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1. Changes in an enzyme ensemble during catalysis observed by high-resolution XFEL crystallography

Author

Smith, Nathan, Dasgupta, Medhanjali, Wych, David C, Dolamore, Cole, Sierra, Raymond G, Lisova, Stella, Marchany-Rivera, Darya, Cohen, Aina E, Boutet, Sébastien, Hunter, Mark S, Kupitz, Christopher, Poitevin, Frédéric, Moss, Frank R, Mittan-Moreau, David W, Brewster, Aaron S, Sauter, Nicholas K, Young, Iris D, Wolff, Alexander M, Tiwari, Virendra K, Kumar, Nivesh, Berkowitz, David B, Hadt, Ryan G, Thompson, Michael C, Follmer, Alec H, Wall, Michael E, and Wilson, Mark A

Subjects
Biochemistry and Cell Biology, Chemical Sciences, Biological Sciences, Bioengineering, Crystallography, X-Ray, Proteins, Catalysis, Molecular Dynamics Simulation, Protein Conformation, Hydrolases
Abstract

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Published
2024

2. A user-friendly plug-and-play cyclic olefin copolymer-based microfluidic chip for room-temperature, fixed-target serial crystallography.

Author

Liu, Zhongrui, Gu, Kevin, Shelby, Megan, Gilbile, Deepshika, Lyubimov, Artem, Russi, Silvia, Cohen, Aina, Narayanasamy, Sankar, Botha, Sabine, Kupitz, Christopher, Sierra, Raymond, Poitevin, Fredric, Gilardi, Antonio, Lisova, Stella, Coleman, Matthew, Frank, Matthias, and Kuhl, Tonya

Subjects
X-ray crystallography, XFELs, cyclic olefin copolymer, fixed targets, microfluidics, room-temperature SFX, sample delivery, serial crystallography, synchrotrons
Abstract

Over the past two decades, serial X-ray crystallography has enabled the structure determination of a wide range of proteins. With the advent of X-ray free-electron lasers (XFELs), ever-smaller crystals have yielded high-resolution diffraction and structure determination. A crucial need to continue advancement is the efficient delivery of fragile and micrometre-sized crystals to the X-ray beam intersection. This paper presents an improved design of an all-polymer microfluidic `chip for room-temperature fixed-target serial crystallography that can be tailored to broadly meet the needs of users at either synchrotron or XFEL light sources. The chips are designed to be customized around different types of crystals and offer users a friendly, quick, convenient, ultra-low-cost and robust sample-delivery platform. Compared with the previous iteration of the chip [Gilbile et al. (2021), Lab Chip, 21, 4831-4845], the new design eliminates cleanroom fabrication. It has a larger imaging area to volume, while maintaining crystal hydration stability for both in situ crystallization or direct crystal slurry loading. Crystals of two model proteins, lysozyme and thaumatin, were used to validate the effectiveness of the design at both synchrotron (lysozyme and thaumatin) and XFEL (lysozyme only) facilities, yielding complete data sets with resolutions of 1.42, 1.48 and 1.70 Å, respectively. Overall, the improved chip design, ease of fabrication and high modifiability create a powerful, all-around sample-delivery tool that structural biologists can quickly adopt, especially in cases of limited sample volume and small, fragile crystals.

Published
2023

3. Room-temperature structural studies of SARS-CoV-2 protein NendoU with an X-ray free-electron laser.

Author

Jernigan, Rebecca J, Logeswaran, Dhenugen, Doppler, Diandra, Nagaratnam, Nirupa, Sonker, Mukul, Yang, Jay-How, Ketawala, Gihan, Martin-Garcia, Jose M, Shelby, Megan L, Grant, Thomas D, Mariani, Valerio, Tolstikova, Alexandra, Sheikh, Michelle Z, Yung, Mimi Cho, Coleman, Matthew A, Zaare, Sahba, Kaschner, Emily K, Rabbani, Mohammad Towshif, Nazari, Reza, Zacks, Michele A, Hayes, Brandon, Sierra, Raymond G, Hunter, Mark S, Lisova, Stella, Batyuk, Alexander, Kupitz, Christopher, Boutet, Sebastien, Hansen, Debra T, Kirian, Richard A, Schmidt, Marius, Fromme, Raimund, Frank, Matthias, Ros, Alexandra, Chen, Julian J-L, Botha, Sabine, and Fromme, Petra

Subjects
Humans, Crystallography, X-Ray, Lasers, Temperature, Electrons, COVID-19, SARS-CoV-2, NSP15, NendoU, X-ray free-electron laser, serial femtosecond crystallography, Prevention, Infectious Diseases, Emerging Infectious Diseases, Vaccine Related, Biodefense, Infection, Chemical Sciences, Biological Sciences, Information and Computing Sciences, Biophysics
Abstract

NendoU from SARS-CoV-2 is responsible for the virus's ability to evade the innate immune system by cleaving the polyuridine leader sequence of antisense viral RNA. Here we report the room-temperature structure of NendoU, solved by serial femtosecond crystallography at an X-ray free-electron laser to 2.6 Å resolution. The room-temperature structure provides insight into the flexibility, dynamics, and other intrinsic properties of NendoU, with indications that the enzyme functions as an allosteric switch. Functional studies examining cleavage specificity in solution and in crystals support the uridine-purine cleavage preference, and we demonstrate that enzyme activity is fully maintained in crystal form. Optimizing the purification of NendoU and identifying suitable crystallization conditions set the benchmark for future time-resolved serial femtosecond crystallography studies. This could advance the design of antivirals with higher efficacy in treating coronaviral infections, since drugs that block allosteric conformational changes are less prone to drug resistance.

Published
2023

4. Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Author

Smith, Nathan, Dasgupta, Medhanjali, Wych, David C, Dolamore, Cole, Sierra, Raymond G, Lisova, Stella, Marchany-Rivera, Darya, Cohen, Aina E, Boutet, Sébastien, Hunter, Mark S, Kupitz, Christopher, Poitevin, Frédéric, Moss, Frank R, Brewster, Aaron S, Sauter, Nicholas K, Young, Iris D, Wolff, Alexander M, Tiwari, Virendra K, Kumar, Nivesh, Berkowitz, David B, Hadt, Ryan G, Thompson, Michael C, Follmer, Alec H, Wall, Michael E, and Wilson, Mark A

Subjects
Biochemistry and Cell Biology, Chemical Sciences, Biological Sciences, enzyme dynamics, isonitrile, serial crystallography, x-ray free electron laser
Abstract

Enzymes populate ensembles of structures with intrinsically different catalytic proficiencies that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography (MISC) at an X-ray free electron laser (XFEL) to observe catalysis in a designed mutant (G150T) isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations and formation of the thioimidate catalytic intermediate selects for catalytically competent substates. A prior proposal for active site cysteine charge-coupled conformational changes in ICH is validated by determining structures of the enzyme over a range of pH values. A combination of large molecular dynamics simulations of the enzyme in crystallo and time-resolved electron density maps shows that ionization of the general acid Asp17 during catalysis causes additional conformational changes that propagate across the dimer interface, connecting the two active sites. These ionization-linked changes in the ICH conformational ensemble permit water to enter the active site in a location that is poised for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

Published
2023

5. Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam

Author

Malla, Tek Narsingh, Zielinski, Kara, Aldama, Luis, Bajt, Sasa, Feliz, Denisse, Hayes, Brendon, Hunter, Mark, Kupitz, Christopher, Lisova, Stella, Knoska, Juraj, Martin-Garcia, Jose Manuel, Mariani, Valerio, Pandey, Suraj, Poudyal, Ishwor, Sierra, Raymond G., Tolstikova, Alexandra, Yefanov, Oleksandr, Yoon, Chung Hong, Ourmazd, Abbas, Fromme, Petra, Schwander, Peter, Barty, Anton, Chapman, Henry N., Stojkovic, Emina A., Batyuk, Alexander, Boutet, Sébastien, Phillips, Jr., George N., Pollack, Lois, and Schmidt, Marius

Published
2023
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6. Time-resolved serial femtosecond crystallography at the European XFEL

