The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.

Membranes are vital structures for cellular life forms. As thin, hydrophobic films, they provide a physical barrier separating the aqueous cytoplasm from the outside world or from the interiors of other cellular compartments. They maintain a selective permeability for the import and export of water-soluble compounds, enabling the living cell to maintain a stable chemical environment for biological processes. Cell membranes are primarily composed of two crucial substances, lipids and proteins. Bacterial membranes can sense environmental changes or communication signals from other cells and they support different cell processes, including cell division, differentiation, protein secretion and supplementary protein functions. The original fluid mosaic model of membrane structure has been recently revised because it has become apparent that domains of different lipid composition are present in both eukaryotic and prokaryotic cell membranes. In this review, we summarize different aspects of phospholipid domain formation in bacterial membranes, mainly in Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. We describe the role of these lipid domains in membrane dynamics and the localization of specific proteins and protein complexes in relation to the regulation of cellular function.

Background: The Gram-positive bacterium Bacillus subtilis is an important producer of high quality industrial enzymes and a few eukaryotic proteins. Most of these proteins are secreted into the growth medium, but successful examples of cytoplasmic protein production are also known. Therefore, one may anticipate that the high protein production potential of B. subtilis can be exploited for protein complexes and membrane proteins to facilitate their functional and structural analysis. The high quality of proteins produced with B. subtilis results from the action of cellular quality control systems that efficiently remove misfolded or incompletely synthesized proteins. Paradoxically, cellular quality control systems also represent bottlenecks for the production of various heterologous proteins at significant concentrations. Conclusion: While inactivation of quality control systems has the potential to improve protein production yields, this could be achieved at the expense of product quality. Mechanisms underlying degradation of secretory proteins are nowadays well understood and often controllable. It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis that limit the production of high quality protein complexes and membrane proteins, and to enhance those systems that facilitate assembly of these proteins.

Summary: The fluid mosaic model of membrane structure has been revised in recent years as it has become evident that domains of different lipid composition are present in eukaryotic and prokaryotic cells. Using membrane binding fluorescent dyes, we demonstrate the presence of lipid spirals extending along the long axis of cells of the rod‐shaped bacterium Bacillus subtilis. These spiral structures are absent from cells in which the synthesis of phosphatidylglycerol is disrupted, suggesting an enrichment in anionic phospholipids. Green fluorescent protein fusions of the cell division protein MinD also form spiral structures and these were shown by fluorescence resonance energy transfer to be coincident with the lipid spirals. These data indicate a higher level of membrane lipid organization than previously observed and a primary role for lipid spirals in determining the site of cell division in bacterial cells.