligned amyloid fibrils on low background graphene

Here we present a new approach to diffraction imaging of amyloid fibrils, combining a free-standing graphene support and single nanofocused X-ray pulses of femtosecond duration from an X-ray free-electron laser. Due to the very low background scattering from the graphene support and mutual alignment of filaments, diffraction from tobacco mosaic virus (TMV) filaments and amyloid protofibrils is obtained to 2.7 Å and 2.4 Å resolution in single diffraction patterns, respectively. Some TMV diffraction patterns exhibit asymmetry that indicates the presence of a limited number of axial rotations in the XFEL focus. Signal-to-noise levels from individual diffraction patterns are enhanced using computational alignment and merging, giving patterns that are superior to those obtainable from synchrotron radiation sources. We anticipate that our approach will be a starting point for further investigations into unsolved structures of filaments and other weakly scattering objects

Comparative transcriptomics analyses reveal the conservation of an ancestral infectious strategy in two bacteriophage genera.

International audienceAlthough the evolution of tailed bacteriophages has increasingly been better understood through comparisons of their DNA sequences, the functional consequences of this evolution on phage infectious strategies have remained unresolved. In this study, we comprehensively compared the transcriptional strategies of two related myoviruses, PAK_P3 and PAK_P4, infecting the same Pseudomonas aeruginosa host strain. Outside of the conservation of their structural clusters, their highly syntenic genomes display only limited DNA similarity. Despite this apparent divergence, we found that both viruses follow a similar infection scheme, relying on a temporal regulation of their gene expression, likely involving the use of antisense transcripts, as well as a rapid degradation of 90% of the host non-ribosomal mRNA, as previously reported for PAK_P3. However, the kinetics of the mRNA degradation is remarkably faster during PAK_P4 infection. Moreover, we found that each virus has evolved specific adaptations, as exemplified by the distinct patterns of their core genes expression as well as the specific manipulation of the expression of iron-related host genes by PAK_P4. This study enhances our understanding of the evolutionary process of virulent phages, which relies on adjusting globally conserved ancestral infection mechanisms.The ISME Journal advance online publication, 12 May 2017; doi:10.1038/ismej.2017.63