25 results on '"Conrad, Chelsie E."'
Search Results
2. Ultrafast structural changes within a photosynthetic reaction centre
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Dods, Robert, Båth, Petra, Morozov, Dmitry, Gagnér, Viktor Ahlberg, Arnlund, David, Luk, Hoi Ling, Kübel, Joachim, Maj, Michał, Vallejos, Adams, Wickstrand, Cecilia, Bosman, Robert, Beyerlein, Kenneth R., Nelson, Garrett, Liang, Mengning, Milathianaki, Despina, Robinson, Joseph, Harimoorthy, Rajiv, Berntsen, Peter, Malmerberg, Erik, Johansson, Linda, Andersson, Rebecka, Carbajo, Sergio, Claesson, Elin, Conrad, Chelsie E., Dahl, Peter, Hammarin, Greger, Hunter, Mark S., Li, Chufeng, Lisova, Stella, Royant, Antoine, Safari, Cecilia, Sharma, Amit, Williams, Garth J., Yefanov, Oleksandr, Westenhoff, Sebastian, Davidsson, Jan, DePonte, Daniel P., Boutet, Sébastien, Barty, Anton, Katona, Gergely, Groenhof, Gerrit, Brändén, Gisela, and Neutze, Richard
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- 2021
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3. Synchronous RNA conformational changes trigger ordered phase transitions in crystals
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Ramakrishnan, Saminathan, Stagno, Jason R., Conrad, Chelsie E., Ding, Jienyu, Yu, Ping, Bhandari, Yuba R., Lee, Yun-Tzai, Pauly, Gary, Yefanov, Oleksandr, Wiedorn, Max O., Knoska, Juraj, Oberthür, Dominik, White, Thomas A., Barty, Anton, Mariani, Valerio, Li, Chufeng, Brehm, Wolfgang, Heinz, William F., Magidson, Valentin, Lockett, Stephen, Hunter, Mark S., Boutet, Sébastien, Zatsepin, Nadia A., Zuo, Xiaobing, Grant, Thomas D., Pandey, Suraj, Schmidt, Marius, Spence, John C. H., Chapman, Henry N., and Wang, Yun-Xing
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- 2021
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4. Segmented flow generator for serial crystallography at the European X-ray free electron laser
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Echelmeier, Austin, Cruz Villarreal, Jorvani, Messerschmidt, Marc, Kim, Daihyun, Coe, Jesse D., Thifault, Darren, Botha, Sabine, Egatz-Gomez, Ana, Gandhi, Sahir, Brehm, Gerrit, Conrad, Chelsie E., Hansen, Debra T., Madsen, Caleb, Bajt, Saša, Meza-Aguilar, J. Domingo, Oberthür, Dominik, Wiedorn, Max O., Fleckenstein, Holger, Mendez, Derek, Knoška, Juraj, Martin-Garcia, Jose M., Hu, Hao, Lisova, Stella, Allahgholi, Aschkan, Gevorkov, Yaroslav, Ayyer, Kartik, Aplin, Steve, Ginn, Helen Mary, Graafsma, Heinz, Morgan, Andrew J., Greiffenberg, Dominic, Klujev, Alexander, Laurus, Torsten, Poehlsen, Jennifer, Trunk, Ulrich, Mezza, Davide, Schmidt, Bernd, Kuhn, Manuela, Fromme, Raimund, Sztuk-Dambietz, Jolanta, Raab, Natascha, Hauf, Steffen, Silenzi, Alessandro, Michelat, Thomas, Xu, Chen, Danilevski, Cyril, Parenti, Andrea, Mekinda, Leonce, Weinhausen, Britta, Mills, Grant, Vagovic, Patrik, Kim, Yoonhee, Kirkwood, Henry, Bean, Richard, Bielecki, Johan, Stern, Stephan, Giewekemeyer, Klaus, Round, Adam R., Schulz, Joachim, Dörner, Katerina, Grant, Thomas D., Mariani, Valerio, Barty, Anton, Mancuso, Adrian P., Weierstall, Uwe, Spence, John C. H., Chapman, Henry N., Zatsepin, Nadia, Fromme, Petra, Kirian, Richard A., and Ros, Alexandra
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- 2020
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5. Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature
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Ishigami, Izumi, Zatsepin, Nadia A., Hikita, Masahide, Conrad, Chelsie E., Nelson, Garrett, Coe, Jesse D., Basu, Shibom, Grant, Thomas D., Seaberg, Matthew H., Sierra, Raymond G., Hunter, Mark S., Fromme, Petra, Fromme, Raimund, Yeh, Syun-Ru, and Rousseau, Denis L.
