All Publications


  • Structural basis of ligand recognition at the human MT1 melatonin receptor (vol 569, pg 284, 2019) NATURE Stauch, B., Johansson, L. C., McCorvy, J. D., Patel, N., Han, G., Huang, X., Gati, C., Batyuk, A., Slocum, S. T., Ishchenko, A., Brehm, W., White, T. A., Michaelian, N., Madsen, C., Zhu, L., Grant, T. D., Grandner, J. M., Shiriaeva, A., Olsen, R. J., Tribo, A. R., Yous, S., Stevens, R. C., Weierstall, U., Katritch, V., Roth, B. L., Liu, W., Cherezov, V. 2019; 569 (7756): E6
  • XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity NATURE Johansson, L. C., Stauch, B., McCorvy, J. D., Han, G., Patel, N., Huang, X., Batyuk, A., Gati, C., Slocum, S. T., Li, C., Grandner, J. M., Hao, S., Olsen, R. J., Tribo, A. R., Zaare, S., Zhu, L., Zatsepin, N. A., Weierstall, U., Yous, S., Stevens, R. C., Liu, W., Roth, B. L., Katritch, V., Cherezov, V. 2019; 569 (7755): 289-+
  • Structural basis of ligand recognition at the human MT1 melatonin receptor NATURE Stauch, B., Johansson, L. C., McCorvy, J. D., Patel, N., Han, G., Huang, X., Gati, C., Batyuk, A., Slocum, S. T., Ishchenko, A., Brehm, W., White, T. A., Michaelian, N., Madsen, C., Zhu, L., Grant, T. D., Grandner, J. M., Shiriaeva, A., Olsen, R. J., Tribo, A. R., Yous, S., Stevens, R. C., Weierstall, U., Katritch, V., Roth, B. L., Liu, W., Cherezov, V. 2019; 569 (7755): 284-+
  • XFEL structures of the human MT2 melatonin receptor reveal the basis of subtype selectivity. Nature Johansson, L. C., Stauch, B., McCorvy, J. D., Han, G. W., Patel, N., Huang, X., Batyuk, A., Gati, C., Slocum, S. T., Li, C., Grandner, J. M., Hao, S., Olsen, R. H., Tribo, A. R., Zaare, S., Zhu, L., Zatsepin, N. A., Weierstall, U., Yous, S., Stevens, R. C., Liu, W., Roth, B. L., Katritch, V., Cherezov, V. 2019

    Abstract

    The human MT1 and MT2 melatonin receptors1,2 are 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 MT2 has also been implicated in type 2 diabetes4,5. Here we report X-ray free electron laser (XFEL) structures of the human MT2 receptor in complex with the agonists 2-phenylmelatonin (2-PMT) and ramelteon6 at resolutions of 2.8A and 3.3A, 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 MT2 with a published structure8 of MT1 reveals 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 MT1 and MT2, but in addition the MT2 structures 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.

    View details for PubMedID 31019305

  • Structural basis of ligand recognition at the human MT1 melatonin receptor. Nature Stauch, B., Johansson, L. C., McCorvy, J. D., Patel, N., Han, G. W., Huang, X., Gati, C., Batyuk, A., Slocum, S. T., Ishchenko, A., Brehm, W., White, T. A., Michaelian, N., Madsen, C., Zhu, L., Grant, T. D., Grandner, J. M., Shiriaeva, A., Olsen, R. H., Tribo, A. R., Yous, S., Stevens, R. C., Weierstall, U., Katritch, V., Roth, B. L., Liu, W., Cherezov, V. 2019

    Abstract

    Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)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 burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 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 MT1, 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 MT1 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.

    View details for PubMedID 31019306

  • Publisher Correction: Structural basis of ligand recognition at the human MT1 melatonin receptor. Nature Stauch, B., Johansson, L. C., McCorvy, J. D., Patel, N., Han, G. W., Huang, X. P., Gati, C., Batyuk, A., Slocum, S. T., Ishchenko, A., Brehm, W., White, T. A., Michaelian, N., Madsen, C., Zhu, L., Grant, T. D., Grandner, J. M., Shiriaeva, A., Olsen, R. H., Tribo, A. R., Yous, S., Stevens, R. C., Weierstall, U., Katritch, V., Roth, B. L., Liu, W., Cherezov, V. 2019

    Abstract

    Change history: In this Letter, the rotation signs around 90°, 135° and 15° were missing and in the HTML, Extended Data Tables 2 and 3 were the wrong tables; these errors have been corrected online.

