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The prostaglandin DP1 receptor plays critical roles in allergic responses, sleep regulation, immune modulation, and vasodilation. Here, authors present atomic structures of DP1 in different states revealing molecular mechanisms of receptor inhibition and activation.

This protocol describes procedures for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature and under cryogenic conditions using Mylar-based film sandwich plates.

The GPCR Network was established in 2010 with the aim of structurally characterizing 15–25 representative human G protein-coupled receptors (GPCRs) within 5 years; so far, more than eight have been determined. Here, Stevens and colleagues provide an overview of this collaborative effort and the challenges remaining in gaining detailed insights into the structure–function relationships of this receptor superfamily.

S1P₅ is a sphingosine-1-phosphate (S1P) receptor implicated in immune and neurodegenerative disorders. Here, authors report a crystal structure of the S1P₅ receptor in complex with a selective inverse agonist, revealing an allosteric subpocket and shedding light on inverse agonism in S1P receptors.

Human leukotriene B4 receptors (BLT1 and BLT2) are members of the GPCR superfamily that respond to a potent pro-inflammatory lipid and chemoattractant LTB4. Here authors determined a crystal structure of the human BLT1 in complex with a selective antagonist MK-D-046 and provide insights into hBLT1 ligand recognition and its mechanism of action.

Cysteinyl leukotriene G protein-coupled receptors CysLT₁ and CysLT₂ regulate pro-inflammatory responses associated with allergic disorders. Here, authors describe four crystal structures of CysLT₂R in complex with three dual CysLT₁R/CysLT₂R antagonists, which shed light on CysLTR ligand selectivity.

Cryo-electron microscopy structures of apo, agonist- and positive allosteric modulator-bound forms of the GB1–GB2 heterodimer of the metabotropic γ-aminobutyric acid (GABA) receptor shed light on the activation mechanism of this receptor.

Crystal structures of two complexes of the angiotensin II receptor AT₂R with distinct tightly bound ligands reveal an active-like state of the receptor, in which helix VIII adopts a non-canonical position that blocks binding of G proteins and β-arrestins.

Structural determination of GAT1 using cryo-electron microscopy provides insights into the biology and pharmacology of this GABA transporter.

A computational screen of an ultra-large virtual library against the structure of the melatonin receptor found nanomolar ligands, and ultimately two selective MT₁ inverse agonists that induced phase advancement of the mouse circadian clock when given at subjective dusk.

The crystal structure of the human κ-opioid receptor in complex with an antagonist, JDTic, is determined, with potential importance for the design of new therapeutic agents.

Antagonists of Smoothened, a class F GPCR involved in the hedgehog pathway, have been developed to treat some cancers. Here Wang et al.report structures of Smoothened in complex with antagonists and an agonist, and describe how mutations may result in resistance to anti-Smoothened treatment.

The crystal structure of the full-length human glucagon receptor reveals the essential role of the 12-residue ‘stalk’ segment and an extracellular loop in the regulation of ligand binding and receptor activation.

Serial femtosecond X-ray crystallography permits the use of very small protein crystals; however, a continuous flow of sample is required. Weierstall et al. design and demonstrate an injector system that can supply microcrystals in the lipidic cubic phase, dramatically reducing the quantities of protein required.

The X-ray crystal structure of the human glucagon receptor, a potential drug target for type 2 diabetes, offers a structural basis for molecular recognition by class B G-protein-coupled receptors.

The MT₁ melatonin receptor differs markedly from 5-HT receptors and shows atypical ligand entry; its structure with various ligands sheds light on receptor specificity.

Structural and functional studies show that the MT₂ melatonin receptor, unlike the MT₁ receptor, contains an extracellular opening for ligand entry, shedding light on receptor subtype specificity.

A structure of the prostaglandin E₂ receptor 3 (EP3) bound to the agonist misoprostol shows a completely enclosed binding pocket with a structured water molecule that coordinates misoprostol's ring structure and explains the receptor's selectivity.

The 1.8 Å high-resolution X-ray crystal structure of the human δ-opioid receptor is presented, with site-directed mutagenesis and functional studies revealing a crucial role for a sodium ion in mediating allosteric control in this receptor.

Opsin has recently been shown to equilibrate phospholipids across the photoreceptor disc membranes. Goren et al.demonstrate that the large conformational changes driven by exposure to light are not required for this activity, and provide insight into the potential mechanisms of lipid translocation.

Diacylglycerol kinase is a small bacterial membrane-bound trimer that catalyses diacylglycerol conversion to phosphatidic acid. Here, the authors solve the crystal structure of the kinase bound to a lipid substrate and an ATP analogue, and show that the active site arose through convergent evolution.

Smoothened receptors (SMO) play a key role in the Hedgehog signalling pathway. Here the authors present the structure of a multi-domain human SMO with a rationally designed stabilizing ligand bound in the transmembrane domain of the receptor, and propose a model for SMO activation.

Two X-ray crystal structures are presented of the human P2Y₁ G-protein-coupled receptor, which is an important target for anti-thrombotic drugs; the structures unexpectedly reveal two ligand-binding sites.

The crystal structure of the human smoothened (SMO) receptor is presented in complex with a small-molecule antitumour agent; this represents the first example of a non-class-A, 7-transmembrane (7TM) receptor structure, revealing different conserved motifs common within class frizzled 7TM receptors and an unusually complex arrangement of long extracellular loops stabilized by disulphide bonds.

Serial femtosecond crystallography of the human δ-opioid receptor in complex with an endomorphin-derived peptide reveals interactions that are important for understanding the pharmacology of opioid peptides and developing analgesics with reduced side effects.

G protein-coupled receptors are a large family of signalling proteins that mediate cellular responses primarily via G proteins or arrestins, and they are targets of one-third of the current clinically used drugs; here, an active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin-1 is determined, revealing unique structural features that may constitute essential elements for arrestin-biased signalling.

The X-ray crystal structure of the human P2Y₁₂ receptor, which regulates platelet activation and thrombus formation, is solved in complex with an antithrombotic drug, providing insights for the development of new drugs.

An X-ray structure of human P2Y₁₂ receptor, a clinical drug target for platelet aggregation inhibitors, is presented in complex with an agonist, providing insight into the δ-group of class A G-protein-coupled receptors.

The crystal structure of CCR2 chemokine receptor in a complex with two different antagonists—one orthosteric the other allosteric—which functionally cooperate to inhibit CCR2.

The crystal structure of the human nociceptin/orphanin FQ peptide receptor in complex with the peptide mimetic antagonist compound-24 is determined, with potential importance for the development of new therapeutic agents.

Serial femtosecond crystallography is a high-throughput, serial crystallography technique for studying macromolecular crystals at an X-ray free-electron laser. This Primer gives an overview of microcrystallization methods, sample delivery and data analysis for applications including membrane proteins, radiation damage-prone systems and time-resolved experiments.

Single-molecule FRET experiments with human A2A adenosine receptor in its apo and agonist-bound states in lipid nanodiscs provide insights into its conformational dynamics and activation.

This protocol describes procedures for the preparation and characterization of microcrystals for serial femtosecond crystallography in lipidic cubic phase (LCP-SFX) for protein structure determination at X-ray free-electron lasers (XFELs).