- Published
- 2017
6. Detection of a Geminate Photoproduct of Bovine Cytochrome c Oxidase by Time-Resolved Serial Femtosecond Crystallography.
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Ishigami, Izumi, Carbajo, Sergio, Zatsepin, Nadia, Hikita, Masahide, Conrad, Chelsie E., Nelson, Garrett, Coe, Jesse, Basu, Shibom, Grant, Thomas, Seaberg, Matthew H., Sierra, Raymond G., Hunter, Mark S., Fromme, Petra, Fromme, Raimund, Rousseau, Denis L., and Yeh, Syun-Ru
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- 2023
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7. Microcrystallization techniques for serial femtosecond crystallography using photosystem II from Thermosynechococcus elongatus as a model system
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Kupitz, Christopher, Grotjohann, Ingo, Conrad, Chelsie E., Roy-Chowdhury, Shatabdi, Fromme, Raimund, and Fromme, Petra
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- 2014
8. Real‐time crystallographic studies of the adenine riboswitch using an X‐ray free‐electron laser
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Stagno, Jason R., Bhandari, Yuba R., Conrad, Chelsie E., Liu, Yu, and Wang, Yun‐Xing
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- 2017
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9. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser
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Kang, Yanyong, Zhou, X. Edward, Gao, Xiang, He, Yuanzheng, Liu, Wei, Ishchenko, Andrii, Barty, Anton, White, Thomas A., Yefanov, Oleksandr, Han, Gye Won, Xu, Qingping, de Waal, Parker W., Ke, Jiyuan, Tan, M. H. Eileen, Zhang, Chenghai, Moeller, Arne, West, Graham M., Pascal, Bruce D., Van Eps, Ned, Caro, Lydia N., Vishnivetskiy, Sergey A., Lee, Regina J., Suino-Powell, Kelly M., Gu, Xin, Pal, Kuntal, Ma, Jinming, Zhi, Xiaoyong, Boutet, Sébastien, Williams, Garth J., Messerschmidt, Marc, Gati, Cornelius, Zatsepin, Nadia A., Wang, Dingjie, James, Daniel, Basu, Shibom, Roy-Chowdhury, Shatabdi, Conrad, Chelsie E., Coe, Jesse, Liu, Haiguang, Lisova, Stella, Kupitz, Christopher, Grotjohann, Ingo, Fromme, Raimund, Jiang, Yi, Tan, Minjia, Yang, Huaiyu, Li, Jun, Wang, Meitian, Zheng, Zhong, Li, Dianfan, Howe, Nicole, Zhao, Yingming, Standfuss, Jörg, Diederichs, Kay, Dong, Yuhui, Potter, Clinton S., Carragher, Bridget, Caffrey, Martin, Jiang, Hualiang, Chapman, Henry N., Spence, John C. H., Fromme, Petra, Weierstall, Uwe, Ernst, Oliver P., Katritch, Vsevolod, Gurevich, Vsevolod V., Griffin, Patrick R., Hubbell, Wayne L., Stevens, Raymond C., Cherezov, Vadim, Melcher, Karsten, and Xu, H. Eric
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- 2015
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10. Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser
