26 results on '"Schertler G"'
Search Results
2. The Structure of Bacteriorhodopsin and Its Relevance to the Visual Opsins and Other Seven-Helix G-Protein Coupled Receptors
- Author
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Henderson, R. and Schertler, G. F. X.
- Published
- 1990
3. Viral and Microbial Threats - a joint position paper by Analytical Research Infrastructures of Europe (ARIE)
- Author
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Cicchetti, G., Daalen, M. van, Gleizes, P.E., Leonard, G., Redlich, B., Schertler, G., Toimil Morales, M.E., Wacklin-Knecht, H., Cicchetti, G., Daalen, M. van, Gleizes, P.E., Leonard, G., Redlich, B., Schertler, G., Toimil Morales, M.E., and Wacklin-Knecht, H.
- Abstract
Contains fulltext : 228245.pdf (publisher's version ) (Open Access)
- Published
- 2020
4. Structure of the micro-opioid receptor-Gi protein complex
- Author
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Koehl, A., Hu, H., Maeda, S., Zhang, Y., Qu, Q., Paggi, J. M., Latorraca, N. R., Hilger, D., Dawson, R., Matile, H., Schertler, G. F. X., Granier, Sébastien, Weis, W. I., Dror, R. O., Manglik, A., Skiniotis, G., Kobilka, B. K., Institut de Génomique Fonctionnelle (IGF), and Université de Montpellier (UM)-Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Montpellier 2 - Sciences et Techniques (UM2)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
[SDV]Life Sciences [q-bio] - Published
- 2018
5. 4D Biology for health and disease
- Author
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Luxembourg Centre for Systems Biomedicine (LCSB): Experimental Neurobiology (Balling Group) [research center], Abrahams, J. P., Apweiler, R., Balling, Rudi, Bertero, M. G., Bujnicki, J. M., Chayen, N. E., Chène, P., Corthals, G. L., Dylag, T., Förster, F., Heck, A. J., Henderson, P. J., Herwig, R., Jehenson, P., Kokalj, S. J., Laue, E., Legrain, P., Martens, L., Migliorini, C., Musacchio, A., Podobnik, M., Schertler, G. F., Schreiber, G., Sixma, T. K., Smit, A. B., Stuart, D., Svergun, D. I., Taussig, M. J., Luxembourg Centre for Systems Biomedicine (LCSB): Experimental Neurobiology (Balling Group) [research center], Abrahams, J. P., Apweiler, R., Balling, Rudi, Bertero, M. G., Bujnicki, J. M., Chayen, N. E., Chène, P., Corthals, G. L., Dylag, T., Förster, F., Heck, A. J., Henderson, P. J., Herwig, R., Jehenson, P., Kokalj, S. J., Laue, E., Legrain, P., Martens, L., Migliorini, C., Musacchio, A., Podobnik, M., Schertler, G. F., Schreiber, G., Sixma, T. K., Smit, A. B., Stuart, D., Svergun, D. I., and Taussig, M. J.
- Abstract
The "4D Biology Workshop for Health and Disease", held on 16-17th of March 2010 in Brussels, aimed at finding the best organising principles for large-scale proteomics, interactomics and structural genomics/biology initiatives, and setting the vision for future high-throughput research and large-scale data gathering in biological and medical science. Major conclusions of the workshop include the following. (i) Development of new technologies and approaches to data analysis is crucial. Biophysical methods should be developed that span a broad range of time/spatial resolution and characterise structures and kinetics of interactions. Mathematics, physics, computational and engineering tools need to be used more in biology and new tools need to be developed. (ii) Database efforts need to focus on improved definitions of ontologies and standards so that system-scale data and associated metadata can be understood and shared efficiently. (iii) Research infrastructures should play a key role in fostering multidisciplinary research, maximising knowledge exchange between disciplines and facilitating access to diverse technologies. (iv) Understanding disease on a molecular level is crucial. System approaches may represent a new paradigm in the search for biomarkers and new targets in human disease. (v) Appropriate education and training should be provided to help efficient exchange of knowledge between theoreticians, experimental biologists and clinicians. These conclusions provide a strong basis for creating major possibilities in advancing research and clinical applications towards personalised medicine.
