obituary John C. H. Spence (1946-2021)

Obituary for John C. H. Spence

Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody

Highly selective monoclonal antibodies recognizing the extracellular 3D epitope of G protein-coupled receptors represent valuable tools for elucidating receptor function and localization in the cell and show promise for a range of therapeutic applications. Here we present the structure of a complex between the human serotonin 2B receptor, captured in an active-like state, and an antibody Fab fragment, bound to the extracellular side of the receptor. The structure uncovers the mechanisms of receptor activation and of extracellular receptor recognition by antibodies

Femtosecond x-ray diffraction from an aerosolized beam of protein nanocrystals

We demonstrate near-atomic-resolution Bragg diffraction from aerosolized single granulovirus crystals using an x-ray free-electron laser. The form of the aerosol injector is nearly identical to conventional liquid-microjet nozzles, but the x-ray-scattering background is reduced by several orders of magnitude by the use of helium carrier gas rather than liquid. This approach provides a route to study the weak diffuse or lattice-transform signal arising from small crystals. The high speed of the particles is particularly well suited to upcoming MHz-repetition-rate x-ray free-electron lasers

High-resolution ab initio three-dimensional X-ray diffraction microscopy

Coherent X-ray diffraction microscopy is a method of imaging non-periodic isolated objects at resolutions only limited, in principle, by the largest scattering angles recorded. We demonstrate X-ray diffraction imaging with high resolution in all three dimensions, as determined by a quantitative analysis of the reconstructed volume images. These images are retrieved from the 3D diffraction data using no a priori knowledge about the shape or composition of the object, which has never before been demonstrated on a non-periodic object. We also construct 2D images of thick objects with infinite depth of focus (without loss of transverse spatial resolution). These methods can be used to image biological and materials science samples at high resolution using X-ray undulator radiation, and establishes the techniques to be used in atomic-resolution ultrafast imaging at X-ray free-electron laser sources.Comment: 22 pages, 11 figures, submitte

Serial macromolecular crystallography at ALBA Synchrotron Light Source

12 pags., 4 figs., 2 tabs. -- Addenda and errata: https://journals.iucr.org/s/issues/2022/03/00/rv5160/rv5160.pdfThe increase in successful adaptations of serial crystallography at synchrotron radiation sources continues. To date, the number of serial synchrotron crystallography (SSX) experiments has grown exponentially, with over 40 experiments reported so far. In this work, we report the first SSX experiments with viscous jets conducted at ALBA beamline BL13-XALOC. Small crystals (15-30 μm) of five soluble proteins (lysozyme, proteinase K, phycocyanin, insulin and α-spectrin-SH3 domain) were suspended in lipidic cubic phase (LCP) and delivered to the X-ray beam with a high-viscosity injector developed at Arizona State University. Complete data sets were collected from all proteins and their high-resolution structures determined. The high quality of the diffraction data collected from all five samples, and the lack of specific radiation damage in the structures obtained in this study, confirm that the current capabilities at the beamline enables atomic resolution determination of protein structures from microcrystals as small as 15 μm using viscous jets at room temperature. Thus, BL13-XALOC can provide a feasible alternative to X-ray free-electron lasers when determining snapshots of macromolecular structures.The following funding is acknowledged: Ayuda de Atracciony Retencion de Talento Investigador" from the Community of Madrid (scholarship No. 2019-T1/BMD-15552); STC Programof the National Science Foundation through BioXFEL (awardNo. 1231306); the Centre for Applied Structural Discovery(CASD) at the Biodesign Institute at Arizona State University; the Spanish Ministry of Science and Innovation, grants EQC2021-007532-P, PID2020-117028GB-I00, BIO2016-77883-C2-2-

Abstract P-4: Robust Method for Background Subtraction in Serial X-ray Diffraction Data

Background: Membrane receptors play an important role in signal transduction across the cell membrane in all living organisms. Their structural studies have been enabled by multiple technological breakthroughs in their heterologous expression, stabilization, crystallization, and crystallographic data collection as well as in cryogenic electron microscopy (cryoEM). During the last decade, serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has enabled structure determination of previously inaccessible proteins, including several G-protein-coupled receptors (GPCR), that produce only micrometer-sized crystals, thus paving the way towards understanding their activation mechanism and rational drug discovery. In addition to experimental difficulties, membrane protein structure determination is also often accompanied by data processing challenges. In particular, the lipidic cubic phase that serves as a carrier for membrane protein microcrystals, as well as various XFEL beam-shaping devices may generate substantial background scattering that could complicate the structure factor extraction from the diffraction images. Methods: In this work, we tested an adaptation of the denoising algorithm via matrix decomposition to XFEL-SFX data. We benchmarked its performance using high-background data from PAL-XFEL and established its applicability to serial crystallography image denoising, as well as compared it to the CrystFEL-based image denoising algorithm. Results: We find that, although the decomposition-based image denoising does not outperform CrystFEL median subtraction, it performs better than the integration without any additional subtraction. We find the non-negative matrix factorization performing better than more traditional singular-value decomposition methods, both in terms of visual interpretability and final data quality. Conclusion: We hope that this work will draw attention to background subtraction methods in structural biology, and will pave the way towards processing of most challenging datasets in structural biology, in particularly, those collected from membrane proteins

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The g-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergen

Segmented flow generator for serial crystallography at the European X-ray free electron laser

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported

Применение программного продукта «Яндекс.Сервер» для организации поиска в электронном каталоге библиотеки

The huge amounts of information accumulated by libraries in recent years put before developers a problem of the organization of fast and qualitative search which decision is possible with the use of modern search tools of Web-technology. The author examines one of these tools the software product “Yandex. Server”, allowing to organize optimum search in the electronic library catalog. The software product “Yandex. Server” gives a chance to carry out optimum search taking into account morphology of Russian and English languages, as well as the various logical conditions that provides effective and flexible search in the electronic library catalog.Накопленные библиотеками за последние годы огромные массивы информации ставят перед разработчиками задачу организации быстрого и качественного поиска, решение которой возможно с использованием современных поисковых инструментов веб-технологии. Автор рассматривает один из таких инструментов - программный продукт «Яндекс. Сервер», позволяющий организовать оптимальный поиск в электронном каталоге библиотеки с учетом морфологии русского и английского языков, а также различных логических условий