TIR (Toll/interleukin-1 receptor) domains are widely distributed in animals, plants and bacteria, and function through self-association and homotypic interactions with other TIR domains [1].Across phyla, these domains feature in proteins with immune functions -TLRs (Toll-like receptors), IL-1Rs (interleukin-1 receptors) and their adaptor proteins in animals; NLRs (nucleotide-binding, leucine-rich repeat receptors) in plants; and antiphage defence proteins in bacteria.Although long assumed to only have protein interaction functions, the TIR domains across kingdoms also feature self-association-dependent enzymatic activities, namely cleavage of nucleotides such as NAD + [2,3].We used an integrated structural biology approach to characterize the signalosomes formed by different TIR domains.We reconstituted large assemblies of the TLR/adaptor TIR domains (not known to have enzymatic activities); the structures of the filamentous assemblies of the TIR domains of TLR adaptor MAL [4], TRAM and the TLR4:MAL complex (unpublished) were determined by cryo-electron microscopy (cryoEM) helical reconstruction, and the structures of crystalline arrays of MyD88 were determined by micro-electron diffraction and serial femtosecond crystallography [5].We further stabilized the active assemblies of enzyme TIR domains from the mammalian protein SARM1 (involved in axon degeneration; octameric complexes) [6] and the bacterial protein AbTir from Acinetobacter baumanii [3] (filamentous assemblies) with NAD + mimics and determined their structures using single-particle cryoEM and helical reconstruction, respectively.We found that all these TIR domain assemblies feature a head-to-tail arrangement of TIR molecules, with the enzyme active site located in the interface between two molecules, explaining the requirement for self-association in enzyme activity.However, such head-to-tail row of molecules in stabilized by another row associating in an antiparallel fashion in enzyme assemblies such as those from SARM1 and plant NLRs, and in a parallel fashion in both scaffold assemblies (in TLR adaptors) and bacterial enzyme assemblies (Fig. 1).In all cases, we validated the observed interactions by structure-guided mutagenesis and functional assays (e.g.[7]).The products of enzymatic reactions have downstream signalling functions in immune pathways or their suppression.Our studies will form the foundation of applications ranging from the treatment of inflammatory disorders and bacterial infections in humans to the prevention of plant diseases.Figure 1.Structures of filamentous TIR-domain assemblies (four TIR domains are shown in each case, but the assemblies are open-ended from left to right of the page).

The European X-ray Free Electron Laser (XFEL) and Linac Coherent Light Source (LCLS) II are extremely intense sources of X-rays capable of generating Serial Femtosecond Crystallography (SFX) data at megahertz (MHz) repetition rates. Previous work has shown that it is possible to use consecutive X-ray pulses to collect diffraction patterns from individual crystals. Here, we exploit the MHz pulse structure of the European XFEL to obtain two complete datasets from the same lysozyme crystal, first hit and the second hit, before it exits the beam. The two datasets, separated by <1 µs, yield up to 2.1 Å resolution structures. Comparisons between the two structures reveal no indications of radiation damage or significant changes within the active site, consistent with the calculated dose estimates. This demonstrates MHz SFX can be used as a tool for tracking sub-microsecond structural changes in individual single crystals, a technique we refer to as multi-hit SFX.

MyD88 and MAL are Toll-like receptor (TLR) adaptors that signal to induce pro-inflammatory cytokine production. We previously observed that the TIR domain of MAL (MAL(TIR)) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88(TIR)). These crystals are too small for conventional X-ray crystallography, but are ideally suited to structure determination by microcrystal electron diffraction (MicroED) and serial femtosecond crystallography (SFX). Here, we present MicroED and SFX structures of the MyD88(TIR) assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MAL(TIR). We demonstrate via mutagenesis that the MyD88(TIR) assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88(TIR). Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques. MAL and MyD88 are downstream adaptors of Toll-like receptors (TLR) and the MAL TIR domain forms filaments in vitro, which in turn nucleate the assembly of crystalline arrays of the MyD88 TIR domain. Here, the authors present the structure of these MyD88 TIR crystalline arrays solved by both microcrystal electron diffraction and serial femtosecond crystallography, and they show with mutagenesis experiments that MyD88 interface residues are important for TLR4 signaling in vivo.

Characterising and understanding the mechanisms involved in cell death are especially important to combating threats to human health, particularly for the study of antimicrobial peptides and their effectiveness against pathogenic fungi. However, imaging these processes often relies on the use of synthetic molecules which bind to specific cellular targets to produce contrast. Here we study yeast cell death, induced by the anti-fungal peptide, NaD1. By treating yeast as a model organism we aim to understand anti-fungal cell death processes without relying on sample modification. Using a quantitative phase imaging technique, ptychography, we were able to produce label free images of yeast cells during death and use them to investigate the mode of action of NaD1. Using this technique we were able to identify a significant phase shift which provided a clear signature of yeast cell death. Additionally, ptychography identifies cell death much earlier than a comparative fluorescence study, providing new insights into the cellular changes that occur during cell death. The results indicate ptychography has great potential as a means of providing additional information about cellular processes which otherwise may be masked by indirect labelling approaches.

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.