- Published
- 2011
6. Protein crystallization at the Laboratory of Molecular Biology: robotics, procedures and developments
- Author
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Gorrec, F.P.M., primary, Perisic, O., additional, Michie, K., additional, Schertler, G., additional, and Lowe, J., additional
- Published
- 2008
- Full Text
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7. Projection structure of frog rhodopsin in two crystal forms.
- Author
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Schertler, G F, primary and Hargrave, P A, additional
- Published
- 1995
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8. Ultrafast structural changes direct the first molecular events of vision.
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Gruhl T, Weinert T, Rodrigues MJ, Milne CJ, Ortolani G, Nass K, Nango E, Sen S, Johnson PJM, Cirelli C, Furrer A, Mous S, Skopintsev P, James D, Dworkowski F, Båth P, Kekilli D, Ozerov D, Tanaka R, Glover H, Bacellar C, Brünle S, Casadei CM, Diethelm AD, Gashi D, Gotthard G, Guixà-González R, Joti Y, Kabanova V, Knopp G, Lesca E, Ma P, Martiel I, Mühle J, Owada S, Pamula F, Sarabi D, Tejero O, Tsai CJ, Varma N, Wach A, Boutet S, Tono K, Nogly P, Deupi X, Iwata S, Neutze R, Standfuss J, Schertler G, and Panneels V
- Subjects
- Animals, Binding Sites radiation effects, Crystallography, Heterotrimeric GTP-Binding Proteins chemistry, Heterotrimeric GTP-Binding Proteins metabolism, Isomerism, Photons, Protein Binding radiation effects, Protein Conformation radiation effects, Retinaldehyde chemistry, Retinaldehyde metabolism, Retinaldehyde radiation effects, Time Factors, Rhodopsin chemistry, Rhodopsin metabolism, Rhodopsin radiation effects, Vision, Ocular physiology, Vision, Ocular radiation effects
- Abstract
Vision is initiated by the rhodopsin family of light-sensitive G protein-coupled receptors (GPCRs)
1 . A photon is absorbed by the 11-cis retinal chromophore of rhodopsin, which isomerizes within 200 femtoseconds to the all-trans conformation2 , thereby initiating the cellular signal transduction processes that ultimately lead to vision. However, the intramolecular mechanism by which the photoactivated retinal induces the activation events inside rhodopsin remains experimentally unclear. Here we use ultrafast time-resolved crystallography at room temperature3 to determine how an isomerized twisted all-trans retinal stores the photon energy that is required to initiate the protein conformational changes associated with the formation of the G protein-binding signalling state. The distorted retinal at a 1-ps time delay after photoactivation has pulled away from half of its numerous interactions with its binding pocket, and the excess of the photon energy is released through an anisotropic protein breathing motion in the direction of the extracellular space. Notably, the very early structural motions in the protein side chains of rhodopsin appear in regions that are involved in later stages of the conserved class A GPCR activation mechanism. Our study sheds light on the earliest stages of vision in vertebrates and points to fundamental aspects of the molecular mechanisms of agonist-mediated GPCR activation., (© 2023. The Author(s).)- Published
- 2023
- Full Text
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9. Efficient production of a functional G protein-coupled receptor in E. coli for structural studies.