Author

Pandey, Suraj, Bean, Richard, Sato, Tokushi, Poudyal, Ishwor, Bielecki, Johan, Cruz Villarreal, Jorvani, Yefanov, Oleksandr, Mariani, Valerio, White, Thomas A, Kupitz, Christopher, Hunter, Mark, Abdellatif, Mohamed H, Bajt, Saša, Bondar, Valerii, Echelmeier, Austin, Doppler, Diandra, Emons, Moritz, Frank, Matthias, Fromme, Raimund, Gevorkov, Yaroslav, Giovanetti, Gabriele, Jiang, Man, Kim, Daihyun, Kim, Yoonhee, Kirkwood, Henry, Klimovskaia, Anna, Knoska, Juraj, Koua, Faisal HM, Letrun, Romain, Lisova, Stella, Maia, Luis, Mazalova, Victoria, Meza, Domingo, Michelat, Thomas, Ourmazd, Abbas, Palmer, Guido, Ramilli, Marco, Schubert, Robin, Schwander, Peter, Silenzi, Alessandro, Sztuk-Dambietz, Jolanta, Tolstikova, Alexandra, Chapman, Henry N, Ros, Alexandra, Barty, Anton, Fromme, Petra, Mancuso, Adrian P, and Schmidt, Marius

Subjects
Biochemistry and Cell Biology, Biological Sciences, Bacterial Proteins, Crystallography, X-Ray, Light, Models, Molecular, Photoreceptors, Microbial, Protein Conformation, Time Factors, Technology, Medical and Health Sciences, Developmental Biology, Biological sciences
Abstract

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. Here, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate the picosecond time range in the photocycle of photoactive yellow protein (PYP) with MHz X-ray pulse rates. We show that difference electron density maps of excellent quality can be obtained. The results connect the previously explored femtosecond PYP dynamics to timescales accessible at synchrotrons. This opens the door to a wide range of time-resolved studies at the EuXFEL.

Published
2020

7. Author Correction: Membrane protein megahertz crystallography at the European XFEL

Author

Gisriel, Chris, Coe, Jesse, Letrun, Romain, Yefanov, Oleksandr M, Luna-Chavez, Cesar, Stander, Natasha E, Lisova, Stella, Mariani, Valerio, Kuhn, Manuela, Aplin, Steve, Grant, Thomas D, Dörner, Katerina, Sato, Tokushi, Echelmeier, Austin, Villarreal, Jorvani Cruz, Hunter, Mark S, Wiedorn, Max O, Knoska, Juraj, Mazalova, Victoria, Roy-Chowdhury, Shatabdi, Yang, Jay-How, Jones, Alex, Bean, Richard, Bielecki, Johan, Kim, Yoonhee, Mills, Grant, Weinhausen, Britta, Meza, Jose D, Al-Qudami, Nasser, Bajt, Saša, Brehm, Gerrit, Botha, Sabine, Boukhelef, Djelloul, Brockhauser, Sandor, Bruce, Barry D, Coleman, Matthew A, Danilevski, Cyril, Discianno, Erin, Dobson, Zachary, Fangohr, Hans, Martin-Garcia, Jose M, Gevorkov, Yaroslav, Hauf, Steffen, Hosseinizadeh, Ahmad, Januschek, Friederike, Ketawala, Gihan K, Kupitz, Christopher, Maia, Luis, Manetti, Maurizio, Messerschmidt, Marc, Michelat, Thomas, Mondal, Jyotirmoy, Ourmazd, Abbas, Previtali, Gianpietro, Sarrou, Iosifina, Schön, Silvan, Schwander, Peter, Shelby, Megan L, Silenzi, Alessandro, Sztuk-Dambietz, Jolanta, Szuba, Janusz, Turcato, Monica, White, Thomas A, Wrona, Krzysztof, Xu, Chen, Abdellatif, Mohamed H, Zook, James D, Spence, John CH, Chapman, Henry N, Barty, Anton, Kirian, Richard A, Frank, Matthias, Ros, Alexandra, Schmidt, Marius, Fromme, Raimund, Mancuso, Adrian P, Fromme, Petra, and Zatsepin, Nadia A

Subjects
Biochemistry and Cell Biology, Biological Sciences
Abstract

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Published
2020

8. Membrane protein megahertz crystallography at the European XFEL

Author

Gisriel, Chris, Coe, Jesse, Letrun, Romain, Yefanov, Oleksandr M, Luna-Chavez, Cesar, Stander, Natasha E, Lisova, Stella, Mariani, Valerio, Kuhn, Manuela, Aplin, Steve, Grant, Thomas D, Dörner, Katerina, Sato, Tokushi, Echelmeier, Austin, Cruz Villarreal, Jorvani, Hunter, Mark S, Wiedorn, Max O, Knoska, Juraj, Mazalova, Victoria, Roy-Chowdhury, Shatabdi, Yang, Jay-How, Jones, Alex, Bean, Richard, Bielecki, Johan, Kim, Yoonhee, Mills, Grant, Weinhausen, Britta, Meza, Jose D, Al-Qudami, Nasser, Bajt, Saša, Brehm, Gerrit, Botha, Sabine, Boukhelef, Djelloul, Brockhauser, Sandor, Bruce, Barry D, Coleman, Matthew A, Danilevski, Cyril, Discianno, Erin, Dobson, Zachary, Fangohr, Hans, Martin-Garcia, Jose M, Gevorkov, Yaroslav, Hauf, Steffen, Hosseinizadeh, Ahmad, Januschek, Friederike, Ketawala, Gihan K, Kupitz, Christopher, Maia, Luis, Manetti, Maurizio, Messerschmidt, Marc, Michelat, Thomas, Mondal, Jyotirmoy, Ourmazd, Abbas, Previtali, Gianpietro, Sarrou, Iosifina, Schön, Silvan, Schwander, Peter, Shelby, Megan L, Silenzi, Alessandro, Sztuk-Dambietz, Jolanta, Szuba, Janusz, Turcato, Monica, White, Thomas A, Wrona, Krzysztof, Xu, Chen, Abdellatif, Mohamed H, Zook, James D, Spence, John CH, Chapman, Henry N, Barty, Anton, Kirian, Richard A, Frank, Matthias, Ros, Alexandra, Schmidt, Marius, Fromme, Raimund, Mancuso, Adrian P, Fromme, Petra, and Zatsepin, Nadia A

Subjects
Biochemistry and Cell Biology, Biological Sciences, 1.1 Normal biological development and functioning, Underpinning research, Generic health relevance, Crystallography, Cyanobacteria, Electrons, Lasers, Membrane Proteins, Models, Molecular, Photosystem I Protein Complex, Static Electricity, Synchrotrons, Thermosynechococcus, X-Rays
Abstract

The world's first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.

Published
2019

9. Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography

Author

Olmos, Jose L, Pandey, Suraj, Martin-Garcia, Jose M, Calvey, George, Katz, Andrea, Knoska, Juraj, Kupitz, Christopher, Hunter, Mark S, Liang, Mengning, Oberthuer, Dominik, Yefanov, Oleksandr, Wiedorn, Max, Heyman, Michael, Holl, Mark, Pande, Kanupriya, Barty, Anton, Miller, Mitchell D, Stern, Stephan, Roy-Chowdhury, Shatabdi, Coe, Jesse, Nagaratnam, Nirupa, Zook, James, Verburgt, Jacob, Norwood, Tyler, Poudyal, Ishwor, Xu, David, Koglin, Jason, Seaberg, Matthew H, Zhao, Yun, Bajt, Saša, Grant, Thomas, Mariani, Valerio, Nelson, Garrett, Subramanian, Ganesh, Bae, Euiyoung, Fromme, Raimund, Fung, Russell, Schwander, Peter, Frank, Matthias, White, Thomas A, Weierstall, Uwe, Zatsepin, Nadia, Spence, John, Fromme, Petra, Chapman, Henry N, Pollack, Lois, Tremblay, Lee, Ourmazd, Abbas, Phillips, George N, and Schmidt, Marius

Subjects
Biochemistry and Cell Biology, Biological Sciences, Rare Diseases, Infectious Diseases, Good Health and Well Being, Anti-Bacterial Agents, Bacterial Proteins, Biocatalysis, Ceftriaxone, Cephalosporin Resistance, Crystallography, X-Ray, Kinetics, Lasers, Models, Molecular, Mycobacterium tuberculosis, Time Factors, beta-Lactamases, Developmental Biology
Abstract

BackgroundEver since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases.ResultsHere, we demonstrate a general method for capturing enzyme catalysis "in action" by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s.ConclusionsMISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

Published
2018

10. Cooperative allostery and structural dynamics of streptavidin at cryogenic- and ambient-temperature

Author

Ayan, Esra, Yuksel, Busra, Destan, Ebru, Ertem, Fatma Betul, Yildirim, Gunseli, Eren, Meryem, Yefanov, Oleksandr M., Barty, Anton, Tolstikova, Alexandra, Ketawala, Gihan K., Botha, Sabine, Dao, E. Han, Hayes, Brandon, Liang, Mengning, Seaberg, Matthew H., Hunter, Mark S., Batyuk, Alexander, Mariani, Valerio, Su, Zhen, Poitevin, Frederic, Yoon, Chun Hong, Kupitz, Christopher, Cohen, Aina, Doukov, Tzanko, Sierra, Raymond G., Dağ, Çağdaş, and DeMirci, Hasan

