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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). We have recently established a procedure for serial femtosecond crystallography (SFX) in lipidic cubic phase (LCP) for protein structure determination at X-ray free-electron lasers (XFELs). LCP-SFX uses the gel-like LCP as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection. LCP is a liquid-crystalline mesophase composed of lipids and water. It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization. Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications. The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room-temperature high-resolution data with minimal effects of radiation damage from sub-10-μm crystals of membrane and soluble proteins that are difficult to crystallize, while eliminating the need for crystal harvesting and cryo-cooling. Compared with SFX methods for microcrystals in solution using liquid injectors, LCP-SFX reduces protein consumption by 2–3 orders of magnitude for data collection at currently available XFELs. The whole procedure typically takes 3–5 d, including the time required for the crystals to grow.

Metabotropic γ-aminobutyric acid receptors (GABA B ) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis 1 . GABA B belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits 2 . Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1–GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABA B . Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface. 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.

The angiotensin II receptors AT 1 R and AT 2 R serve as key components of the renin–angiotensin–aldosterone system. AT 1 R has a central role in the regulation of blood pressure, but the function of AT 2 R is unclear and it has a variety of reported effects. To identify the mechanisms that underlie the differences in function and ligand selectivity between these receptors, here we report crystal structures of human AT 2 R bound to an AT 2 R-selective ligand and to an AT 1 R/AT 2 R dual ligand, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins or β-arrestins, in agreement with the lack of signalling responses in standard cellular assays. Structure–activity relationship, docking and mutagenesis studies revealed the crucial interactions for ligand binding and selectivity. Our results thus provide insights into the structural basis of the distinct functions of the angiotensin receptors, and may guide the design of new selective ligands. Crystal structures of two complexes of the angiotensin II receptor AT 2 R 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. A new state for GPCRs The angiotensin receptors AT 1 R and AT 2 R are G-protein-coupled receptors (GPCRs) with important roles in blood pressure regulation. Although AT 2 R is an important drug target for cardioprotection and for treating neuropathic pain and is believed to counteract several effects mediated by AT 1 R, its structure and function are not well understood. In this work, the authors report several crystal structures of AT 2 R in complex with two tightly bound ligands. These structures show a significant conformational rearrangement of the transmembrane helices to an active-like state that is similar to other class A GPCRs, save for one remarkable difference. In the active-like conformation, helix VIII adopts a non-canonical position, which not only stabilizes the state but also blocks the canonical signalling pathway of GPCRs by preventing binding of the G protein and β-arrestin. This challenges the notion of differentiating these ligands as 'agonists' or 'antagonists', or terming the state as 'active', as it precludes signalling partner interactions.

Escherichia coli use a transmembrane sensor protein to sense nitrate in their external environment and initiate a biochemical response. Gushchin et al. compared crystal structures of portions of the NarQ receptor that included the transmembrane helices in ligand-bound or unbound states. The structures suggest a signaling mechanism by which piston- and lever-like movements are transmitted to response regulator proteins within the cell. Such two-component systems are very common in bacteria and, if better understood, might provide targets for antimicrobial therapies. Science , this issue p. eaah6345 Crystal structures show how sensing of nitrate occurs in bacteria. One of the major and essential classes of transmembrane (TM) receptors, present in all domains of life, is sensor histidine kinases, parts of two-component signaling systems (TCSs). The structural mechanisms of TM signaling by these sensors are poorly understood. We present crystal structures of the periplasmic sensor domain, the TM domain, and the cytoplasmic HAMP domain of the Escherichia coli nitrate/nitrite sensor histidine kinase NarQ in the ligand-bound and mutated ligand-free states. The structures reveal that the ligand binding induces rearrangements and pistonlike shifts of TM helices. The HAMP domain protomers undergo leverlike motions and convert these pistonlike motions into helical rotations. Our findings provide the structural framework for complete understanding of TM TCS signaling and for development of antimicrobial treatments targeting TCSs.

Melatonin ( N -acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms 1 by synchronization to environmental cues and is involved in diverse physiological processes 2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function 3 . Melatonin is formed in the pineal gland in a light-regulated manner 4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness 5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT 1 ) and type 1B (MT 2 ) 3 , 6 . Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden 7 . Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids 8 , 9 , and is one of the most popular supplements in the United States 10 . Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT 1 in complex with four agonists: the insomnia drug ramelteon 11 , two melatonin analogues, and the mixed melatonin–serotonin antidepressant agomelatine 12 , 13 . The structure of MT 2 is described in an accompanying paper 14 . Although the MT 1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT 1 , access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT 1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors. The MT 1 melatonin receptor differs markedly from 5-HT receptors and shows atypical ligand entry; its structure with various ligands sheds light on receptor specificity.

Despite remarkable progress, mainly due to the development of LCP and ‘bicelle’ crystallization, lack of structural information remains a bottleneck in membrane protein (MP) research. A major reason is the absence of complete understanding of the mechanism of crystallization. Here we present small-angle scattering studies of the evolution of the “bicelle” crystallization matrix in the course of MP crystal growth. Initially, the matrix corresponds to liquid-like bicelle state. However, after adding the precipitant, the crystallization matrix transforms to jelly-like state. The data suggest that this final phase is composed of interconnected ribbon-like bilayers, where crystals grow. A small amount of multilamellar phase appears, and its volume increases concomitantly with the volume of growing crystals. We suggest that the lamellar phase surrounds the crystals and is critical for crystal growth, which is also common for LCP crystallization. The study discloses mechanisms of “bicelle” MP crystallization and will support rational design of crystallization.

Cysteinyl leukotriene G protein-coupled receptors CysLT 1 and CysLT 2 regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT 1 R has been used for treating asthma and associated diseases for over two decades, CysLT 2 R has recently started to emerge as a potential drug target against atopic asthma, brain injury and central nervous system disorders, as well as several types of cancer. Here, we describe four crystal structures of CysLT 2 R in complex with three dual CysLT 1 R/CysLT 2 R antagonists. The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular determinants of CysLTR ligand selectivity and specific effects of disease-related single nucleotide variants. Cysteinyl leukotriene G protein-coupled receptors CysLT 1 and CysLT 2 regulate pro-inflammatory responses associated with allergic disorders. Here, authors describe four crystal structures of CysLT 2 R in complex with three dual CysLT 1 R/CysLT 2 R antagonists, which shed light on CysLTR ligand selectivity.

The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs. 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.

Light-driven proton pumps are present in many organisms. Here, we present a high-resolution structure of a proteorhodopsin from a permafrost bacterium, Exiguobacterium sibiricum rhodopsin (ESR). Contrary to the proton pumps of known structure, ESR possesses three unique features. First, ESR's proton donor is a lysine side chain that is situated very close to the bulk solvent. Second, the α-helical structure in the middle of the helix F is replaced by 3 ₁₀- and π-helix–like elements that are stabilized by the Trp-154 and Asn-224 side chains. This feature is characteristic for the proteorhodopsin family of proteins. Third, the proton release region is connected to the bulk solvent by a chain of water molecules already in the ground state. Despite these peculiarities, the positions of water molecule and amino acid side chains in the immediate Schiff base vicinity are very well conserved. These features make ESR a very unusual proton pump. The presented structure sheds light on the large family of proteorhodopsins, for which structural information was not available previously.

Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20-25�C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.