- Author
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Abiko LA, Rogowski M, Gautier A, Schertler G, and Grzesiek S
- Subjects
- Escherichia coli Proteins chemistry, Escherichia coli Proteins isolation & purification, Gene Expression, Genetic Vectors genetics, Models, Molecular, Nuclear Magnetic Resonance, Biomolecular methods, Protein Binding, Protein Stability, Receptors, G-Protein-Coupled chemistry, Receptors, G-Protein-Coupled isolation & purification, Recombinant Proteins, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins biosynthesis, Escherichia coli Proteins genetics, Receptors, G-Protein-Coupled biosynthesis, Receptors, G-Protein-Coupled genetics
- Abstract
G protein-coupled receptors (GPCRs) are transmembrane signal transducers which regulate many key physiological process. Since their discovery, their analysis has been limited by difficulties in obtaining sufficient amounts of the receptors in high-quality, functional form from heterologous expression hosts. Albeit highly attractive because of its simplicity and the ease of isotope labeling for NMR studies, heterologous expression of functional GPCRs in E. coli has proven particularly challenging due to the absence of the more evolved protein expression and folding machinery of higher eukaryotic hosts. Here we first give an overview on the previous strategies for GPCR E. coli expression and then describe the development of an optimized robust protocol for the E. coli expression and purification of two mutants of the turkey β
1 -adrenergic receptor (β1 AR) uniformly or selectively labeled in15 N or2 H,15 N. These mutants had been previously optimized for thermal stability using insect cell expression and used successfully in crystallographic and NMR studies. The same sequences were then used for E. coli expression. Optimization of E. coli expression was achieved by a quantitative analysis of losses of receptor material at each step of the solubilization and purification procedure. Final yields are 0.2-0.3 mg receptor per liter culture. Whereas both expressed mutants are well folded and competent for orthosteric ligand binding, the less stable YY-β1 AR mutant also comprises the two native tyrosines Y5.58 and Y7.53 , which enable G protein binding. High-quality1 H-15 N TROSY spectra were obtained for E. coli-expressed YY-β1 AR in three different functional states (antagonist, agonist, and agonist + G protein-mimicking nanobody-bound), which are identical to spectra obtained of the same forms of the receptor expressed in insect cells. NdeI and AgeI restriction sites introduced into the expression plasmid allow for the easy replacement of the receptor gene by other GPCR genes of interest, and the provided quantitative workflow analysis may guide the respective adaptation of the purification protocol.- Published
- 2021
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10. Soluble dimeric prion protein ligand activates Adgrg6 receptor but does not rescue early signs of demyelination in PrP-deficient mice.
- Author
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Henzi A, Senatore A, Lakkaraju AKK, Scheckel C, Mühle J, Reimann R, Sorce S, Schertler G, Toyka KV, and Aguzzi A
- Subjects
- Animals, Cell Line, Demyelinating Diseases genetics, Female, Immunoglobulin Fc Fragments chemistry, Immunoglobulin Fc Fragments genetics, Male, Mice, Mice, Inbred C57BL, Peptide Fragments chemistry, Peptide Fragments genetics, Prion Proteins genetics, Recombinant Proteins chemistry, Recombinant Proteins pharmacology, Recombinant Proteins therapeutic use, Sciatic Nerve metabolism, Transcriptome, Demyelinating Diseases drug therapy, Peptide Fragments therapeutic use, Prion Proteins chemistry, Receptors, G-Protein-Coupled agonists
- Abstract
The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT2Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT2Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the early molecular signs demyelination was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2020
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11. Advances in long-wavelength native phasing at X-ray free-electron lasers.
- Author
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Nass K, Cheng R, Vera L, Mozzanica A, Redford S, Ozerov D, Basu S, James D, Knopp G, Cirelli C, Martiel I, Casadei C, Weinert T, Nogly P, Skopintsev P, Usov I, Leonarski F, Geng T, Rappas M, Doré AS, Cooke R, Nasrollahi Shirazi S, Dworkowski F, Sharpe M, Olieric N, Bacellar C, Bohinc R, Steinmetz MO, Schertler G, Abela R, Patthey L, Schmitt B, Hennig M, Standfuss J, Wang M, and Milne CJ
- Abstract
Long-wavelength pulses from the Swiss X-ray free-electron laser (XFEL) have been used for de novo protein structure determination by native single-wavelength anomalous diffraction (native-SAD) phasing of serial femtosecond crystallography (SFX) data. In this work, sensitive anomalous data-quality indicators and model proteins were used to quantify improvements in native-SAD at XFELs such as utilization of longer wavelengths, careful experimental geometry optimization, and better post-refinement and partiality correction. Compared with studies using shorter wavelengths at other XFELs and older software versions, up to one order of magnitude reduction in the required number of indexed images for native-SAD was achieved, hence lowering sample consumption and beam-time requirements significantly. Improved data quality and higher anomalous signal facilitate so-far underutilized de novo structure determination of challenging proteins at XFELs. Improvements presented in this work can be used in other types of SFX experiments that require accurate measurements of weak signals, for example time-resolved studies., (© Karol Nass et al. 2020.)