Published
2022
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11. Resolution extension by image summing in serial femtosecond crystallography of two-dimensional membrane-protein crystals

Author

Casadei, Cecilia M, Tsai, Ching-Ju, Barty, Anton, Hunter, Mark S, Zatsepin, Nadia A, Padeste, Celestino, Capitani, Guido, Benner, W Henry, Boutet, Sébastien, Hau-Riege, Stefan P, Kupitz, Christopher, Messerschmidt, Marc, Ogren, John I, Pardini, Tom, Rothschild, Kenneth J, Sala, Leonardo, Segelke, Brent, Williams, Garth J, Evans, James E, Li, Xiao-Dan, Coleman, Matthew, Pedrini, Bill, and Frank, Matthias

Subjects
Inorganic Chemistry, Chemical Sciences, Physical Sciences, Bioengineering, serial crystallography, free-electron lasers, membrane proteins, two-dimensional crystals, Atomic, Molecular, Nuclear, Particle and Plasma Physics, Condensed Matter Physics, Physical Chemistry (incl. Structural), Physical chemistry, Condensed matter physics
Abstract

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump-probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.

Published
2018

12. Megahertz serial crystallography

Author

Wiedorn, Max O, Oberthür, Dominik, Bean, Richard, Schubert, Robin, Werner, Nadine, Abbey, Brian, Aepfelbacher, Martin, Adriano, Luigi, Allahgholi, Aschkan, Al-Qudami, Nasser, Andreasson, Jakob, Aplin, Steve, Awel, Salah, Ayyer, Kartik, Bajt, Saša, Barák, Imrich, Bari, Sadia, Bielecki, Johan, Botha, Sabine, Boukhelef, Djelloul, Brehm, Wolfgang, Brockhauser, Sandor, Cheviakov, Igor, Coleman, Matthew A, Cruz-Mazo, Francisco, Danilevski, Cyril, Darmanin, Connie, Doak, R Bruce, Domaracky, Martin, Dörner, Katerina, Du, Yang, Fangohr, Hans, Fleckenstein, Holger, Frank, Matthias, Fromme, Petra, Gañán-Calvo, Alfonso M, Gevorkov, Yaroslav, Giewekemeyer, Klaus, Ginn, Helen Mary, Graafsma, Heinz, Graceffa, Rita, Greiffenberg, Dominic, Gumprecht, Lars, Göttlicher, Peter, Hajdu, Janos, Hauf, Steffen, Heymann, Michael, Holmes, Susannah, Horke, Daniel A, Hunter, Mark S, Imlau, Siegfried, Kaukher, Alexander, Kim, Yoonhee, Klyuev, Alexander, Knoška, Juraj, Kobe, Bostjan, Kuhn, Manuela, Kupitz, Christopher, Küpper, Jochen, Lahey-Rudolph, Janine Mia, Laurus, Torsten, Le Cong, Karoline, Letrun, Romain, Xavier, P Lourdu, Maia, Luis, Maia, Filipe RNC, Mariani, Valerio, Messerschmidt, Marc, Metz, Markus, Mezza, Davide, Michelat, Thomas, Mills, Grant, Monteiro, Diana CF, Morgan, Andrew, Mühlig, Kerstin, Munke, Anna, Münnich, Astrid, Nette, Julia, Nugent, Keith A, Nuguid, Theresa, Orville, Allen M, Pandey, Suraj, Pena, Gisel, Villanueva-Perez, Pablo, Poehlsen, Jennifer, Previtali, Gianpietro, Redecke, Lars, Riekehr, Winnie Maria, Rohde, Holger, Round, Adam, Safenreiter, Tatiana, Sarrou, Iosifina, Sato, Tokushi, Schmidt, Marius, Schmitt, Bernd, Schönherr, Robert, Schulz, Joachim, Sellberg, Jonas A, Seibert, M Marvin, and Seuring, Carolin

Subjects
Biochemistry and Cell Biology, Chemical Sciences, Biological Sciences
Abstract

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.

Published
2018

13. Structural enzymology using X-ray free electron lasers

Author

Kupitz, Christopher, Olmos, Jose L, Holl, Mark, Tremblay, Lee, Pande, Kanupriya, Pandey, Suraj, Oberthür, Dominik, Hunter, Mark, Liang, Mengning, Aquila, Andrew, Tenboer, Jason, Calvey, George, Katz, Andrea, Chen, Yujie, Wiedorn, Max O, Knoska, Juraj, Meents, Alke, Majriani, Valerio, Norwood, Tyler, Poudyal, Ishwor, Grant, Thomas, Miller, Mitchell D, Xu, Weijun, Tolstikova, Aleksandra, Morgan, Andrew, Metz, Markus, Martin-Garcia, Jose M, Zook, James D, Roy-Chowdhury, Shatabdi, Coe, Jesse, Nagaratnam, Nirupa, Meza, Domingo, Fromme, Raimund, Basu, Shibom, Frank, Matthias, White, Thomas, Barty, Anton, Bajt, Sasa, Yefanov, Oleksandr, Chapman, Henry N, Zatsepin, Nadia, Nelson, Garrett, Weierstall, Uwe, Spence, John, Schwander, Peter, Pollack, Lois, Fromme, Petra, Ourmazd, Abbas, Phillips, George N, and Schmidt, Marius

Subjects
Inorganic Chemistry, Chemical Sciences, Physical Sciences, Infectious Diseases, Good Health and Well Being, Physical chemistry, Condensed matter physics, Particle and high energy physics
Abstract

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

Published
2017

14. Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein

Author

Pande, Kanupriya, Hutchison, Christopher DM, Groenhof, Gerrit, Aquila, Andy, Robinson, Josef S, Tenboer, Jason, Basu, Shibom, Boutet, Sébastien, DePonte, Daniel P, Liang, Mengning, White, Thomas A, Zatsepin, Nadia A, Yefanov, Oleksandr, Morozov, Dmitry, Oberthuer, Dominik, Gati, Cornelius, Subramanian, Ganesh, James, Daniel, Zhao, Yun, Koralek, Jake, Brayshaw, Jennifer, Kupitz, Christopher, Conrad, Chelsie, Roy-Chowdhury, Shatabdi, Coe, Jesse D, Metz, Markus, Xavier, Paulraj Lourdu, Grant, Thomas D, Koglin, Jason E, Ketawala, Gihan, Fromme, Raimund, Šrajer, Vukica, Henning, Robert, Spence, John CH, Ourmazd, Abbas, Schwander, Peter, Weierstall, Uwe, Frank, Matthias, Fromme, Petra, Barty, Anton, Chapman, Henry N, Moffat, Keith, van Thor, Jasper J, and Schmidt, Marius

Subjects
Biological Sciences, Chemical Sciences, Theoretical and Computational Chemistry, Bacterial Proteins, Crystallography, Isomerism, Light, Photochemical Processes, Photons, Photoreceptors, Microbial, Protein Conformation, Time Factors, General Science & Technology
Abstract

A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes after photon absorption. The initial step is often the photoisomerization of a conjugated chromophore. Isomerization occurs on ultrafast time scales and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans-to-cis isomerization of the chromophore in photoactive yellow protein. Femtosecond hard x-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on photoactive yellow protein microcrystals over a time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.

Published
2016

15. Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

Author

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, MH 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 CH, 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

Subjects
1.1 Normal biological development and functioning, Underpinning research, Generic health relevance, Animals, Arrestin, Binding Sites, Crystallography, X-Ray, Disulfides, Humans, Lasers, Mice, Models, Molecular, Multiprotein Complexes, Protein Binding, Reproducibility of Results, Rhodopsin, Signal Transduction, X-Rays, General Science & Technology
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.