    View details for PubMedID 31048811

  • Publisher Correction: Crystal structure of misoprostol bound to the labor inducer prostaglandin E2 receptor. Nature chemical biology Audet, M., White, K. L., Breton, B., Zarzycka, B., Han, G. W., Lu, Y., Gati, C., Batyuk, A., Popov, P., Velasquez, J., Manahan, D., Hu, H., Weierstall, U., Liu, W., Shui, W., Katritch, V., Cherezov, V., Hanson, M. A., Stevens, R. C. 2018

    Abstract

    In the version of this article originally published, the present address for Petr Popov was incorrectly listed as 'Koltech Institute of Science & Technology, Moscow, Russia'. The correct present address is 'Skolkovo Institute of Science and Technology, Moscow, Russia'. The error has been corrected in the HTML and PDF versions of the paper.

    View details for PubMedID 30573766

  • Crystal structure of misoprostol bound to the labor inducer prostaglandin E2 receptor. Nature chemical biology Audet, M., White, K. L., Breton, B., Zarzycka, B., Han, G. W., Lu, Y., Gati, C., Batyuk, A., Popov, P., Velasquez, J., Manahan, D., Hu, H., Weierstall, U., Liu, W., Shui, W., Katritch, V., Cherezov, V., Hanson, M. A., Stevens, R. C. 2018

    Abstract

    Misoprostol is a life-saving drug in many developing countries for women at risk of post-partum hemorrhaging owing to its affordability, stability, ease of administration and clinical efficacy. However, misoprostol lacks receptor and tissue selectivities, and thus its use is accompanied by a number of serious side effects. The development of pharmacological agents combining the advantages of misoprostol with improved selectivity is hindered by the absence of atomic details of misoprostol action in labor induction. Here, we present the 2.5A resolution crystal structure of misoprostol free-acid form bound to the myometrium labor-inducing prostaglandin E2 receptor 3 (EP3). The active state structure reveals a completely enclosed binding pocket containing a structured water molecule that coordinates misoprostol's ring structure. Modeling of selective agonists in the EP3 structure reveals rationales for selectivity. These findings will provide the basis for the next generation of uterotonic drugs that will be suitable for administration in low resource settings.

    View details for PubMedID 30510194

  • Structure of the 30S ribosomal decoding complex at ambient temperature. RNA (New York, N.Y.) Dao, E. H., Poitevin, F., Sierra, R. G., Gati, C., Rao, Y., Ciftci, H. I., Aksit, F., McGurk, A., Obrinski, T., Mgbam, P., Hayes, B., DE Lichtenberg, C., Pardo-Avila, F., Corsepius, N., Zhang, L., Seaberg, M. H., Hunter, M. S., Liang, M., Koglin, J. E., Wakatsuki, S., Demirci, H. 2018

    Abstract

    The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New lightsources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 A resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA-protein complexes at near-physiological temperatures.

    View details for PubMedID 30139800

  • Aminoglycoside ribosome interactions reveal novel conformational states at ambient temperature. Nucleic acids research O'Sullivan, M. E., Poitevin, F., Sierra, R. G., Gati, C., Dao, E. H., Rao, Y., Aksit, F., Ciftci, H., Corsepius, N., Greenhouse, R., Hayes, B., Hunter, M. S., Liang, M., McGurk, A., Mbgam, P., Obrinsky, T., Pardo-Avila, F., Seaberg, M. H., Cheng, A. G., Ricci, A. J., DeMirci, H. 2018