- Published
- 2020
- Full Text
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12. Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the gβ subunit.
- Author
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Tsai CJ, Marino J, Adaixo R, Pamula F, Muehle J, Maeda S, Flock T, Taylor NM, Mohammed I, Matile H, Dawson RJ, Deupi X, Stahlberg H, and Schertler G
- Subjects
- Animals, Cattle, Cryoelectron Microscopy, GTP-Binding Protein beta Subunits metabolism, Multiprotein Complexes ultrastructure, Protein Binding, Rhodopsin metabolism, GTP-Binding Protein alpha Subunits ultrastructure, GTP-Binding Protein beta Subunits ultrastructure, GTP-Binding Protein gamma Subunits ultrastructure, Rhodopsin ultrastructure
- Abstract
One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gβ subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gβ as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway., Competing Interests: CT, JM, RA, FP, JM, SM, TF, NT, IM, XD, HS No competing interests declared, HM, RD Employee of Hoffmann-La Roche Ltd, GS declares that he is a co-founder and scientific advisor of the company leadXpro AG and InterAx Biotech AG, and that he has been a member of the MAX IV Scientific Advisory Committee during the time when the research has been performed., (© 2019, Tsai et al.)
- Published
- 2019
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13. Perspective: Opportunities for ultrafast science at SwissFEL.
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Abela R, Beaud P, van Bokhoven JA, Chergui M, Feurer T, Haase J, Ingold G, Johnson SL, Knopp G, Lemke H, Milne CJ, Pedrini B, Radi P, Schertler G, Standfuss J, Staub U, and Patthey L
- Abstract
We present the main specifications of the newly constructed Swiss Free Electron Laser, SwissFEL, and explore its potential impact on ultrafast science. In light of recent achievements at current X-ray free electron lasers, we discuss the potential territory for new scientific breakthroughs offered by SwissFEL in Chemistry, Biology, and Materials Science, as well as nonlinear X-ray science.
- Published
- 2018
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14. Serial millisecond crystallography for routine room-temperature structure determination at synchrotrons.
- Author
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Weinert T, Olieric N, Cheng R, Brünle S, James D, Ozerov D, Gashi D, Vera L, Marsh M, Jaeger K, Dworkowski F, Panepucci E, Basu S, Skopintsev P, Doré AS, Geng T, Cooke RM, Liang M, Prota AE, Panneels V, Nogly P, Ermler U, Schertler G, Hennig M, Steinmetz MO, Wang M, and Standfuss J
- Abstract
Historically, room-temperature structure determination was succeeded by cryo-crystallography to mitigate radiation damage. Here, we demonstrate that serial millisecond crystallography at a synchrotron beamline equipped with high-viscosity injector and high frame-rate detector allows typical crystallographic experiments to be performed at room-temperature. Using a crystal scanning approach, we determine the high-resolution structure of the radiation sensitive molybdenum storage protein, demonstrate soaking of the drug colchicine into tubulin and native sulfur phasing of the human G protein-coupled adenosine receptor. Serial crystallographic data for molecular replacement already converges in 1,000-10,000 diffraction patterns, which we collected in 3 to maximally 82 minutes. Compared with serial data we collected at a free-electron laser, the synchrotron data are of slightly lower resolution, however fewer diffraction patterns are needed for de novo phasing. Overall, the data we collected by room-temperature serial crystallography are of comparable quality to cryo-crystallographic data and can be routinely collected at synchrotrons.Serial crystallography was developed for protein crystal data collection with X-ray free-electron lasers. Here the authors present several examples which show that serial crystallography using high-viscosity injectors can also be routinely employed for room-temperature data collection at synchrotrons.
- Published
- 2017
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15. Structural role of the T94I rhodopsin mutation in congenital stationary night blindness.