Published
2015

17. Ambient Temperature Bacterial Large Ribosomal Subunit Structure Enabled by Serial Femtosecond X-ray Crystallography

Author

Tosun, Bilge, primary, Rao, Yashas, additional, Destan, Ebru, additional, Dao, E. Han, additional, Ertem, Fatma Betul, additional, Yilmaz, Merve, additional, Yapici, Ilkin, additional, Gul, Mehmet, additional, Ayan, Esra, additional, Johnson, Jerome Austin, additional, Shafiei, Alaleh, additional, Kulakman, Cahine, additional, Hayes, Brandon, additional, Liang, Mengning, additional, Yoon, Chun Hong, additional, Su, Zhen, additional, Hunter, Mark Steven, additional, Kupitz, Christopher J., additional, Poitevin, Frederic P., additional, Ciftci, Halil Ibrahim, additional, Sierra, Raymond George, additional, Gregory, Steven, additional, Milon, Pohl, additional, Kurkcuoglu, Ozge, additional, and DeMirci, Hasan, additional

Published
2023
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18. RNA structures and dynamics with Å resolution revealed by x-ray free-electron lasers

Author

Zielinski, Kara A., primary, Sui, Shuo, additional, Pabit, Suzette A., additional, Rivera, Daniel A., additional, Wang, Tong, additional, Hu, Qingyue, additional, Kashipathy, Maithri M., additional, Lisova, Stella, additional, Schaffer, Chris B., additional, Mariani, Valerio, additional, Hunter, Mark S., additional, Kupitz, Christopher, additional, Moss, Frank R., additional, Poitevin, Frédéric P., additional, Grant, Thomas D., additional, and Pollack, Lois, additional

Published
2023
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19. Changes in an Enzyme Ensemble During Catalysis Observed by High Resolution XFEL Crystallography

Author

Smith, Nathan, primary, Dasgupta, Medhanjali, additional, Wych, David C., additional, Dolamore, Cole, additional, Sierra, Raymond G., additional, Lisova, Stella, additional, Marchany-Rivera, Darya, additional, Cohen, Aina E., additional, Boutet, Sébastien, additional, Hunter, Mark S., additional, Kupitz, Christopher, additional, Poitevin, Frédéric, additional, Moss, Frank R., additional, Brewster, Aaron S., additional, Sauter, Nicholas K., additional, Young, Iris D., additional, Wolff, Alexander M., additional, Tiwari, Virendra K., additional, Kumar, Nivesh, additional, Berkowitz, David B., additional, Hadt, Ryan G., additional, Thompson, Michael C., additional, Follmer, Alec H., additional, Wall, Michael E., additional, and Wilson, Mark A., additional

Published
2023
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20. Atomic structure of granulin determined from native nanocrystalline granulovirus using an X-ray free-electron laser

Author

Gati, Cornelius, Oberthuer, Dominik, Yefanov, Oleksandr, Bunker, Richard D., Stellato, Francesco, Chiu, Elaine, Yeh, Shin-Mei, Aquila, Andrew, Basu, Shibom, Bean, Richard, Beyerlein, Kenneth R., Botha, Sabine, Boutet, Sébastien, DePonte, Daniel P., Doak, R. Bruce, Fromme, Raimund, Galli, Lorenzo, Grotjohann, Ingo, James, Daniel R., Kupitz, Christopher, Lomb, Lukas, Messerschmidt, Marc, Nass, Karol, Rendek, Kimberly, Shoeman, Robert L., Wang, Dingjie, Weierstall, Uwe, White, Thomas A., Williams, Garth J., Zatsepin, Nadia A., Fromme, Petra, Spence, John C. H., Goldie, Kenneth N., Jehle, Johannes A., Metcalf, Peter, Barty, Anton, and Chapman, Henry N.

Published
2017

21. Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

Author

National Science Foundation (US), National Institutes of Health (US), Department of Energy (US), Max Planck Institute for Medical Research, Comunidad de Madrid, Ministerio de Ciencia, Innovación y Universidades (España), Junta de Andalucía, Doppler, Diandra [0000-0002-3586-3936], Sonker, Mukul [0000-0001-5816-8410], Egatz-Gomez, Ana [0000-0002-6080-943X], Manna, Abhik [0000-0001-8681-6702], Cruz Villarreal, Jorvani [0000-0002-6762-7348], Pey, Angel L. [0000-0001-7706-3243], Ruiz-Fresneda, Miguel A. [0000-0001-6349-6566], Pacheco-Garcia, Juan Luis [0000-0001-6043-7536], Hermoso, Juan A. [0000-0002-1862-8950], Batyuk, Alexander [0000-0002-9393-2880], Fromme, Raimund [0000-0003-4835-1080], Kirian, Richard A. [0000-0001-7197-3086], Martín-García, José M. [0000-0002-4558-3858], Ros, Alexandra [0000-0001-7709-8331], Doppler, Diandra, Sonker, Mukul, Egatz-Gomez, Ana, Grieco, Alice, Zaare, Sahba, Jernigan, Rebecca, Meza-Aguilar, Jose Domingo, Rabbani, Mohammad T., Manna, Abhik, Alvarez, Roberto C., Karpos, Konstantinos, Cruz Villarreal, Jorvani, Nelson, Garrett, Yang, Jay-How, Carrion, Jackson, Morin, Katherine, Ketawala, Gihan K, Pey, Angel L., Ruiz-Fresneda, Miguel A., Pacheco-Garcia, Juan Luis, Hermoso, Juan A., Nazari, Reza, Sierra, Raymond, Hunter, Mark S., Batyuk, Alexander, Kupitz, Christopher J., Sublett, Robert E., Lisova, Stella, Mariani, Valerio, Boutet, Sébastien, Fromme, Raimund, Grant, Thomas D., Botha, Sabine, Fromme, Petra, Kirian, Richard A., Martín-García, José M., Ros, Alexandra, National Science Foundation (US), National Institutes of Health (US), Department of Energy (US), Max Planck Institute for Medical Research, Comunidad de Madrid, Ministerio de Ciencia, Innovación y Universidades (España), Junta de Andalucía, Doppler, Diandra [0000-0002-3586-3936], Sonker, Mukul [0000-0001-5816-8410], Egatz-Gomez, Ana [0000-0002-6080-943X], Manna, Abhik [0000-0001-8681-6702], Cruz Villarreal, Jorvani [0000-0002-6762-7348], Pey, Angel L. [0000-0001-7706-3243], Ruiz-Fresneda, Miguel A. [0000-0001-6349-6566], Pacheco-Garcia, Juan Luis [0000-0001-6043-7536], Hermoso, Juan A. [0000-0002-1862-8950], Batyuk, Alexander [0000-0002-9393-2880], Fromme, Raimund [0000-0003-4835-1080], Kirian, Richard A. [0000-0001-7197-3086], Martín-García, José M. [0000-0002-4558-3858], Ros, Alexandra [0000-0001-7709-8331], Doppler, Diandra, Sonker, Mukul, Egatz-Gomez, Ana, Grieco, Alice, Zaare, Sahba, Jernigan, Rebecca, Meza-Aguilar, Jose Domingo, Rabbani, Mohammad T., Manna, Abhik, Alvarez, Roberto C., Karpos, Konstantinos, Cruz Villarreal, Jorvani, Nelson, Garrett, Yang, Jay-How, Carrion, Jackson, Morin, Katherine, Ketawala, Gihan K, Pey, Angel L., Ruiz-Fresneda, Miguel A., Pacheco-Garcia, Juan Luis, Hermoso, Juan A., Nazari, Reza, Sierra, Raymond, Hunter, Mark S., Batyuk, Alexander, Kupitz, Christopher J., Sublett, Robert E., Lisova, Stella, Mariani, Valerio, Boutet, Sébastien, Fromme, Raimund, Grant, Thomas D., Botha, Sabine, Fromme, Petra, Kirian, Richard A., Martín-García, José M., and Ros, Alexandra

Abstract

Droplet injection strategies are a promising tool to reduce the large amount of sample consumed in serial femtosecond crystallography (SFX) measurements at X-ray free electron lasers (XFELs) with continuous injection approaches. Here, we demonstrate a new modular microfluidic droplet injector (MDI) design that was successfully applied to deliver microcrystals of the human NAD(P)H:quinone oxidoreductase 1 (NQO1) and phycocyanin. We investigated droplet generation conditions through electrical stimulation for both protein samples and implemented hardware and software components for optimized crystal injection at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Stanford Linac Coherent Light Source (LCLS). Under optimized droplet injection conditions, we demonstrate that up to 4-fold sample consumption savings can be achieved with the droplet injector. In addition, we collected a full data set with droplet injection for NQO1 protein crystals with a resolution up to 2.7 Å, leading to the first room-temperature structure of NQO1 at an XFEL. NQO1 is a flavoenzyme associated with cancer, Alzheimer's and Parkinson's disease, making it an attractive target for drug discovery. Our results reveal for the first time that residues Tyr128 and Phe232, which play key roles in the function of the protein, show an unexpected conformational heterogeneity at room temperature within the crystals. These results suggest that different substates exist in the conformational ensemble of NQO1 with functional and mechanistic implications for the enzyme's negative cooperativity through a conformational selection mechanism. Our study thus demonstrates that microfluidic droplet injection constitutes a robust sample-conserving injection method for SFX studies on protein crystals that are difficult to obtain in amounts necessary for continuous injection, including the large sample quantities required for time-resolved mix-and-inject studies.