    Abstract

    The bacterial 30S ribosomal subunit is a primary antibiotic target. Despite decades of discovery, the mechanisms by which antibiotic binding induces ribosomal dysfunction are not fully understood. Ambient temperature crystallographic techniques allow more biologically relevant investigation of how local antibiotic binding site interactions trigger global subunit rearrangements that perturb protein synthesis. Here, the structural effects of 2-deoxystreptamine (paromomycin and sisomicin), a novel sisomicin derivative, N1-methyl sulfonyl sisomicin (N1MS) and the non-deoxystreptamine (streptomycin) aminoglycosides on the ribosome at ambient and cryogenic temperatures were examined. Comparative studies led to three main observations. First, individual aminoglycoside-ribosome interactions in the decoding center were similar for cryogenic versus ambient temperature structures. Second, analysis of a highly conserved GGAA tetraloop of h45 revealed aminoglycoside-specific conformational changes, which are affected by temperature only for N1MS. We report the h44-h45 interface in varying states, i.e. engaged, disengaged and in equilibrium. Third, we observe aminoglycoside-induced effects on 30S domain closure, including a novel intermediary closure state, which is also sensitive to temperature. Analysis of three ambient and five cryogenic crystallography datasets reveal a correlation between h44-h45 engagement and domain closure. These observations illustrate the role of ambient temperature crystallography in identifying dynamic mechanisms of ribosomal dysfunction induced by local drug-binding site interactions. Together, these data identify tertiary ribosomal structural changes induced by aminoglycoside binding that provides functional insight and targets for drug design.

    View details for PubMedID 30113694

  • Structural biology of G protein-coupled receptors: new opportunities from XFELs and cryoEM CURRENT OPINION IN STRUCTURAL BIOLOGY Ishchenko, A., Gati, C., Cherezov, V. 2018; 51: 44–52

    Abstract

    G protein-coupled receptors mediate cell signaling and regulate the majority of sensory and physiological processes in the human body. Recent breakthroughs in cryo-electron microscopy and X-ray free electron lasers have accelerated structural studies of difficult-to-crystallize receptors and their signaling complexes, and have opened up new opportunities in understanding conformational dynamics and visualizing the process of receptor activation with unprecedented spatial and temporal resolution. Here, we summarize major milestones and challenges associated with the application of these techniques and outline future directions in their development with a focus on membrane protein structural biology.

    View details for PubMedID 29554543

    View details for PubMedCentralID PMC6139287

  • Cryo-EM structure of the human neutral amino acid transporter ASCT2 NATURE STRUCTURAL & MOLECULAR BIOLOGY Garaeva, A. A., Oostergetel, G. T., Gati, C., Guskov, A., Paulino, C., Slotboom, D. J. 2018; 25 (6): 515-+

    Abstract

    Human ASCT2 belongs to the SLC1 family of secondary transporters and is specific for the transport of small neutral amino acids. ASCT2 is upregulated in cancer cells and serves as the receptor for many retroviruses; hence, it has importance as a potential drug target. Here we used single-particle cryo-EM to determine a structure of the functional and unmodified human ASCT2 at 3.85-Å resolution. ASCT2 forms a homotrimeric complex in which each subunit contains a transport and a scaffold domain. Prominent extracellular extensions on the scaffold domain form the predicted docking site for retroviruses. Relative to structures of other SLC1 members, ASCT2 is in the most extreme inward-oriented state, with the transport domain largely detached from the central scaffold domain on the cytoplasmic side. This domain detachment may be required for substrate binding and release on the intracellular side of the membrane.

    View details for PubMedID 29872227

  • Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II NATURE METHODS Sierra, R. G., Gati, C., Laksmono, H., Dao, E. H., Gul, S., Fuller, F., Kern, J., Chatterjee, R., Ibrahim, M., Brewster, A. S., Young, I. D., Michels-Clark, T., Aquila, A., Liang, M., Hunter, M. S., Koglin, J. E., Boutet, S., Junco, E. A., Hayes, B., Bogan, M. J., Hampton, C. Y., Puglisi, E. V., Sauter, N. K., Stan, C. A., Zouni, A., Yano, J., Yachandra, V. K., Soltis, S. M., Puglisi, J. D., DeMirci, H. 2016; 13 (1): 59-?

    Abstract

    We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

    View details for DOI 10.1038/NMETH.3667

    View details for Web of Science ID 000367463600028

    View details for PubMedCentralID PMC4890631

  • Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II. Nature methods Sierra, R. G., Gati, C., Laksmono, H., Dao, E. H., Gul, S., Fuller, F., Kern, J., Chatterjee, R., Ibrahim, M., Brewster, A. S., Young, I. D., Michels-Clark, T., Aquila, A., Liang, M., Hunter, M. S., Koglin, J. E., Boutet, S., Junco, E. A., Hayes, B., Bogan, M. J., Hampton, C. Y., Puglisi, E. V., Sauter, N. K., Stan, C. A., Zouni, A., Yano, J., Yachandra, V. K., Soltis, S. M., Puglisi, J. D., DeMirci, H. 2016; 13 (1): 59–62

    Abstract

    We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).

