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Cytochrome coxidase (CcO) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme bgroup in their active sites, CcO has a unique binuclear center (BNC) composed of a copper atom (CuB) and a heme a3iron, where O2binds and is reduced to water. CO is a versatile O2surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine CcO (bCcO) revealed that photolyzing CO from the heme a3iron leads to a metastable intermediate (CuB-CO), where CO is bound to CuB, before it escapes out of the BNC. Here, with a pump-probe based time-resolved serial femtosecond X-ray crystallography, we detected a geminate photoproduct of the bCcO–CO complex, where CO is dissociated from the heme a3iron and moved to a temporary binding site midway between the CuBand the heme a3iron, while the locations of the two metal centers and the conformation of Helix-X, housing the proximal histidine ligand of the heme a3iron, remain in the CO complex state. This new structure, combined with other reported structures of bCcO, allows for a clearer definition of the ligand dissociation trajectory as well as the associated protein dynamics.

The human MT1and MT2melatonin receptors1,2are G-protein-coupled receptors (GPCRs) that help to regulate circadian rhythm and sleep patterns3. Drug development efforts have targeted both receptors for the treatment of insomnia, circadian rhythm and mood disorders, and cancer3, and MT2has also been implicated in type 2 diabetes4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT2receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon6at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants: H2085.46A (superscripts represent the Ballesteros–Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-PMT. Comparison of the structures of MT2with a published structure8of MT1reveals that, despite conservation of the orthosteric ligand-binding site residues, there are notable conformational variations as well as differences in [3H]melatonin dissociation kinetics that provide insights into the selectivity between melatonin receptor subtypes. A membrane-buried lateral ligand entry channel is observed in both MT1and MT2, but in addition the MT2structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data that support a prominent role for intramembrane ligand entry in both receptors, and suggest that there might also be an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents.

We report on the analysis of virtual powder-diffraction patterns from serial femtosecond crystallography (SFX) data collected at an X-ray free-electron laser. Different approaches to binning and normalizing these patterns are discussed with respect to the microstructural characteristics which each highlights. Analysis of SFX data from a powder of Pr0.5Ca0.5MnO3in this way finds evidence of other trace phases in its microstructure which was not detectable in a standard powder-diffraction measurement. Furthermore, a comparison between two virtual powder pattern integration strategies is shown to yield different diffraction peak broadening, indicating sensitivity to different types of microstrain. This paper is a first step in developing new data analysis methods for microstructure characterization from serial crystallography data.