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Abstract Sample consumption for serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) remains a major limitation preventing broader use of this powerful technology in macromolecular crystallography. This drawback is exacerbated in the case of time-resolved (TR)-SFX experiments, where the amount of sample required per reaction time point is multiplied by the number of time points investigated. Thus, in order to reduce the limitation of sample consumption, here we demonstrate the implementation of segmented droplet generation in conjunction with a mix-and-inject approach for TR studies on NAD(P)H:quinone oxidoreductase 1 (NQO1). We present the design and application of mix-and-inject segmented droplet injectors for the Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument at the European XFEL (EuXFEL) with a synchronized droplet injection approach that allows liquid phase protein crystal injection. We carried out TR-crystallography experiments with this approach for a 305 ms and a 1190 ms time point in the reaction of NQO1 with its coenzyme NADH. With this successful TR-SFX approach, up to 97% of the sample has been conserved compared to continuous crystal suspension injection with a gas dynamic virtual nozzle. Furthermore, the obtained structural information for the reaction of NQO1 with NADH is an important part of the future elucidation of the reaction mechanism of this crucial therapeutic enzyme. 

Let \(\Sigma\) be a closed subset of \(\mathbb{R}^{n+1}\) which is parabolic Ahlfors-David regular and assume that \(\Sigma\) satisfies a 2-sided corkscrew condition. Assume, in addition, that \(\Sigma\) is either time-forwards Ahlfors-David regular, time-backwards Ahlfors-David regular, or parabolic uniform rectifiable. We then first prove that \(\Sigma\) satisfies a weak synchronized two cube condition. Based on this we are able to revisit the argument of Nyström and Strömqvist (2009) and prove that \(\Sigma\) contain suniform big pieces of Lip(1,1/2) graphs. When \(\Sigma\) is parabolic uniformly rectifiable the construction can be refined and in this case we prove that \(\Sigma\) contains uniform big pieces of regular parabolic Lip(1,1/2) graphs. Similar results hold if \(\Omega\subset\mathbb{R}^{n+1}\) is a connected component of \(\mathbb{R}^{n+1}\setminus\Sigma\) and in this context we also give a parabolic counterpart of the main result of Azzam et al. (2017) by proving that if \(\Omega\) is a one-sided parabolic chord arc domain, and if \(\Sigma\) is parabolic uniformly rectifiable, then \(\Omega\) is in fact a parabolic chord arc domain. Our results give a flexible parabolic version of the classical (elliptic) result of David and Jerison (1990) concerning the existence of uniform big pieces of Lipschitz graphs for sets satisfying a two disc condition. 

Francisella tularensisis an extremely infectious pathogen and a category A bioterrorism agent. It causes the highly contagious zoonosis, Tularemia. Currently, FDA approved vaccines against tularemia are unavailable.F.tularensisouter membrane protein A (FopA) is a well-studied virulence determinant and protective antigen against tularemia. It is a major outer membrane protein (Omp) ofF.tularensis. However, FopA-based therapeutic intervention is hindered due to lack of complete structural information for membrane localized mature FopA. In our study, we established recombinant expression, monodisperse purification, crystallization and X-ray diffraction (~6.5 Å) of membrane localized mature FopA. Further, we performed bioinformatics and biophysical experiments to unveil its structural organization in the outer membrane. FopA consists of 393 amino acids and has less than 40% sequence identity to known bacterial Omps. Using comprehensive sequence alignments and structure predictions together with existing partial structural information, we propose a two-domain organization for FopA. Circular dichroism spectroscopy and heat modifiability assay confirmed FopA has a β-barrel domain consistent with alphafold2’s prediction of an eight stranded β-barrel at the N-terminus. Small angle X-ray scattering (SAXS) and native-polyacrylamide gel electrophoresis revealed FopA purified in detergent micelles is predominantly dimeric. Molecular density derived from SAXS at 31 Å shows putative dimeric N-terminal β-barrels surrounded by detergent corona and connected to C-terminal domains via flexible linker. Disorder analysis predicts N- and C-terminal domains are interspersed by a long intrinsically disordered region and alphafold2 predicts this region to be largely unstructured. Taken together, we propose a dimeric, two-domain organization of FopA in the outer membrane: the N-terminal β-barrel is membrane embedded, provides dimerization interface and tethers to membrane extrinsic C-terminal domain via long flexible linker. Structure determination of membrane localized mature FopA is essential to understand its role in pathogenesis and develop anti-tularemia therapeutics. Our results pave the way towards it.