- Author
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Singhal A, Guo Y, Matkovic M, Schertler G, Deupi X, Yan EC, and Standfuss J
- Subjects
- Binding Sites, Catalytic Domain, Darkness, Genetic Association Studies, Humans, Models, Biological, Models, Molecular, Protein Binding, Protein Conformation, Protein Stability, Schiff Bases chemistry, Structure-Activity Relationship, Thermodynamics, Eye Diseases, Hereditary genetics, Genetic Diseases, X-Linked genetics, Mutation, Myopia genetics, Night Blindness genetics, Rhodopsin chemistry, Rhodopsin genetics
- Abstract
Congenital stationary night blindness (CSNB) is an inherited and non-progressive retinal dysfunction. Here, we present the crystal structure of CSNB-causing T94I
2.61 rhodopsin in the active conformation at 2.3 Å resolution. The introduced hydrophobic side chain prolongs the lifetime of the G protein activating metarhodopsin-II state by establishing a direct van der Waals contact with K2967.43 , the site of retinal attachment. This is in stark contrast to the light-activated state of the CSNB-causing G90D2.57 mutation, where the charged mutation forms a salt bridge with K2967.43 To find the common denominator between these two functional modifications, we combined our structural data with a kinetic biochemical analysis and molecular dynamics simulations. Our results indicate that both the charged G90D2.57 and the hydrophobic T94I2.61 mutation alter the dark state by weakening the interaction between the Schiff base (SB) and its counterion E1133.28 We propose that this interference with the tight regulation of the dim light photoreceptor rhodopsin increases background noise in the visual system and causes the loss of night vision characteristic for CSNB patients., (© 2016 The Authors.)- Published
- 2016
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16. Lipidic cubic phase injector is a viable crystal delivery system for time-resolved serial crystallography.
- Author
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Nogly P, Panneels V, Nelson G, Gati C, Kimura T, Milne C, Milathianaki D, Kubo M, Wu W, Conrad C, Coe J, Bean R, Zhao Y, Båth P, Dods R, Harimoorthy R, Beyerlein KR, Rheinberger J, James D, DePonte D, Li C, Sala L, Williams GJ, Hunter MS, Koglin JE, Berntsen P, Nango E, Iwata S, Chapman HN, Fromme P, Frank M, Abela R, Boutet S, Barty A, White TA, Weierstall U, Spence J, Neutze R, Schertler G, and Standfuss J
- Subjects
- Crystallography, X-Ray instrumentation, Feasibility Studies, Protein Conformation, Synchrotrons, Time Factors, Viscosity, X-Ray Absorption Spectroscopy instrumentation, X-Ray Absorption Spectroscopy methods, Bacteriorhodopsins chemistry, Crystallography, X-Ray methods, Lasers, Lipids chemistry
- Abstract
Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
- Published
- 2016
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17. Batch crystallization of rhodopsin for structural dynamics using an X-ray free-electron laser.
- Author
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Wu W, Nogly P, Rheinberger J, Kick LM, Gati C, Nelson G, Deupi X, Standfuss J, Schertler G, and Panneels V
- Subjects
- Animals, Cattle, Crystallization, Lasers statistics & numerical data, Rhodopsin chemistry, X-Ray Diffraction methods
- Abstract
Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.
- Published
- 2015
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18. Time-resolved structural studies with serial crystallography: A new light on retinal proteins.
- Author
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Panneels V, Wu W, Tsai CJ, Nogly P, Rheinberger J, Jaeger K, Cicchetti G, Gati C, Kick LM, Sala L, Capitani G, Milne C, Padeste C, Pedrini B, Li XD, Standfuss J, Abela R, and Schertler G
- Abstract
Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination.
- Published
- 2015
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19. Lipidic cubic phase serial millisecond crystallography using synchrotron radiation.
- Author
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Nogly P, James D, Wang D, White TA, Zatsepin N, Shilova A, Nelson G, Liu H, Johansson L, Heymann M, Jaeger K, Metz M, Wickstrand C, Wu W, Båth P, Berntsen P, Oberthuer D, Panneels V, Cherezov V, Chapman H, Schertler G, Neutze R, Spence J, Moraes I, Burghammer M, Standfuss J, and Weierstall U
- Abstract
Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.