Published
2023

22. Heterogeneity in the M. tuberculosis β-Lactamase Inhibition by Sulbactam

Author

Schmidt, Marius, primary, Malla, Tek Narsingh, additional, Zielinski, Kara, additional, Aldama, Luis, additional, Bajt, Sasa, additional, Feliz, Denisse, additional, Hayes, Brandon, additional, Hunter, Mark, additional, Kupitz, Christopher, additional, Lisova, Stella, additional, Knoska, Juraj, additional, Martin-Garcia, Jose, additional, Mariani, Valerio, additional, Pandey, Suraj, additional, Poudyal, Ishwor, additional, Sierra, Raymond, additional, Tolstikova, Alexandra, additional, Yefanov, Oleksandr, additional, Yoon, Ching Hong, additional, Ourmazd, Abbas, additional, Fromme, Petra, additional, Schwander, Peter, additional, Barty, Anton, additional, Chapman, Henry, additional, Stojković, Emina, additional, Batyuk, Alexander, additional, Boutet, Sébastien, additional, Phillips, George, additional, and Pollack, Lois, additional

Published
2023
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23. Modular droplet injector for sample conservation providing new structural insight for the conformational heterogeneity in the disease-associated NQO1 enzyme

Author

Doppler, Diandra, primary, Sonker, Mukul, additional, Egatz-Gomez, Ana, additional, Grieco, Alice, additional, Zaare, Sahba, additional, Jernigan, Rebecca, additional, Meza-Aguilar, Jose Domingo, additional, Rabbani, Mohammad T., additional, Manna, Abhik, additional, Alvarez, Roberto C., additional, Karpos, Konstantinos, additional, Cruz Villarreal, Jorvani, additional, Nelson, Garrett, additional, Yang, Jay-How, additional, Carrion, Jackson, additional, Morin, Katherine, additional, Ketawala, Gihan K., additional, Pey, Angel L., additional, Ruiz-Fresneda, Miguel Angel, additional, Pacheco-Garcia, Juan Luis, additional, Hermoso, Juan A., additional, Nazari, Reza, additional, Sierra, Raymond, additional, Hunter, Mark S., additional, Batyuk, Alexander, additional, Kupitz, Christopher J., additional, Sublett, Robert E., additional, Lisova, Stella, additional, Mariani, Valerio, additional, Boutet, Sébastien, additional, Fromme, Raimund, additional, Grant, Thomas D., additional, Botha, Sabine, additional, Fromme, Petra, additional, Kirian, Richard A., additional, Martin-Garcia, Jose Manuel, additional, and Ros, Alexandra, additional

Published
2023
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24. Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

Author

Sonker, Mukul, primary, Doppler, Diandra, additional, Egatz-Gomez, Ana, additional, Zaare, Sahba, additional, Rabbani, Mohammad T., additional, Manna, Abhik, additional, Cruz Villarreal, Jorvani, additional, Nelson, Garrett, additional, Ketawala, Gihan K., additional, Karpos, Konstantinos, additional, Alvarez, Roberto C., additional, Nazari, Reza, additional, Thifault, Darren, additional, Jernigan, Rebecca, additional, Oberthür, Dominik, additional, Han, Huijong, additional, Sierra, Raymond, additional, Hunter, Mark S., additional, Batyuk, Alexander, additional, Kupitz, Christopher J., additional, Sublett, Robert E., additional, Poitevin, Frederic, additional, Lisova, Stella, additional, Mariani, Valerio, additional, Tolstikova, Alexandra, additional, Boutet, Sebastien, additional, Messerschmidt, Marc, additional, Meza-Aguilar, J. Domingo, additional, Fromme, Raimund, additional, Martin-Garcia, Jose M., additional, Botha, Sabine, additional, Fromme, Petra, additional, Grant, Thomas D., additional, Kirian, Richard A., additional, and Ros, Alexandra, additional

Published
2022
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25. Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein

Author

Tenboer, Jason, Basu, Shibom, Zatsepin, Nadia, Pande, Kanupriya, Milathianaki, Despina, Frank, Matthias, Hunter, Mark, Boutet, Sébastien, Williams, Garth J., Koglin, Jason E., Oberthuer, Dominik, Heymann, Michael, Kupitz, Christopher, Conrad, Chelsie, Coe, Jesse, Roy-Chowdhury, Shatabdi, Weierstall, Uwe, James, Daniel, Wang, Dingjie, Grant, Thomas, Barty, Anton, Yefanov, Oleksandr, Scales, Jennifer, Gati, Cornelius, Seuring, Carolin, Srajer, Vukica, Henning, Robert, Schwander, Peter, Fromme, Raimund, Ourmazd, Abbas, Moffat, Keith, Van Thor, Jasper J., Spence, John C. H., Fromme, Petra, Chapman, Henry N., and Schmidt, Marius

Published
2014

26. Endogenous Retroviral Elements Generate Pathologic Neutrophils in Pulmonary Arterial Hypertension

Author

Taylor, Shalina, primary, Isobe, Sarasa, additional, Cao, Aiqin, additional, Contrepois, Kévin, additional, Benayoun, Bérénice A., additional, Jiang, Lihua, additional, Wang, Lingli, additional, Melemenidis, Stavros, additional, Ozen, Mehmet O., additional, Otsuki, Shoichiro, additional, Shinohara, Tsutomu, additional, Sweatt, Andrew J., additional, Kaplan, Jordan, additional, Moonen, Jan-Renier, additional, Marciano, David P., additional, Gu, Mingxia, additional, Miyagawa, Kazuya, additional, Hayes, Brandon, additional, Sierra, Raymond G., additional, Kupitz, Christopher J., additional, Del Rosario, Patricia A., additional, Hsi, Andrew, additional, Thompson, A. A. Roger, additional, Ariza, Maria E., additional, Demirci, Utkan, additional, Zamanian, Roham T., additional, Haddad, Francois, additional, Nicolls, Mark R., additional, Snyder, Michael P., additional, and Rabinovitch, Marlene, additional

Published
2022
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27. 7 Å resolution in protein two-dimensional-crystal X-ray diffraction at Linac Coherent Light Source

Author

Pedrini, Bill, Tsai, Ching-Ju, Capitani, Guido, Padeste, Celestino, Hunter, Mark S., Zatsepin, Nadia A., Barty, Anton, Benner, W. Henry, Boutet, Sébastien, Feld, Geoffrey K., Hau-Riege, Stefan P., Kirian, Richard A., Kupitz, Christopher, Messerschmitt, Marc, Ogren, John I., Pardini, Tommaso, Segelke, Brent, Williams, Garth J., Spence, John C. H., Abela, Rafael, Coleman, Matthew, Evans, James E., Schertler, Gebhard F. X., Frank, Matthias, and Li, Xiao-Dan

Published
2014

29. Serial Femtosecond Crystallography of G Protein-Coupled Receptors

Author

Liu, Wei, Wacker, Daniel, Gati, Cornelius, Han, Gye Won, James, Daniel, Wang, Dingjie, Nelson, Garrett, Weierstall, Uwe, Katritch, Vsevolod, Barty, Anton, Zatsepin, Nadia A., Li, Dianfan, Messerschmidt, Marc, Boutet, Sébastien, Williams, Garth J., Koglin, Jason E., Seibert, M. Marvin, Wang, Chong, Shah, Syed T. A., Basu, Shibom, Fromme, Raimund, Kupitz, Christopher, Rendek, Kimberley N., Grotjohann, Ingo, Fromme, Petra, Kirian, Richard A., Beyerlein, Kenneth R., White, Thomas A., Chapman, Henry N., Caffrey, Martin, Spence, John C. H., Stevens, Raymond C., and Cherezov, Vadim

Published
2013
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30. Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