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Kupitz, Christopher, Basu, Shibom, Grotjohann, Ingo, Fromme, Raimund, Zatsepin, Nadia A., Rendek, Kimberly N., Hunter, Mark S., Shoeman, Robert L., White, Thomas A., Wang, Dingjie, James, Daniel, Yang, Jay-How, Cobb, Danielle E., Reeder, Brenda, Sierra, Raymond G., Liu, Haiguang, Barty, Anton, Aquila, Andrew L., Deponte, Daniel, Kirian, Richard A., Bari, Sadia, Bergkamp, Jesse J., Beyerlein, Kenneth R., Bogan, Michael J., Caleman, Carl, Chao, Tzu-Chiao, Conrad, Chelsie E., Davis, Katherine M., Fleckenstein, Holger, Galli, Lorenzo, Hau-Riege, Stefan P., Kassemeyer, Stephan, Laksmono, Hartawan, Liang, Mengning, Lomb, Lukas, Marchesini, Stefano, Martin, Andrew V., Messerschmidt, Marc, Milathianaki, Despina, Nass, Karol, Ros, Alexandra, Roy-Chowdhury, Shatabdi, Schmidt, Kevin, Seibert, Marvin, Steinbrener, Jan, Stellato, Francesco, Yan, Lifen, Yoon, Chunhong, Moore, Thomas A., Moore, Ana L., Pushkar, Yulia, Williams, Garth J., Boutet, Sébastien, Doak, R. Bruce, Weierstall, Uwe, Frank, Matthias, Chapman, Henry N., Spence, John C. H., and Fromme, Petra
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- 2014
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11. Macromolecular diffractive imaging using imperfect crystals
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Ayyer, Kartik, Yefanov, Oleksandr M., Oberthür, Dominik, Roy-Chowdhury, Shatabdi, Galli, Lorenzo, Mariani, Valerio, Basu, Shibom, Coe, Jesse, Conrad, Chelsie E., Fromme, Raimund, Schaffer, Alexander, Dörner, Katerina, James, Daniel, Kupitz, Christopher, Metz, Markus, Nelson, Garrett, Xavier, Paulraj Lourdu, Beyerlein, Kenneth R., Schmidt, Marius, Sarrou, Iosifina, Spence, John C. H., Weierstall, Uwe, White, Thomas A., Yang, Jay-How, Zhao, Yun, Liang, Mengning, Aquila, Andrew, Hunter, Mark S., Robinson, Joseph S., Koglin, Jason E., Boutet, Sébastien, Fromme, Petra, Barty, Anton, and Chapman, Henry N.
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- 2016
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12. Structural basis for bifunctional peptide recognition at human delta-opioid receptor. IF 13.338
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Fenalti, Gustavo, Zatsepin, Nadia, Betti, Cecilia, Giguere, Patrick, Han, Gye Won, Ishchenko, Andrii, Liu, Wei, Guillemyn, Karel, Zhang, Haitao, James, Daniel, Wang, Dingjie, Weierstall, Uwe, Spence, John CH, Boutet, Sébastien, Messerschmidt, Marc, Williams, Garth J, Gati, Cornelius, Yefanov, Oleksandr M, White, Thomas A, Oberthuer, Dominik, Metz, Markus, Yoon, Chun Hong, Barty, Anton, Chapman, Henry N, Basu, Shibom, Coe, Jesse, Conrad, Chelsie E, Fromme, Raimund, Fromme, Petra, Tourwe, Dirk, Schiller, Peter, Roth, Bryan L, Ballet, Steven, Katritch, Vsevolod, Stevens, Raymond C, Cherezov, Vadim, Chemistry, Faculty of Sciences and Bioengineering Sciences, and Organic Chemistry
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SERIAL FEMTOSECOND CRYSTALLOGRAPHY ,MU AGONIST/DELTA ANTAGONIST ,POTENT ,TOLERANCE ,DEPENDENCE ,MORPHINE ,BOND ,DMT - Abstract
Bifunctional mu- and delta-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human delta-OR bound to the bifunctional delta-OR antagonist and mu-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics.
- Published
- 2015
13. Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature.
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Izumi Ishigami, Zatsepin, Nadia A., Masahide Hikita, Conrad, Chelsie E., Nelson, Garrett, Coe, Jesse D., Shibom Basu, Grant, Thomas D., Seaberg, Matthew H., Sierra, Raymond G., Hunter, Mark S., Fromme, Petra, Fromme, Raimund, Syun-Ru Yeh, and Rousseau, Denis L.