- Published
- 2015
- Full Text
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20. Femtosecond X-ray diffraction from two-dimensional protein crystals.
- Author
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Frank M, Carlson DB, Hunter MS, Williams GJ, Messerschmidt M, Zatsepin NA, Barty A, Benner WH, Chu K, Graf AT, Hau-Riege SP, Kirian RA, Padeste C, Pardini T, Pedrini B, Segelke B, Seibert MM, Spence JC, Tsai CJ, Lane SM, Li XD, Schertler G, Boutet S, Coleman M, and Evans JE
- Abstract
X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.
- Published
- 2014
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21. Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II.
- Author
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Deupi X, Edwards P, Singhal A, Nickle B, Oprian D, Schertler G, and Standfuss J
- Subjects
- Animals, Binding Sites, Cattle, GTP-Binding Protein alpha Subunits, HEK293 Cells, Humans, Ions, Models, Molecular, Mutant Proteins chemistry, Mutation genetics, Protein Stability, Retinaldehyde chemistry, Spectrum Analysis, GTP-Binding Proteins metabolism, Rhodopsin chemistry, Rhodopsin metabolism
- Abstract
G protein-coupled receptors (GPCR) are seven transmembrane helix proteins that couple binding of extracellular ligands to conformational changes and activation of intracellular G proteins, GPCR kinases, and arrestins. Constitutively active mutants are ubiquitously found among GPCRs and increase the inherent basal activity of the receptor, which often correlates with a pathological outcome. Here, we have used the M257Y(6.40) constitutively active mutant of the photoreceptor rhodopsin in combination with the specific binding of a C-terminal fragment from the G protein alpha subunit (GαCT) to trap a light activated state for crystallization. The structure of the M257Y/GαCT complex contains the agonist all-trans-retinal covalently bound to the native binding pocket and resembles the G protein binding metarhodopsin-II conformation obtained by the natural activation mechanism; i.e., illumination of the prebound chromophore 11-cis-retinal. The structure further suggests a molecular basis for the constitutive activity of 6.40 substitutions and the strong effect of the introduced tyrosine based on specific interactions with Y223(5.58) in helix 5, Y306(7.53) of the NPxxY motif and R135(3.50) of the E(D)RY motif, highly conserved residues of the G protein binding site.
- Published
- 2012
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22. Structure of rhodopsin.
- Author
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Schertler GF
- Subjects
- Animals, Bacteriorhodopsins chemistry, Crystallography, GTP-Binding Proteins, Humans, Microscopy, Electron, Molecular Structure, Protein Structure, Secondary, Receptors, Cell Surface, Rhodopsin chemistry
- Abstract
Two-dimensional crystals of rhodopsin were studied to determine the arrangement of the transmembrane alpha helices. A combination of electron cryo-microscopy, image processing and electron crystallography was used to extract amplitudes and phases from images, and a three-dimensional map to a resolution of 7.5 A was calculated. Density peaks for all seven transmembrane helices were observed and the helix axes for all seven helices could be estimated. Near the intracellular side, which interacts with the G protein transducin, we observed three layers of helices arranged differently from bacteriorhodopsin. The arrangement opens up towards the extracellular side forming a cavity that serves as the binding pocket for the retinal. This cavity is closed towards the intracellular side by the long and highly tilted helix 3, and must be closed towards the extracellular side by the loop linking helices 4 and 5 that is linked by a disulphide bridge to the extracellular end of helix 3.
- Published
- 1998
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23. Arrangement of rhodopsin transmembrane alpha-helices.
