51 research outputs found
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
Structural dynamics and functional cooperativity of human NQO1 by ambient temperature serial crystallography and simulations
The human NQO1 (hNQO1) is a flavin adenine nucleotide (FAD)-dependent
oxidoreductase that catalyzes the two-electron reduction of quinones to hydroquinones,
being essential for the antioxidant defense system, stabilization of
tumor suppressors, and activation of quinone-based chemotherapeutics. Moreover,
it is overexpressed in several tumors, which makes it an attractive cancer
drug target. To decipher new structural insights into the flavin reductive halfreaction
of the catalytic mechanism of hNQO1, we have carried serial crystallography
experiments at new ID29 beamline of the ESRF to determine, to the
best of our knowledge, the first structure of the hNQO1 in complex with
NADH. We have also performed molecular dynamics simulations of free
hNQO1 and in complex with NADH. This is the first structural evidence that
the hNQO1 functional cooperativity is driven by structural communication
between the active sites through long-range propagation of cooperative effects
across the hNQO1 structure. Both structural results and MD simulations have
supported that the binding of NADH significantly decreases protein dynamics and stabilizes hNQO1 especially at the dimer core and interface. Altogether,
these results pave the way for future time-resolved studies, both at x-ray freeelectron
lasers and synchrotrons, of the dynamics of hNQO1 upon binding to
NADH as well as during the FAD cofactor reductive half-reaction. This knowledge
will allow us to reveal unprecedented structural information of the relevance
of the dynamics during the catalytic function of hNQO1.European Union NextGenerationEU/PRTR (Grant number CNS2022-135713)MCIN/AEI/10.13039/501100011033/ERDF (Grant number
MCIN/AEI/PID2022-136369NB-I00)Ayuda de Atracción y Retención de Talento Investigador from the Community of Madrid (Grant number 2019-T1/BMD-15552)ERDF/Spanish Ministry of Science, Innovation
and Universities-State Research Agency (Grant number
RTI2018-096246-B-I00)Consejería de Economía, Conocimiento,
Empresas y Universidad, Junta de Andalucía
(Grant number P18-RT-2413)ERDF/Counseling of Economic
Transformation, Industry, Knowledge and Universities
(Grant number B-BIO-84-UGR20)Gobierno de
Aragon (Grant number E35-23R
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
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