G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G proteinindependent 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 TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms § Correspondence to H. Eric Xu: Eric.Xu@vai.org. * These authors contributed equally.Contributions: Y.K. initiated the project, developed the expression and purification methods for rhodopsin-arrestin complex, and bulk-purified expression constructs and proteins used in LCP crystallization for the SFX method; X.E.Z. collected the synchrotron data, helped with the SFX data collection, processed the data, and solved the structures; X.G. expressed and purified rhodopsinarrestin complexes, characterized their binding and thermal stability, discovered the initial crystallization conditions with 9.7 MAG, prepared most crystals for synchrotron data collection, prepared all crystals for the final data collection by SFX, helped with SFX data collection, and established the initial cross-linking method for the rhodopsin-arrestin complex; Y.H. designed and performed Tango assays and disulfide bond cross-linking experiments; C.Z. developed the mammalian expression methods; P.W.dW helped with XFEL data processing and performed computational experiments; J.K., M.H.E.T., K. M. S-P., K. P., J. M., Y.J., X.Y.Z., and Q.C. performed cell culture, mutagenesis, protein purification, rhodopsin-arrestin binding experiments; W.L. and A.I. grew crystals and collected synchrotron data at APS and SFX data at LCLS, G.W.H. and Q.X. determined and validated the structure. Z.Z. and V.K. constructed the full model, the phosphorylated rhodopsin-arrestin model, and help writing the paper; D.W., S.L., D.J., C.K., Sh.B., and N.A. Z. helped with XFEL data collection and initial data analysis; S.B., M.M., and G.J.W. set up the XFEL experiment, performed the data collection, and commented on the paper. A.B., T.W., C.G., O.Y., and H.C. helped with XFEL data collection and data analysis, processed the data and helped with structure validation. G.M. W., B.P., and P.G. performed HDX experiments and helped with manuscript writing. J.L. helped initiate this collaborative project and with writing the paper. M.W. collected the 7.7 Å dataset at Swiss Light Source. A.M.,...

The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC)1. Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1–agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state2, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a ‘twin toggle switch’ of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros–Weinstein numbering3) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties.

Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals, providing a continuously-renewed source of material for serial femtosecond crystallography. Data collected from sub-10 μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere1. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S0 to S4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed2 technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S1 state and after double laser excitation (putative S3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn4CaO5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn3CaOx cubane in the S2 to S3 transition, as predicted by spectroscopic and computational studies3,4. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.