- Author
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Unger VM, Hargrave PA, Baldwin JM, and Schertler GF
- Subjects
- Animals, Anura, Crystallography, Rhodopsin ultrastructure, Protein Conformation, Rhodopsin chemistry
- Abstract
Rhodopsins, the photoreceptors in rod cells, are G-protein-coupled receptors with seven hydrophobic segments containing characteristic conserved sequence patterns that define a large family. Members of the family are expected to share a conserved transmembrane structure. Direct evidence for the arrangement of seven alpha-helices was obtained from a 9A projection map of bovine rhodopsin. Structural constraints inferred from a comparison of G-protein-coupled receptor sequences were used to assign the seven hydrophobic stretches in the sequence to features in the projection map. A low-resolution three-dimensional structure of bovine rhodopsin and two projection structures of frog rhodopsin confirmed the position of the three least tilted helices, 4, 6 and 7. A more elongated peak of density for helix 5 indicated that it is tilted or bent, but helices 1, 2 and 3 were not resolved. Here we have used electron micrographs of frozen-hydrated two-dimensional frog rhodopsin crystals to determine the structure of frog rhodopsin. Seven rods of density in the map are used to estimate tilt angles for the seven helices. Density visible on the extracellular side of the membrane suggests a folded domain. Density extends from helix 6 on the intracellular side, and a short connection between helices 1 and 2, and possibly a part of the carboxy terminus, are visible.
- Published
- 1997
- Full Text
- View/download PDF
24. Low resolution structure of bovine rhodopsin determined by electron cryo-microscopy.
- Author
-
Unger VM and Schertler GF
- Subjects
- Animals, Bacteriorhodopsins chemistry, Cattle, Crystallization, Freezing, Microscopy, Electron methods, Models, Structural, Rhodopsin chemistry, Rhodopsin ultrastructure
- Abstract
The visual pigment rhodopsin is a member of the G protein-coupled receptor family. Electron cryo-microscopy was used to determine the three-dimensional structure of bovine rhodopsin from tilted two-dimensional crystals embedded in vitrified water. The effective resolution in a map obtained from the 23 best crystals was about 9.5 A horizontally and approximately 47 A normal to the plane of the membrane. Four clearly resolved tracks of density in the map correspond to four alpha-helices oriented nearly perpendicular to the plane of the membrane. One of these helices appears to be more tilted than anticipated from the projection structure published previously. The remaining three helices are presumably more highly tilted, given that they form a continuous "arc-shaped" feature and could not be resolved to the same extent. The overall density distribution in the low resolution map shows an arrangement of the helices in which the "arc-shaped" feature is extended by a fourth, less tilted helix. The band of these four tilted helices is flanked by a straight helix on the outer side and a pair of straight helices on its inner side.
- Published
- 1995
- Full Text
- View/download PDF
25. Projection structure of rhodopsin.
- Author
-
Schertler GF, Villa C, and Henderson R
- Subjects
- Animals, Cattle, Crystallization, Image Processing, Computer-Assisted, Lipid Bilayers, Microscopy, Electron, Rhodopsin ultrastructure
- Abstract
Light absorption by the visual pigment rhodopsin triggers, through G-protein coupling, a cascade of events in the outer segment of the rod cell of the vertebrate retina that results in membrane hyperpolarization and nerve excitation. Rhodopsin, which contains 348 amino acids, has seven helices that cross the disk membrane and its amino terminus is extracellular. A wealth of biochemical data is available for rhodopsin: 11-cis retinal is bound to lysine 296 in helix VII; glutamic acid 113 on helix III is the counterion to the protonated Schiff's base; a disulphide bridge, cystine 110-187, connects helix III to the second extracellular loop e2 (refs 13, 14); the carboxy terminus has two palmitoylated cysteines forming a cytoplasmic loop i4 (ref. 15); three intracellular loops i2, i3 and i4 mediate activation of the heterotrimeric G protein transducin; glutamic acid 135 and arginine 136 at the cytoplasmic end of helix III affect binding of transducin. But to provide a framework to interpret these data, not only for rhodopsin but for other G-protein-coupled receptors, requires the structure to be determined. Here we present a projection map of rhodopsin showing the configuration of the helices.
- Published
- 1993
- Full Text
- View/download PDF
26. Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.
- Author
-
Schertler GF, Lozier R, Michel H, and Oesterhelt D
- Subjects
- Bacteriorhodopsins genetics, Crystallization, Halobacterium metabolism, Kinetics, Microspectrophotometry, Mutation, Photochemistry, Protein Conformation, Bacteriorhodopsins chemistry
- Abstract
The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.
- Published
- 1991
- Full Text
- View/download PDF
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