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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

The DEFECTIVE EMBRYO AND MERISTEMS 1 (DEM1) gene encodes a protein of unknown biochemical function required for meristem formation and seedling development in tomato, but it was unclear whether DEM1's primary role was in cell division or alternatively, in defining the identity of meristematic cells. Genome sequence analysis indicates that flowering plants possess at least two DEM genes. Arabidopsis has two DEM genes, DEM1 and DEM2, which we show are expressed in developing embryos and meristems in a punctate pattern that is typical of genes involved in cell division. Homozygous dem1 dem2 double mutants were not recovered, and plants carrying a single functional DEM1 allele and no functional copies of DEM2, i.e. DEM1/dem1 dem2/dem2 plants, exhibit normal development through to the time of flowering but during male reproductive development, chromosomes fail to align on the metaphase plate at meiosis II and result in abnormal numbers of daughter cells following meiosis. Additionally, these plants show defects in both pollen and embryo sac development, and produce defective male and female gametes. In contrast, dem1/dem1 DEM2/dem2 plants showed normal levels of fertility, indicating that DEM2 plays a more important role than DEM1 in gamete viability. The increased importance of DEM2 in gamete viability correlated with higher mRNA levels of DEM2 compared to DEM1 in most tissues examined and particularly in the vegetative shoot apex, developing siliques, pollen and sperm. We also demonstrate that gamete viability depends not only on the number of functional DEM alleles inherited following meiosis, but also on the number of functional DEM alleles in the parent plant that undergoes meiosis. Furthermore, DEM1 interacts with RAS-RELATED NUCLEAR PROTEIN 1 (RAN1) in yeast two-hybrid and pull-down binding assays, and we show that fluorescent proteins fused to DEM1 and RAN1 co-localize transiently during male meiosis and pollen development. In eukaryotes, RAN is a highly conserved GTPase that plays key roles in cell cycle progression, spindle assembly during cell division, reformation of the nuclear envelope following cell division, and nucleocytoplasmic transport. Our results demonstrate that DEM proteins play an essential role in cell division in plants, most likely through an interaction with RAN1.

The ROCO family is a family of GTPases characterized by a central ROC-COR tandem domain. Interest in the structure and function of ROCO proteins has increased with the identification of their important roles in human disease. Nevertheless, the functions of most ROCO proteins are still unknown. In the present study, we characterized the structure, evolution, and expression of ROCOs in four species of brown algae. Brown algae have a larger number of ROCO proteins than other organisms reported to date. Phylogenetic analyses showed that ROCOs have an ancient origin, likely originated in prokaryotes. ROCOs in brown algae clustered into four groups and showed no strong relationship with red algae or green algae. Brown algal ROCOs retain the ancestral LRR-ROC-COR domain arrangement, which is found in prokaryotes, plants and some basal metazoans. Remarkably, individual LRR motifs in ROCO genes are each encoded by separate exons and exhibit intense exon shuffling and diversifying selection. Furthermore, the tandem LRR exons exhibit alternative splicing to generate multiple transcripts. Both exon shuffling and alternative splicing of LRR repeats may be important mechanisms for generating diverse ligand-binding specificities as immune receptors. Besides their potential immune role, expression analysis shows that many ROCO genes are responsive to other stress conditions, suggesting they could participate in multiple signal pathways, not limited to the immune response. Our results substantially enhance our understanding of the structure and function of this mysterious gene family. [ABSTRACT FROM AUTHOR]

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.

The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.

Axons are an essential component of the nervous system, and axon degeneration is an early feature of many neurodegenerative disorders. The NAD+ metabolome plays an essential role in regulating axonal integrity. Axonal levels of NAD+ and its precursor NMN are controlled in large part by the NAD+ synthesizing survival factor NMNAT2 and the pro-neurodegenerative NADase SARM1, whose activation triggers axon destruction. SARM1 has emerged as a promising axon-specific target for therapeutic intervention, and its function, regulation, structure, and role in neurodegenerative diseases have been extensively characterized in recent years. In this review, we first introduce the key molecular players involved in the SARM1-dependent axon degeneration program. Next, we summarize recent major advances in our understanding of how SARM1 is kept inactive in healthy neurons and how it becomes activated in injured or diseased neurons, which has involved important insights from structural biology. Finally, we discuss the role of SARM1 in neurodegenerative disorders and environmental neurotoxicity and its potential as a therapeutic target. [ABSTRACT FROM AUTHOR]