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CRYSTAL structure ,CYTOCHROMES ,OXIDASES ,X-ray crystallography ,ENZYMES - Abstract
Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a
3 iron atom, with a bent Fe-C-O angle of ~142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near CuB , which, in turn, moves closer to the heme a3 iron by ~0.38 Å. Structural comparison reveals that ligand binding to the heme a3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a3 , thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry. [ABSTRACT FROM AUTHOR]- Published
- 2017
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14. Serial femtosecond crystallography: A revolution in structural biology.
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Martin-Garcia, Jose M., Conrad, Chelsie E., Coe, Jesse, Roy-Chowdhury, Shatabdi, and Fromme, Petra
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CRYSTALLOGRAPHY , *SYNCHROTRON radiation sources , *ELECTRONIC data processing , *RADIATION damage , *SINGLE crystals - Abstract
Macromolecular crystallography at synchrotron sources has proven to be the most influential method within structural biology, producing thousands of structures since its inception. While its utility has been instrumental in progressing our knowledge of structures of molecules, it suffers from limitations such as the need for large, well-diffracting crystals, and radiation damage that can hamper native structural determination. The recent advent of X-ray free electron lasers (XFELs) and their implementation in the emerging field of serial femtosecond crystallography (SFX) has given rise to a remarkable expansion upon existing crystallographic constraints, allowing structural biologists access to previously restricted scientific territory. SFX relies on exceptionally brilliant, micro-focused X-ray pulses, which are femtoseconds in duration, to probe nano/micrometer sized crystals in a serial fashion. This results in data sets comprised of individual snapshots, each capturing Bragg diffraction of single crystals in random orientations prior to their subsequent destruction. Thus structural elucidation while avoiding radiation damage, even at room temperature, can now be achieved. This emerging field has cultivated new methods for nanocrystallogenesis, sample delivery, and data processing. Opportunities and challenges within SFX are reviewed herein. [ABSTRACT FROM AUTHOR]
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- 2016
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15. Serial femtosecond X-ray diffraction of enveloped virus microcrystals.
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Lawrence, Robert M., Conrad, Chelsie E., Zatsepin, Nadia A., Grant, Thomas D., Haiguang Liu, James, Daniel, Nelson, Garrett, Subramanian, Ganesh, Aquila, Andrew, Hunter, Mark S., Liang, Mengning, Boutet, Sébastien, Coe, Jesse, Spence, John C. H., Weierstall, Uwe, Wei Liu, Fromme, Petra, Cherezov, Vadim, and Hogue, Brenda G.
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X-ray diffraction ,CRYSTALLOGRAPHY ,FEMTOSECOND lasers ,FREE electron lasers ,PROTEIN structure - Abstract
Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ~700Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ~40Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis suggests this results from molecular transforms of individual particles. Viral proteins undergo structural changes during entry and infection, which could, in principle, be studied with SFX. This is an important step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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16. Microfluidic sorting of protein nanocrystals by size for X-ray free-electron laser diffraction.