Author

National Science Foundation (US), National Institutes of Health (US), Department of Energy (US), Max Planck Institute for Medical Research, Sonker, Mukul, Doppler, Diandra, Egatz-Gomez, Ana, Zaare, Sahba, Rabbani, Mohammad T., Manna, Abhik, Cruz Villarreal, Jorvani, Nelson, Garrett, Ketawala, Gihan K., Karpos, Konstantinos, Alvarez, Roberto C., Nazari, Reza, Thifault, Darren, Jernigan, Rebecca, Oberthür, Dominik, Han, Huijong, Sierra, Raymond, Hunter, Mark S., Batyuk, Alexander, Kupitz, Christopher J., Sublett, Robert E., Poitevin, Frederic, Lisova, Stella, Mariani, Valerio, Tolstikova, Alexandra, Boutet, Sebastien, Messerschmidt, Marc, Meza-Aguilar, J Domingo, Fromme, Raimund, Martín-García, Jose Manuel, Botha, Sabine, Fromme, Petra, Grant, Thomas D., Kirian, Richard A., Ros, Alexandra, National Science Foundation (US), National Institutes of Health (US), Department of Energy (US), Max Planck Institute for Medical Research, Sonker, Mukul, Doppler, Diandra, Egatz-Gomez, Ana, Zaare, Sahba, Rabbani, Mohammad T., Manna, Abhik, Cruz Villarreal, Jorvani, Nelson, Garrett, Ketawala, Gihan K., Karpos, Konstantinos, Alvarez, Roberto C., Nazari, Reza, Thifault, Darren, Jernigan, Rebecca, Oberthür, Dominik, Han, Huijong, Sierra, Raymond, Hunter, Mark S., Batyuk, Alexander, Kupitz, Christopher J., Sublett, Robert E., Poitevin, Frederic, Lisova, Stella, Mariani, Valerio, Tolstikova, Alexandra, Boutet, Sebastien, Messerschmidt, Marc, Meza-Aguilar, J Domingo, Fromme, Raimund, Martín-García, Jose Manuel, Botha, Sabine, Fromme, Petra, Grant, Thomas D., Kirian, Richard A., and Ros, Alexandra

Abstract

With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.

Published
2022

31. Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography

Author

Ertem, Fatma Betül; Güven, Ömür; Büyükdağ, Cengizhan; Göçenler, Oktay; Ayan, Esra; Yüksel, Büşra; Gül, Mehmet; Usta, Gözde; Çakılkaya, Barış; Johnson, J. Austin; Demirci, Hasan; Dağ, Çağdaş; Demirci, Hasan (ORCID 0000-0002-9135-5397 & YÖK ID 307350), Dao, E. Han; Su, Zhen; Poitevin, Frederic; Yoon, Chun Hong; Kupitz, Christopher; Hayes, Brandon; Liang, Mengning; Hunter, Mark S.; Batyuk, Alexander; Sierra, Raymond G.; Ketawala, Gihan; Botha, Sabine, Koç Üniversitesi İş Bankası Enfeksiyon Hastalıkları Uygulama ve Araştırma Merkezi (EHAM) / Koç University İşbank Center for Infectious Diseases (KU-IS CID), Graduate School of Sciences and Engineering; College of Sciences, Department of Molecular Biology and Genetics, Ertem, Fatma Betül; Güven, Ömür; Büyükdağ, Cengizhan; Göçenler, Oktay; Ayan, Esra; Yüksel, Büşra; Gül, Mehmet; Usta, Gözde; Çakılkaya, Barış; Johnson, J. Austin; Demirci, Hasan; Dağ, Çağdaş; Demirci, Hasan (ORCID 0000-0002-9135-5397 & YÖK ID 307350), Dao, E. Han; Su, Zhen; Poitevin, Frederic; Yoon, Chun Hong; Kupitz, Christopher; Hayes, Brandon; Liang, Mengning; Hunter, Mark S.; Batyuk, Alexander; Sierra, Raymond G.; Ketawala, Gihan; Botha, Sabine, Koç Üniversitesi İş Bankası Enfeksiyon Hastalıkları Uygulama ve Araştırma Merkezi (EHAM) / Koç University İşbank Center for Infectious Diseases (KU-IS CID), Graduate School of Sciences and Engineering; College of Sciences, and Department of Molecular Biology and Genetics

Abstract

The SARS-CoV-2 main protease of (Mpro) is an important target for SARS-CoV-2 related drug repurposing and development studies. Here, we describe the steps for structural characterization of SARS-CoV-2 Mpro, starting from plasmid preparation and protein purification. We detail the steps for crystallization using the sitting drop, microbatch (under oil) approach. Finally, we cover data collection and structure determination using serial femtosecond crystallography., National Science Foundation (NSF) Science and Technology Centers; BioXFEL; National Institutes of Health (NIH); National Institute of General Medical Sciences (NIGMS); Scientific and Technological Research Council of Turkey (TÜBİTAK); 2232 International Fellowship for Outstanding Researchers Program of TÜBİTAK; 2244 Industrial PhD Program of TÜBİTAK; 1001 TÜBİTAK Supporting Scientific and Technological Research Projects Program; Linac Coherent Light Source (LCLS); Stanford Synchrotron Light Source (SSRL); SLAC National Accelerator Laboratory; U.S. Department of Energy, Office of Science, Basic Energy Sciences

Published
2022

32. Cooperative allostery and structural dynamics of streptavidin at cryogenic- and ambient-temperature

Author

Dağ, Çağdaş; Ayan, Esra; Yüksel, Büşra; Destan, Ebru; Ertem, Fatma Betül; Yıldırım, Günseli; Eren, Meryem; Demirci, Hasan (ORCID 0000-0002-9135-5397 & YÖK ID 307350), Yefanov, Oleksandr M.; Barty, Anton; Tolstikova, Alexandra; Ketawala, Gihan K.; Botha, Sabine; Dao, E. Han; Hayes, Brandon; Liang, Mengning; Seaberg, Matthew H.; Hunter, Mark S.; Batyuk, Alexander; Mariani, Valerio; Su, Zhen; Poitevin, Frederic; Yoon, Chun Hong; Kupitz, Christopher; Cohen, Aina; Doukov, Tzanko; Sierra, Raymond G., Graduate School of Sciences and Engineering; College of Engineering, Department of Molecular Biology and Genetics, Dağ, Çağdaş; Ayan, Esra; Yüksel, Büşra; Destan, Ebru; Ertem, Fatma Betül; Yıldırım, Günseli; Eren, Meryem; Demirci, Hasan (ORCID 0000-0002-9135-5397 & YÖK ID 307350), Yefanov, Oleksandr M.; Barty, Anton; Tolstikova, Alexandra; Ketawala, Gihan K.; Botha, Sabine; Dao, E. Han; Hayes, Brandon; Liang, Mengning; Seaberg, Matthew H.; Hunter, Mark S.; Batyuk, Alexander; Mariani, Valerio; Su, Zhen; Poitevin, Frederic; Yoon, Chun Hong; Kupitz, Christopher; Cohen, Aina; Doukov, Tzanko; Sierra, Raymond G., Graduate School of Sciences and Engineering; College of Engineering, and Department of Molecular Biology and Genetics

Abstract

Ayan et al. report two structures of the protein streptavidin - one at ambient temperature determined using serial femtosecond crystallography and a second one determined at cryogenic temperature. These results provide insights into the structural dynamics of apo streptavidin and reveal a cooperative allostery between monomers for binding to biotin, and the findings are supported by GNM analysis. Multimeric protein assemblies are abundant in nature. Streptavidin is an attractive protein that provides a paradigm system to investigate the intra- and intermolecular interactions of multimeric protein complexes. Also, it offers a versatile tool for biotechnological applications. Here, we present two apo-streptavidin structures, the first one is an ambient temperature Serial Femtosecond X-ray crystal (Apo-SFX) structure at 1.7 angstrom resolution and the second one is a cryogenic crystal structure (Apo-Cryo) at 1.1 angstrom resolution. These structures are mostly in agreement with previous structural data. Combined with computational analysis, these structures provide invaluable information about structural dynamics of apo streptavidin. Collectively, these data further reveal a novel cooperative allostery of streptavidin which binds to substrate via water molecules that provide a polar interaction network and mimics the substrate biotin which displays one of the strongest affinities found in nature., National Science Foundation (NSF) Science and Technology Centers; 2232 International Fellowship for Outstanding Researchers Program; 1001 Scientific and Technological Research Projects Funding Program; Scientific and Technological Research Council of Turkey (TÜBİTAK); U.S. Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences (OBES); National Institutes of Health, National Institute of General Medical Sciences (NIGMS)