    View details for PubMedID 26619013

  • Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser NATURE Kang, Y., Zhou, X. E., Gao, X., He, Y., Liu, W., Ishchenko, A., Barty, A., Sathish, D., Yefanov, O., Han, G. W., Xu, Q., de Waal, P. W., Ke, J., Tan, M. H., Zhang, C., Moeller, A., West, G. M., Pascal, B. D., Van Eps, N., Caro, L. N., Vishnivetskiy, S. A., Lee, R. J., Suino-Powell, K. M., Gu, X., Pal, K., Ma, J., Zhi, X., Boutet, S., Williams, G. J., Messerschmidt, M., Gati, C., Zatsepin, N. A., Wang, D., James, D., Basu, S., Roy-Chowdhury, S., Conrad, C. E., Coe, J., Liu, H., Lisova, S., Kupitz, C., Grotjohann, I., Fromme, R., Jiang, Y., Tan, M., Yang, H., Li, J., Wang, M., Zheng, Z., Li, D., Howe, N., Zhao, Y., Standfuss, J., Diederichs, K., Dong, Y., Potter, C. S., Carragher, B., Caffrey, M., Jiang, H., Chapman, H. N., Spence, J. C., Fromme, P., Weierstall, U., Ernst, O. P., Katritch, V., Gurevich, V. V., Griffin, P. R., Hubbell, W. L., Stevens, R. C., Cherezov, V., Melcher, K., Xu, H. E. 2015; 523 (7562): 561-?

    Abstract

    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.

    View details for DOI 10.1038/nature14656

    View details for Web of Science ID 000358655200038

    View details for PubMedCentralID PMC4521999

  • Structure of the Angiotensin Receptor Revealed by Serial Femtosecond Crystallography CELL Zhang, H., Unal, H., Gati, C., Han, G. W., Liu, W., Zatsepin, N. A., James, D., Wang, D., Nelson, G., Weierstall, U., Sawaya, M. R., Xu, Q., Messerschmidt, M., Williams, G. J., Boutet, S., Yefanov, O. M., White, T. A., Wang, C., Ishchenko, A., Tirupula, K. C., Desnoyer, R., Coe, J., Conrad, C. E., Fromme, P., Stevens, R. C., Katritch, V., Karnik, S. S., Cherezov, V. 2015; 161 (4): 833-844

    Abstract

    Angiotensin II type 1 receptor (AT(1)R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT(1)R blockers (ARBs), the structural basis for AT(1)R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT(1)R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT(1)R-ZD7155 complex structure revealed key structural features of AT(1)R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT(1)R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT(1)R structure-function relationship and structure-based drug design.

    View details for DOI 10.1016/j.cell.2015.04.011

    View details for Web of Science ID 000354175200016

    View details for PubMedID 25913193

    View details for PubMedCentralID PMC4427029

  • Serial femtosecond X-ray diffraction of 30S ribosomal subunit microcrystals in liquid suspension at ambient temperature using an X-ray free-electron laser ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS Demirci, H., Sierra, R. G., Laksmono, H., Shoeman, R. L., Botha, S., Barends, T. R., Nass, K., Schlichting, I., Doak, R. B., Gati, C., Williams, G. J., Boutet, S., Messerschmidt, M., Jogl, G., Dahlberg, A. E., Gregory, S. T., Bogan, M. J. 2013; 69: 1066-1069

    Abstract

    High-resolution ribosome structures determined by X-ray crystallography have provided important insights into the mechanism of translation. Such studies have thus far relied on large ribosome crystals kept at cryogenic temperatures to reduce radiation damage. Here, the application of serial femtosecond X-ray crystallography (SFX) using an X-ray free-electron laser (XFEL) to obtain diffraction data from ribosome microcrystals in liquid suspension at ambient temperature is described. 30S ribosomal subunit microcrystals diffracted to beyond 6 Å resolution, demonstrating the feasibility of using SFX for ribosome structural studies. The ability to collect diffraction data at near-physiological temperatures promises to provide fundamental insights into the structural dynamics of the ribosome and its functional complexes.

    View details for DOI 10.1107/S174430911302099X

    View details for Web of Science ID 000323719700026

    View details for PubMedID 23989164

    View details for PubMedCentralID PMC3758164