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Abdallah, Bahige G., Zatsepin, Nadia A., Roy-Chowdhury, Shatabdi, Coe, Jesse, Conrad, Chelsie E., Dörner, Katerina, Sierra, Raymond G., Stevenson, Hilary P., Camacho-Alanis, Fernanda, Grant, Thomas D., Nelson, Garrett, James, Daniel, Calero, Guillermo, Wachter, Rebekka M., Spence, John C. H., Weierstall, Uwe, Fromme, Petra, and Ros, Alexandra
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MICROFLUIDICS ,CRYSTALLOIDS (Botany) ,NANOCRYSTALS ,FREE electron lasers ,X-ray diffraction - Abstract
The advent and application of the X-ray free-electron laser (XFEL) has uncovered the structures of proteins that could not previously be solved using traditional crystallography. While this new technology is powerful, optimization of the process is still needed to improve data quality and analysis efficiency. One area is sample heterogeneity, where variations in crystal size (among other factors) lead to the requirement of large data sets (and thus 10-100 mg of protein) for determining accurate structure factors. To decrease sample dispersity, we developed a high-throughput microfluidic sorter operating on the principle of dielectrophoresis, whereby polydisperse particles can be transported into various fluid streams for size fractionation. Using this microsorter, we isolated several milliliters of photosystem I nanocrystal fractions ranging from 200 to 600 nm in size as characterized by dynamic light scattering, nanoparticle tracking, and electron microscopy. Sorted nanocrystals were delivered in a liquid jet via the gas dynamic virtual nozzle into the path of the XFEL at the Linac Coherent Light Source. We obtained diffraction to 4A° resolution, indicating that the small crystals were not damaged by the sorting process. We also observed the shape transforms of photosystem I nanocrystals, demonstrating that our device can optimize data collection for the shape transform-based phasing method. Using simulations, we show that narrow crystal size distributions can significantly improve merged data quality in serial crystallography. From this proof-of-concept work, we expect that the automated size-sorting of protein crystals will become an important step for sample production by reducing the amount of protein needed for a high quality final structure and the development of novel phasing methods that exploit inter-Bragg reflection intensities or use variations in beam intensity for radiation damage-induced phasing. This method will also permit an analysis of the dependence of crystal quality on crystal size. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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17. From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography.
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Dods, Robert, Båth, Petra, Arnlund, David, Beyerlein, Kenneth R., Nelson, Garrett, Liang, Mengling, Harimoorthy, Rajiv, Berntsen, Peter, Malmerberg, Erik, Johansson, Linda, Andersson, Rebecka, Bosman, Robert, Carbajo, Sergio, Claesson, Elin, Conrad, Chelsie E., Dahl, Peter, Hammarin, Greger, Hunter, Mark S., Li, Chufeng, and Lisova, Stella
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CRYSTAL structure , *CRYSTALLOGRAPHY , *MEMBRANE proteins , *ELECTRONS , *MICROCRYSTALLINE polymers - Abstract
Summary Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RC vir ). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RC vir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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18. Detection of a geminate photoproduct of bovine cytochrome c oxidase by time-resolved serial femtosecond crystallography.
- Author
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Ishigami I, Carbajo S, Zatsepin N, Hikita M, Conrad CE, Nelson G, Coe J, Basu S, Grant T, Seaberg MH, Sierra RG, Hunter MS, Fromme P, Fromme R, Rousseau DL, and Yeh SR
- Abstract
Cytochrome c oxidase (C c O) is a large membrane-bound hemeprotein that catalyzes the reduction of dioxygen to water. Unlike classical dioxygen binding hemeproteins with a heme b group in their active sites, C c O has a unique binuclear center (BNC) comprised of a copper atom (Cu
B ) and a heme a3 iron, where O2 binds and is reduced to water. CO is a versatile O2 surrogate in ligand binding and escape reactions. Previous time-resolved spectroscopic studies of the CO complexes of bovine C c O (bC c O) revealed that photolyzing CO from the heme a3 iron leads to a metastable intermediate (CuB -CO), where CO is bound to CuB , before it escapes out of the BNC. Here, with a time-resolved serial femtosecond X-ray crystallography-based pump-probe method, we detected a geminate photoproduct of the bC c O-CO complex, where CO is dissociated from the heme a3 iron and moved to a temporary binding site midway between the CuB and the heme a3 iron, while the locations of the two metal centers and the conformation of the Helix-X, housing the proximal histidine ligand of the heme a3 iron, remain in the CO complex state. This new structure, combined with other reported structures of bC c O, allows the full definition of the ligand dissociation trajectory, as well as the associated protein dynamics.- Published
- 2023
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19. Developing methods to study conformational changes in RNA crystals using a photocaged ligand.
