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Free-electron lasers are capable of high repetition rates and it is assumed that protein crystals often do not survive the first X-ray pulse. Here the authors address these issues with a demonstration of multi-hit serial crystallography in which multiple FEL pulses interact with the sample without destroying it.

One of the outstanding analytical problems in X-ray single-particle imaging (SPI) is the classification of structural heterogeneity, which is especially difficult given the low signal-to-noise ratios of individual patterns and the fact that even identical objects can yield patterns that vary greatly when orientation is taken into consideration. Proposed here are two methods which explicitly account for this orientation-induced variation and can robustly determine the structural landscape of a sample ensemble. The first, termed common-line principal component analysis (PCA), provides a rough classification which is essentially parameter free and can be run automatically on any SPI dataset. The second method, utilizing variation auto-encoders (VAEs), can generate 3D structures of the objects at any point in the structural landscape. Both these methods are implemented in combination with the noise-tolerant expand–maximize–compress (EMC) algorithm and its utility is demonstrated by applying it to an experimental dataset from gold nanoparticles with only a few thousand photons per pattern. Both discrete structural classes and continuous deformations are recovered. These developments diverge from previous approaches of extracting reproducible subsets of patterns from a dataset and open up the possibility of moving beyond the study of homogeneous sample sets to addressing open questions on topics such as nanocrystal growth and dynamics, as well as phase transitions which have not been externally triggered.

Due to the pulsed nature of X-ray free electron laser (XFEL) instruments the majority of protein crystals, which are injected using continuous jet injection techniques are wasted. Here, the authors present a microfluidic device to deliver aqueous protein crystal laden droplets segmented with an immiscible oil and demonstrate that with this device an approx. 60% reduction in sample waste was achieved for data collection of 3-deoxy-D-manno-octulosonate 8-phosphate synthase crystals at the EuXFEL.

Abstract Background We aimed to evaluate the clinical robustness of a commercially developed data-driven respiratory gating algorithm based on principal component analysis, for use in routine PET imaging. Methods One hundred fifty-seven adult FDG PET examinations comprising a total of 1149 acquired bed positions were used for the assessment. These data are representative of FDG scans currently performed at our institution. Data were acquired for 4 min/bed position (3 min/bed for legs). The data-driven gating (DDG) algorithm was applied to each bed position, including those where minimal respiratory motion was expected. The algorithm provided a signal-to-noise measure of respiratory-like frequencies within the data, denoted as R. Qualitative evaluation was performed by visual examination of the waveforms, with each waveform scored on a 3-point scale by two readers and then averaged (score S of 0 = no respiratory signal, 1 = some respiratory-like signal but indeterminate, 2 = acceptable signal considered to be respiratory). Images were reconstructed using quiescent period gating and compared with non-gated images reconstructed with a matched number of coincidences. If present, the SUVmax of a well-defined lesion in the thorax or abdomen was measured and compared between the two reconstructions. Results There was a strong (r = 0.86) and significant correlation between R and scores S. Eighty-six percent of waveforms with R ≥ 15 were scored as acceptable for respiratory gating. On average, there were 1.2 bed positions per patient examination with R ≥ 15. Waveforms with high R and S were found to originate from bed positions corresponding to the thorax and abdomen: 90% of waveforms with R ≥ 15 had bed centres in the range 5.6 cm superior to 27 cm inferior from the dome of the liver. For regions where respiratory motion was expected to be minimal, R tended to be

The analysis of a single-particle imaging (SPI) experiment performed at the AMO beamline at LCLS as part of the SPI initiative is presented here. A workflow for the three-dimensional virus reconstruction of the PR772 bacteriophage from measured single-particle data is developed. It consists of several well defined steps including single-hit diffraction data classification, refined filtering of the classified data, reconstruction of three-dimensional scattered intensity from the experimental diffraction patterns by orientation determination and a final three-dimensional reconstruction of the virus electron density without symmetry constraints. The analysis developed here revealed and quantified nanoscale features of the PR772 virus measured in this experiment, with the obtained resolution better than 10 nm, with a clear indication that the structure was compressed in one direction and, as such, deviates from ideal icosahedral symmetry.

Liquid microjets are a common means of delivering protein crystals to the focus of X-ray free-electron lasers (FELs) for serial femtosecond crystallography measurements. The high X-ray intensity in the focus initiates an explosion of the microjet and sample. With the advent of X-ray FELs with megahertz rates, the typical velocities of these jets must be increased significantly in order to replenish the damaged material in time for the subsequent measurement with the next X-ray pulse. This work reports the results of a megahertz serial diffraction experiment at the FLASH FEL facility using 4.3 nm radiation. The operation of gas-dynamic nozzles that produce liquid microjets with velocities greater than 80 m s−1 was demonstrated. Furthermore, this article provides optical images of X-ray-induced explosions together with Bragg diffraction from protein microcrystals exposed to trains of X-ray pulses repeating at rates of up to 4.5 MHz. The results indicate the feasibility for megahertz serial crystallography measurements with hard X-rays and give guidance for the design of such experiments.

Serial diffraction data collected at the Linac Coherent Light Source from crystalline amyloid fibrils delivered in a liquid jet show that the fibrils are well oriented in the jet. At low fibril concentrations, diffraction patterns are recorded from single fibrils; these patterns are weak and contain only a few reflections. Methods are developed for determining the orientation of patterns in reciprocal space and merging them in three dimensions. This allows the individual structure amplitudes to be calculated, thus overcoming the limitations of orientation and cylindrical averaging in conventional fibre diffraction analysis. The advantages of this technique should allow structural studies of fibrous systems in biology that are inaccessible using existing techniques.

Pontoscolex corethrurus (Müller, 1857) plays an important role in tropical soil ecosystems and has been widely used as an animal model for a large variety of ecological studies in particular due to its common presence and generally high abundance in human-disturbed tropical soils. In this study we describe the complete mitochondrial genome of the peregrine earthworm P. corethrurus. This is the first record of a mitochondrial genome within the Rhinodrilidae family. Its mitochondrial genome is 14 835 bp in length containing 37 genes (13 protein-coding genes (PCG) 2 rRNA genes and 22 tRNA genes). It has the same gene content and structure as in other sequenced earthworms but unusual among invertebrates it hasseveral overlapping open reading frames. All genes are encoded on the same strand. Most of the PCGs use ATG as the start codon except for ND3 which uses GTG as the start codon. The A+T content of the mitochondrial genome is 59.9% (31.8% A 28.1% T 14.6% G and 25.6% for C). The annotated genome sequence has been deposited in GenBank under the accession number KT988053.