MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography.

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 (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). 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 MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques

MyD88 TIR domain higher-order assembly interactions revealed by microcrystal electron diffraction and serial femtosecond crystallography.

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 (MALTIR) forms filaments in vitro and induces formation of crystalline higher-order assemblies of the MyD88 TIR domain (MyD88TIR). 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 MyD88TIR assembly, which reveal a two-stranded higher-order assembly arrangement of TIR domains analogous to that seen previously for MALTIR. We demonstrate via mutagenesis that the MyD88TIR assembly interfaces are critical for TLR4 signaling in vivo, and we show that MAL promotes unidirectional assembly of MyD88TIR. Collectively, our studies provide structural and mechanistic insight into TLR signal transduction and allow a direct comparison of the MicroED and SFX techniques

Determining Orientations of Optical Transition Dipole Moments Using Ultrafast X-ray Scattering

Identification of the initially prepared, optically active state remains a challenging problem in many studies of ultrafast photoinduced processes. We show that the initially excited electronic state can be determined using the anisotropic component of ultrafast time-resolved X-ray scattering signals. The concept is demonstrated using the time-dependent X-ray scattering of <i>N</i>-methyl morpholine in the gas phase upon excitation by a 200 nm linearly polarized optical pulse. Analysis of the angular dependence of the scattering signal near time zero renders the orientation of the transition dipole moment in the molecular frame and identifies the initially excited state as the 3p_<i>z</i> Rydberg state, thus bypassing the need for further experimental studies to determine the starting point of the photoinduced dynamics and clarifying inconsistent computational results

The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 angstrom resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 angstrom resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.Peer reviewe

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

Enzyme intermediates captured on the fly by mix-and-inject serial crystallography

Background Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Results Here, we demonstrate a general method for capturing enzyme catalysis in action by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2s. Conclusions MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.This work was supported by the National Science Foundation (NSF)-Science and Technology Center (STC) BioXFEL through award STC-1231306, and in part by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DE-SC0002164 (to A.O., algorithm design and development) and by the NSF under contract number 1551489 (to A.O., underlying analytical models). Portions of this research were performed at the Linac Coherent Light Source (LCLS). Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Basic Energy Sciences under contract DE-AC02-76SF00515. This material is based upon work supported by the NSF Graduate Research Fellowship Program to J.L.O. under grant no. 1450681. The work was also supported by funds from the National Institutes of Health grants R01 GM117342-01 and R01 GM095583, by funds from the Biodesign Center for Applied Structural Discovery at Arizona State University, and the US Department of Energy through Lawrence Livermore National Laboratory under contract DE-AC52-07NA27344. Part of this work was also supported by program-oriented funds of the Helmholtz Association

Nanoelectronic Investigation Reveals the Electrochemical Basis of Electrical Conductivity in Shewanella and Geobacter.

The electrical conductivity measured in Shewanella and Geobacter spp. is an intriguing physical property that is the fundamental basis for possible extracellular electron transport (EET) pathways. There is considerable debate regarding the origins of the electrical conductivity reported in these microbial cellular structures, which is essential for deciphering the EET mechanism. Here, we report systematic on-chip nanoelectronic investigations of both Shewanella and Geobacter spp. under physiological conditions to elucidate the complex basis of electrical conductivity of both individual microbial cells and biofilms. Concurrent electrical and electrochemical measurements of living Shewanella at both few-cell and the biofilm levels indicate that the apparent electrical conductivity can be traced to electrochemical-based electron transfer at the cell/electrode interface. We further show that similar results and conclusions apply to the Geobacter spp. Taken together, our study offers important insights into previously proposed physical models regarding microbial conductivities as well as EET pathways for Shewanella and Geobacter spp

Nanoelectronic Investigation Reveals the Electrochemical Basis of Electrical Conductivity in Shewanella and Geobacter.

The electrical conductivity measured in Shewanella and Geobacter spp. is an intriguing physical property that is the fundamental basis for possible extracellular electron transport (EET) pathways. There is considerable debate regarding the origins of the electrical conductivity reported in these microbial cellular structures, which is essential for deciphering the EET mechanism. Here, we report systematic on-chip nanoelectronic investigations of both Shewanella and Geobacter spp. under physiological conditions to elucidate the complex basis of electrical conductivity of both individual microbial cells and biofilms. Concurrent electrical and electrochemical measurements of living Shewanella at both few-cell and the biofilm levels indicate that the apparent electrical conductivity can be traced to electrochemical-based electron transfer at the cell/electrode interface. We further show that similar results and conclusions apply to the Geobacter spp. Taken together, our study offers important insights into previously proposed physical models regarding microbial conductivities as well as EET pathways for Shewanella and Geobacter spp