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Lee HK, Conrad CE, Magidson V, Heinz WF, Pauly G, Yu P, Ramakrishnan S, Stagno JR, and Wang YX
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Crystallographic observation of structural changes in real time requires that those changes be uniform both spatially and temporally. A primary challenge with time-resolved ligand-mixing diffraction experiments is asynchrony caused by variable factors, such as efficiency of mixing, rate of diffusion, crystal size, and subsequently, conformational heterogeneity. One method of minimizing such variability is use of a photolabile caged ligand, which can fully saturate the crystal environment (spatially), and whose photoactivation can rapidly (temporally) trigger the reaction in a controlled manner. Our recently published results on a ligand-mixing experiment using time-resolved X-ray crystallography (TRX) with an X-ray free electron laser (XFEL) demonstrated that large conformational changes upon ligand binding resulted in a solid-to-solid phase transition (SSPT), while maintaining Bragg diffraction. Here we investigate this SSPT by polarized video microscopy (PVM) after light-triggered release of a photo-caged adenine (pcADE). In general, the mean transition times and transition widths of the SSPT were less dependent on crystal size than what was observed in previous PVM studies with direct ADE mixing. Instead, the photo-induced transition appears to be heavily influenced by the equilibrium between caged and uncaged ADE due to relatively low sample exposure and uncaging efficiency. Nevertheless, we successfully demonstrate a method for the characterization of phase transitions in RNA crystals that are inducible with a photocaged ligand. The transition data for three crystals of different sizes were then applied to kinetic analysis by fitting to the known four-state model associated with ligand-induced conformational changes, revealing an apparent concentration of uncaged ADE in crystal of 0.43-0.46 mM. These results provide further insight into approaches to study time-resolved ligand-induced conformational changes in crystals, and in particular, highlight the feasibility of triggering phase transitions using a light-inducible system. Developing such approaches may be paramount for the rapidly emerging field of time-resolved crystallography., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lee, Conrad, Magidson, Heinz, Pauly, Yu, Ramakrishnan, Stagno and Wang.)
- Published
- 2022
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20. Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation.
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Martin-Garcia JM, Conrad CE, Nelson G, Stander N, Zatsepin NA, Zook J, Zhu L, Geiger J, Chun E, Kissick D, Hilgart MC, Ogata C, Ishchenko A, Nagaratnam N, Roy-Chowdhury S, Coe J, Subramanian G, Schaffer A, James D, Ketwala G, Venugopalan N, Xu S, Corcoran S, Ferguson D, Weierstall U, Spence JCH, Cherezov V, Fromme P, Fischetti RF, and Liu W
- Abstract
Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5-20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A
2A adenosine receptor (A2A AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A2A AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A2A AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5-20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.- Published
- 2017
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21. A novel inert crystal delivery medium for serial femtosecond crystallography.
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Conrad CE, Basu S, James D, Wang D, Schaffer A, Roy-Chowdhury S, Zatsepin NA, Aquila A, Coe J, Gati C, Hunter MS, Koglin JE, Kupitz C, Nelson G, Subramanian G, White TA, Zhao Y, Zook J, Boutet S, Cherezov V, Spence JC, Fromme R, Weierstall U, and Fromme P
- Abstract
Serial femtosecond crystallography (SFX) has opened a new era in crystallo-graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.
- Published
- 2015
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22. Structure of the Angiotensin receptor revealed by serial femtosecond crystallography.
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Zhang H, Unal H, Gati C, Han GW, Liu W, Zatsepin NA, James D, Wang D, Nelson G, Weierstall U, Sawaya MR, Xu Q, Messerschmidt M, Williams GJ, Boutet S, Yefanov OM, White TA, Wang C, Ishchenko A, Tirupula KC, Desnoyer R, Coe J, Conrad CE, Fromme P, Stevens RC, Katritch V, Karnik SS, and Cherezov V
- Subjects
- Amino Acid Sequence, Angiotensin II Type 1 Receptor Blockers chemistry, Crystallography, X-Ray, Humans, Molecular Sequence Data, Mutagenesis, Naphthyridines chemistry, Naphthyridines pharmacology, Receptor, Angiotensin, Type 1 genetics, Receptor, Angiotensin, Type 1 metabolism, Sequence Alignment, Angiotensin II Type 1 Receptor Blockers pharmacology, Receptor, Angiotensin, Type 1 chemistry
- 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., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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23. Structural basis for bifunctional peptide recognition at human δ-opioid receptor.