Published
2022

34. High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography

Author

Boutet, Sébastien, Lomb, Lukas, Williams, Garth J., Barends, Thomas R. M., Aquila, Andrew, Doak, R. Bruce, Weierstall, Uwe, DePonte, Daniel P., Steinbrener, Jan, Shoeman, Robert L., Messerschmidt, Marc, Barty, Anton, White, Thomas A., Kassemeyer, Stephan, Kirian, Richard A., Seibert, M. Marvin, Montanez, Paul A., Kenney, Chris, Herbst, Ryan, Hart, Philip, Pines, Jack, Haller, Gunther, Gruner, Sol M., Philipp, Hugh T., Tate, Mark W., Hromalik, Marianne, Koerner, Lucas J., van Bakel, Niels, Morse, John, Ghonsalves, Wilfred, Arnlund, David, Bogan, Michael J., Caleman, Carl, Fromme, Raimund, Hampton, Christina Y., Hunter, Mark S., Johansson, Linda C., Katona, Gergely, Kupitz, Christopher, Liang, Mengning, Martin, Andrew V., Nass, Karol, Redecke, Lars, Stellato, Francesco, Timneanu, Nicusor, Wang, Dingjie, Zatsepin, Nadia A., Schafer, Donald, Defever, James, Neutze, Richard, Fromme, Petra, Spence, John C. H., Chapman, Henry N., and Schlichting, Ilme

Published
2012
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35. Protocol for structure determination of SARS-CoV-2 main protease at near-physiological-temperature by serial femtosecond crystallography

Author

Ertem, Fatma Betul, primary, Guven, Omur, additional, Buyukdag, Cengizhan, additional, Gocenler, Oktay, additional, Ayan, Esra, additional, Yuksel, Busra, additional, Gul, Mehmet, additional, Usta, Gozde, additional, Cakılkaya, Barıs, additional, Johnson, J. Austin, additional, Dao, E. Han, additional, Su, Zhen, additional, Poitevin, Frederic, additional, Yoon, Chun Hong, additional, Kupitz, Christopher, additional, Hayes, Brandon, additional, Liang, Mengning, additional, Hunter, Mark S., additional, Batyuk, Alexander, additional, Sierra, Raymond G., additional, Ketawala, Gihan, additional, Botha, Sabine, additional, Dağ, Çağdaş, additional, and DeMirci, Hasan, additional

Published
2022
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36. The room temperature crystal structure of a bacterial phytochrome determined by serial femtosecond crystallography

Author

Edlund, Petra, Takala, Heikki, Claesson, Elin, Henry, Léocadie, Dods, Robert, Lehtivuori, Heli, Panman, Matthijs, Pande, Kanupriya, White, Thomas, Nakane, Takanori, Berntsson, Oskar, Gustavsson, Emil, Båth, Petra, Modi, Vaibhav, Roy-Chowdhury, Shatabdi, Zook, James, Berntsen, Peter, Pandey, Suraj, Poudyal, Ishwor, Tenboer, Jason, Kupitz, Christopher, Barty, Anton, Fromme, Petra, Koralek, Jake D., Tanaka, Tomoyuki, Spence, John, Liang, Mengning, Hunter, Mark S., Boutet, Sebastien, Nango, Eriko, Moffat, Keith, Groenhof, Gerrit, Ihalainen, Janne, Stojković, Emina A., Schmidt, Marius, and Westenhoff, Sebastian

Published
2016
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37. Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

Author

Guven, Omur, primary, Gul, Mehmet, additional, Ayan, Esra, additional, Johnson, J Austin, additional, Cakilkaya, Baris, additional, Usta, Gozde, additional, Ertem, Fatma Betul, additional, Tokay, Nurettin, additional, Yuksel, Busra, additional, Gocenler, Oktay, additional, Buyukdag, Cengizhan, additional, Botha, Sabine, additional, Ketawala, Gihan, additional, Su, Zhen, additional, Hayes, Brandon, additional, Poitevin, Frederic, additional, Batyuk, Alexander, additional, Yoon, Chun Hong, additional, Kupitz, Christopher, additional, Durdagi, Serdar, additional, Sierra, Raymond G., additional, and DeMirci, Hasan, additional

Published
2021
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38. Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing

Author

Durdagi, Serdar, primary, Dağ, Çağdaş, additional, Dogan, Berna, additional, Yigin, Merve, additional, Avsar, Timucin, additional, Buyukdag, Cengizhan, additional, Erol, Ismail, additional, Ertem, Fatma Betul, additional, Calis, Seyma, additional, Yildirim, Gunseli, additional, Orhan, Muge D., additional, Guven, Omur, additional, Aksoydan, Busecan, additional, Destan, Ebru, additional, Sahin, Kader, additional, Besler, Sabri O., additional, Oktay, Lalehan, additional, Shafiei, Alaleh, additional, Tolu, Ilayda, additional, Ayan, Esra, additional, Yuksel, Busra, additional, Peksen, Ayse B., additional, Gocenler, Oktay, additional, Yucel, Ali D., additional, Can, Ozgur, additional, Ozabrahamyan, Serena, additional, Olkan, Alpsu, additional, Erdemoglu, Ece, additional, Aksit, Fulya, additional, Tanisali, Gokhan, additional, Yefanov, Oleksandr M., additional, Barty, Anton, additional, Tolstikova, Alexandra, additional, Ketawala, Gihan K., additional, Botha, Sabine, additional, Dao, E. Han, additional, Hayes, Brandon, additional, Liang, Mengning, additional, Seaberg, Matthew H., additional, Hunter, Mark S., additional, Batyuk, Alex, additional, Mariani, Valerio, additional, Su, Zhen, additional, Poitevin, Frederic, additional, Yoon, Chun Hong, additional, Kupitz, Christopher, additional, Sierra, Raymond G., additional, Snell, Edward H., additional, and DeMirci, Hasan, additional

Published
2021
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39. Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser

Author

Kupitz, Christopher, Basu, Shibom, Grotjohann, Ingo, Fromme, Raimund, Zatsepin, Nadia A., Rendek, Kimberly N., and Hunter, Mark S.

Subjects
Crystallography -- Research, Free electron lasers -- Research, Photosystem II -- Research, Environmental issues, Science and technology, Zoology and wildlife conservation
Abstract

Femtosecond X-ray pulses were used to obtain diffraction data on photosystem II, revealing conformational changes as the complex transitions from the dark S.sub.1 state to the double-pumped S.sub.3 state; the time-resolved serial femtosecond crystallography technique enables structural determination of protein conformations that are highly prone to traditional radiation damage. An X-ray snapshot of photosystem II structure It has recently been shown that extremely short and intense radiation pulses from X-ray free-electron lasers can be used to obtain diffraction data on nanometre- to micrometre-sized protein crystals before the crystal suffers radiation damage. The hope is that this 'serial femtosecond crystallography' (SFX) approach will produce structures of proteins and protein complexes that do not yield well-ordered macroscopic crystals. These authors collected time-resolved SFX data on small crystals of photosystem II of photosynthesis during its transition from the 'dark' S1 state to the double-excited S3 state. At present the resolution of this technique is moderate, but it is sufficient to reveal significant conformational changes at the Mn.sub.4CaO.sub.5 cluster at the heart of the oxygen evolving complex and at the electron acceptor site. Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth's oxygenic atmosphere.sup.1. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S.sub.0 to S.sub.4, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed.sup.2 technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S.sub.1 state and after double laser excitation (putative S.sub.3 state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn.sub.4CaO.sub.5 core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the 'dangler' Mn) and the Mn.sub.3CaO.sub.x cubane in the S.sub.2 to S.sub.3 transition, as predicted by spectroscopic and computational studies.sup.3,4. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules., Author(s): Christopher Kupitz [sup.1] , Shibom Basu [sup.1] , Ingo Grotjohann [sup.1] , Raimund Fromme [sup.1] , Nadia A. Zatsepin [sup.2] , Kimberly N. Rendek [sup.1] , Mark S. Hunter [...]

Published
2014
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40. PROTEIN STRUCTURE: Femtosecond structural dynamics drives the trans/cis isomerization in photoactive yellow protein

Author

Pande, Kanupriya, Hutchison, Christopher D. M., Groenhof, Gerrit, Aquila, Andy, Robinson, Josef S., Tenboer, Jason, Basu, Shibom, Boutet, Sébastien, DePonte, Daniel P., Liang, Mengning, White, Thomas A., Zatsepin, Nadia A., Yefanov, Oleksandr, Morozov, Dmitry, Oberthuer, Dominik, Gati, Cornelius, Subramanian, Ganesh, James, Daniel, Zhao, Yun, Koralek, Jake, Brayshaw, Jennifer, Kupitz, Christopher, Conrad, Chelsie, Roy-Chowdhury, Shatabdi, Coe, Jesse D., Metz, Markus, Xavier, Paulraj Lourdu, Grant, Thomas D., Koglin, Jason E., Ketawala, Gihan, Fromme, Raimund, Šrajer, Vukica, Henning, Robert, Spence, John C. H., Ourmazd, Abbas, Schwander, Peter, Weierstall, Uwe, Frank, Matthias, Fromme, Petra, Barty, Anton, Chapman, Henry N., Moffat, Keith, van Thor, Jasper J., and Schmidt, Marius

Published
2016
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41. Macromolecular diffractive imaging using imperfect crystals

Author

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.

