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

Attaining atomic resolution from in situ data collection at room temperature using counter-diffusion-based low-cost microchips

Sample handling and manipulation for cryoprotection currently remain critical factors in X-ray structural determination. While several microchips for macromolecular crystallization have been proposed during the last two decades to partially overcome crystal-manipulation issues, increased background noise originating from the scattering of chip-fabrication materials has so far limited the attainable resolution of diffraction data. Here, the conception and use of low-cost, X-ray-transparent microchips for in situ crystallization and direct data collection, and structure determination at atomic resolution close to 1.0 Å , is presented. The chips are fabricated by a combination of either OSTEMER and Kapton or OSTEMER and Mylar materials for the implementation of counter-diffusion crystallization experiments. Both materials produce a sufficiently low scattering background to permit atomic resolution diffraction data collection at room temperature and the generation of 3D structural models of the tested model proteins lysozyme, thaumatin and glucose isomerase. Although the high symmetry of the three model protein crystals produced almost complete data sets at high resolution, the potential of in-line data merging and scaling of the multiple crystals grown along the microfluidic channels is also presented and discussed

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

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

Macromolecular Serial Crystallography

Within the structural biology field, X-ray crystallography prevails as the dominant technique to determine the structures of macromolecules, producing, as of November 2020, more than 150,000 structures since its inception (https://www [...]Jose M. Martin-Garcia was funded by the Community of Madrid through the “Atracción y Retención de Talento” Grant (Ref: 2019-T1BMD-1552), and Shibom Basu was funded by The European Molecular Biology Laboratory (EMBL).Peer reviewe

Room temperature structures beyond 1.5 Å by serial femtosecond crystallography

About 2.5 × 106 snapshots on microcrystals of photoactive yellow protein (PYP) from a recent serial femtosecond crystallographic (SFX) experiment were reanalyzed to maximum resolution. The resolution is pushed to 1.46 Å, and a PYP structural model is refined at that resolution. The result is compared to other PYP models determined at atomic resolution around 1 Å and better at the synchrotron. By comparing subtleties such as individual isotropic temperature factors and hydrogen bond lengths, we were able to assess the quality of the SFX data at that resolution. We also show that the determination of anisotropic temperature factor ellipsoids starts to become feasible with the SFX data at resolutions better than 1.5 Å

Long-wavelength native-SAD phasing : opportunities and challenges

Native single-wavelength anomalous dispersion (SAD) is an attractive experimental phasing technique as it exploits weak anomalous signals from intrinsic light scatterers (Z 2R-TTL) and is applied in the structure determination of an 86 kDa helicase Sen1 protein at beamline BL-1A of the KEK Photon Factory, Japan. Furthermore, X-ray absorption at long wavelengths was controlled by shaping a lysozyme crystal into spheres of defined thicknesses using a deep-UV laser, and a systematic comparison between wavelengths of 2.7 and 3.3 Å is reported for native SAD. The potential of laser-shaping technology and other challenges for an optimized native-SAD experiment at wavelengths >3 Å are discussed.publishe

Femto- and attosecond serial crystallography -time-resolved studies of dynamics in biological systems

Serial Femtosecond X-ray nanocrystallography (SFX) is a recently developed technique for protein structure determination that is based on collection of X-ray diffraction from nano- and microcrystals using a free electron laser (FEL). SFX is especially valuable for challenging proteins as membrane proteins, because the time-consuming process of growing larger crystals can be avoided. In general structural analysis with the use of free electron X-ray laser is a powerful method to overcome the radiation damage problem and to perform time-resolved structure analysis. Recent results of studies on the membrane protein complex Photosystem II using time resolved SFX will form the basis for future time-resolved studies of biomolecules. Furthermore an introduction of the concept of attosecond serial crystallography, which is currently under development, and its potential advantages over SFX will be given. Its capability for new insights in the water oxidation process in Photosystem II and its value for structural and functionals analysis of biological processes in general will be discussed