- Author
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Fenalti G, Zatsepin NA, Betti C, Giguere P, Han GW, Ishchenko A, Liu W, Guillemyn K, Zhang H, James D, Wang D, Weierstall U, Spence JC, Boutet S, Messerschmidt M, Williams GJ, Gati C, Yefanov OM, White TA, Oberthuer D, Metz M, Yoon CH, Barty A, Chapman HN, Basu S, Coe J, Conrad CE, Fromme R, Fromme P, Tourwé D, Schiller PW, Roth BL, Ballet S, Katritch V, Stevens RC, and Cherezov V
- Subjects
- Binding Sites, Crystallography, X-Ray, HEK293 Cells, Humans, Models, Molecular, Oligopeptides chemistry, Protein Structure, Tertiary, Receptors, Opioid, delta antagonists & inhibitors, Tetrahydroisoquinolines chemistry, Receptors, Opioid, delta chemistry
- Abstract
Bifunctional μ- and δ-opioid receptor (OR) ligands are potential therapeutic alternatives, with diminished side effects, to alkaloid opiate analgesics. We solved the structure of human δ-OR bound to the bifunctional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt and Tic. The observed receptor-peptide interactions are critical for understanding of the pharmacological profiles of opioid peptides and for development of improved analgesics.
- Published
- 2015
- Full Text
- View/download PDF
24. Crystallization of Photosystem II for Time-Resolved Structural Studies Using an X-ray Free Electron Laser.
- Author
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Coe J, Kupitz C, Basu S, Conrad CE, Roy-Chowdhury S, Fromme R, and Fromme P
- Subjects
- Crystallization instrumentation, Crystallization methods, Crystallography, X-Ray instrumentation, Electrons, Equipment Design, Lasers, Models, Molecular, Photosystem II Protein Complex isolation & purification, Protein Conformation, Crystallography, X-Ray methods, Photosystem II Protein Complex chemistry, Synechococcus chemistry
- Abstract
Photosystem II (PSII) is a membrane protein supercomplex that executes the initial reaction of photosynthesis in higher plants, algae, and cyanobacteria. It captures the light from the sun to catalyze a transmembrane charge separation. In a series of four charge separation events, utilizing the energy from four photons, PSII oxidizes two water molecules to obtain dioxygen, four protons, and four electrons. The light reactions of photosystems I and II (PSI and PSII) result in the formation of an electrochemical transmembrane proton gradient that is used for the production of ATP. Electrons that are subsequently transferred from PSI via the soluble protein ferredoxin to ferredoxin-NADP(+) reductase that reduces NADP(+) to NADPH. The products of photosynthesis and the elemental oxygen evolved sustain all higher life on Earth. All oxygen in the atmosphere is produced by the oxygen-evolving complex in PSII, a process that changed our planet from an anoxygenic to an oxygenic atmosphere 2.5 billion years ago. In this chapter, we provide recent insight into the mechanisms of this process and methods used in probing this question., (© 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
25. Microcrystallization techniques for serial femtosecond crystallography using photosystem II from Thermosynechococcus elongatus as a model system.
- Author
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Kupitz C, Grotjohann I, Conrad CE, Roy-Chowdhury S, Fromme R, and Fromme P
- Subjects
- Cyanobacteria, Nanoparticles chemistry, Protein Conformation, Crystallization methods, Crystallography, X-Ray methods, Electrons, Lasers, Photosystem II Protein Complex chemistry, X-Ray Diffraction methods
- Abstract
Serial femtosecond crystallography (SFX) is a new emerging method, where X-ray diffraction data are collected from a fully hydrated stream of nano- or microcrystals of biomolecules in their mother liquor using high-energy, X-ray free-electron lasers. The success of SFX experiments strongly depends on the ability to grow large amounts of well-ordered nano/microcrystals of homogeneous size distribution. While methods to grow large single crystals have been extensively explored in the past, method developments to grow nano/microcrystals in sufficient amounts for SFX experiments are still in their infancy. Here, we describe and compare three methods (batch, free interface diffusion (FID) and FID centrifugation) for growth of nano/microcrystals for time-resolved SFX experiments using the large membrane protein complex photosystem II as a model system., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
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