Published
2016
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43. Cooperative Allostery and Structural Dynamics of Streptavidin at Cryogenic- and Ambient-temperature

Author

Ayan, Esra, primary, Yuksel, Busra, additional, Destan, Ebru, additional, Ertem, Fatma Betul, additional, Yildirim, Gunseli, additional, Eren, Meryem, additional, Yefanov, Oleksandr M., additional, Barty, Anton, additional, Tolstikova, Alexandra, additional, Ketawala, Gihan K., additional, Botha, Sabine, additional, Dao, E. Han, additional, Hayes, Brandon, additional, Liang, Mengning, additional, Seaberg, Matthew H., additional, Hunter, Mark S., additional, Batyuk, Alex, additional, Mariani, Valerio, additional, Su, Zhen, additional, Poitevin, Frederic, additional, Yoon, Chun Hong, additional, Kupitz, Christopher, additional, Cohen, Aina, additional, Doukov, Tzanko, additional, Sierra, Raymond G., additional, Dağ, Çağdaş, additional, and DeMirci, Hasan, additional

Published
2021
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44. The Macromolecular Femtosecond Crystallography Instrument at the Linac Coherent Light Source1

Author

Sierra, Raymond G., Batyuk, Alexander, Sun, Zhibin, Aquila, Andrew, Hunter, Mark S., Lane, Thomas J., Liang, Mengning, Yoon, Chun Hong, Alonso-Mori, Roberto, Armenta, Rebecca, Castagna, Jean-Charles, Hollenbeck, Michael, Osier, Ted O., Hayes, Matt, Aldrich, Jeff, Curtis, Robin, Koglin, Jason E., Rendahl, Theodore, Rodriguez, Evan, Carbajo, Sergio, Guillet, Serge, Paul, Rob, Hart, Philip, Nakahara, Kazutaka, Carini, Gabriella, DeMirci, Hasan, Dao, E. Han, Hayes, Brandon M., Rao, Yashas P., Chollet, Matthieu, Feng, Yiping, Fuller, Franklin D., Kupitz, Christopher, Sato, Takahiro, Seaberg, Matthew H., Song, Sanghoon, van Driel, Tim B., Yavas, Hasan, Zhu, Diling, Cohen, Aina E., Wakatsuki, Soichi, and Boutet, Sébastien

Subjects
serial femtosecond crystallography, protein crystallography, time-resolved crystallography, X-ray free-electron laser, room-temperature crystallography, X-ray FEL, Research Papers
Abstract

A description of the Macromolecular Femtosecond Crystallography instrument at the Linac Coherent Light Source is given. Scientific capabilities are highlighted, performance parameters are presented along with some commissioning results., The Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS) is the seventh and newest instrument at the world’s first hard X-ray free-electron laser. It was designed with a primary focus on structural biology, employing the ultrafast pulses of X-rays from LCLS at atmospheric conditions to overcome radiation damage limitations in biological measurements. It is also capable of performing various time-resolved measurements. The MFX design consists of a versatile base system capable of supporting multiple methods, techniques and experimental endstations. The primary techniques supported are forward scattering and crystallography, with capabilities for various spectroscopic methods and time-resolved measurements. The location of the MFX instrument allows for utilization of multiplexing methods, increasing user access to LCLS by running multiple experiments simultaneously.

Published
2019

45. STRUCTURAL BIOLOGY: Time-resolved serial crystallography captures high-resolution intermediates of photoactive yellow protein

Author

Tenboer, Jason, Basu, Shibom, Zatsepin, Nadia, Pande, Kanupriya, Milathianaki, Despina, Frank, Matthias, Hunter, Mark, Boutet, Sébastien, Williams, Garth J., Koglin, Jason E., Oberthuer, Dominik, Heymann, Michael, Kupitz, Christopher, Conrad, Chelsie, Coe, Jesse, Roy-Chowdhury, Shatabdi, Weierstall, Uwe, James, Daniel, Wang, Dingjie, Grant, Thomas, Barty, Anton, Yefanov, Oleksandr, Scales, Jennifer, Gati, Cornelius, Seuring, Carolin, Srajer, Vukica, Henning, Robert, Schwander, Peter, Fromme, Raimund, Ourmazd, Abbas, Moffat, Keith, Van Thor, Jasper J., Spence, John C. H., Fromme, Petra, Chapman, Henry N., and Schmidt, Marius

Published
2014
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46. Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing

Author

Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Department of Materials Science and Engineering, Demirci, Hasan (ORCID 0000-0002-9135-5397 & YÖK ID 307350); Dağ, Çağdaş; Yığın, Merve; Büyükdağ, Cengizhan; Ertem, Fatma Betül; Yıldırım, Günseli; Destan, Ebru; Güven, Ömür; Ayan, Esra; Yüksel, Büşra; Pekşen, Ayşe Buket; Göçenler, Oktay; Yücel, Ali Doğa; Can, Özgür; Ozabrahamyan, Serena; Shafiei, Alaleh; Akşit, Fulya; Tanısalı, Gökhan; Besler, Sabri Özkan, Durdağı, Serdar; Doğan, Berna; Avşar, Timuçin; Erol, İsmail; Çalış, Şeyma; Orhan, Müge D.; Aksoydan, Busecan; Şahin, Kader; Oktay, Lalehan; Tolu, İlayda; Olkan, Alpsu; Erdemoğlu, Ece; Yefanov, Oleksandr M.; Dao, E. Han; Hayes, Brandon; Liang, Mengning; Seaberg, Matthew H.; Hunter, Mark S.; Batyuk, Alex; Mariani, Valerio; Su, Zhen; Poitevin, Frederic; Yoon, Chun Hong; Kupitz, Christopher; Sierra, Raymond G.; Snell, Edward H., Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Department of Materials Science and Engineering, Demirci, Hasan (ORCID 0000-0002-9135-5397 & YÖK ID 307350); Dağ, Çağdaş; Yığın, Merve; Büyükdağ, Cengizhan; Ertem, Fatma Betül; Yıldırım, Günseli; Destan, Ebru; Güven, Ömür; Ayan, Esra; Yüksel, Büşra; Pekşen, Ayşe Buket; Göçenler, Oktay; Yücel, Ali Doğa; Can, Özgür; Ozabrahamyan, Serena; Shafiei, Alaleh; Akşit, Fulya; Tanısalı, Gökhan; Besler, Sabri Özkan, and Durdağı, Serdar; Doğan, Berna; Avşar, Timuçin; Erol, İsmail; Çalış, Şeyma; Orhan, Müge D.; Aksoydan, Busecan; Şahin, Kader; Oktay, Lalehan; Tolu, İlayda; Olkan, Alpsu; Erdemoğlu, Ece; Yefanov, Oleksandr M.; Dao, E. Han; Hayes, Brandon; Liang, Mengning; Seaberg, Matthew H.; Hunter, Mark S.; Batyuk, Alex; Mariani, Valerio; Su, Zhen; Poitevin, Frederic; Yoon, Chun Hong; Kupitz, Christopher; Sierra, Raymond G.; Snell, Edward H.

Abstract

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2.

Published
2021

47. Near-physiological-temperature serial crystallography reveals conformations of SARS-CoV-2 main protease active site for improved drug repurposing

Author

Demirci, Hasan (ORCID 0000-0002-9135-5397 & YÖK ID 307350); Dağ, Çağdaş; Yığın, Merve; Büyükdağ, Cengizhan; Ertem, Fatma Betül; Yıldırım, Günseli; Destan, Ebru; Güven, Ömür; Ayan, Esra; Yüksel, Büşra; Pekşen, Ayşe Buket; Göçenler, Oktay; Yücel, Ali Doğa; Can, Özgür; Ozabrahamyan, Serena; Shafiei, Alaleh; Akşit, Fulya; Tanısalı, Gökhan; Besler, Sabri Özkan, Durdağı, Serdar; Doğan, Berna; Avşar, Timuçin; Erol, İsmail; Çalış, Şeyma; Orhan, Müge D.; Aksoydan, Busecan; Şahin, Kader; Oktay, Lalehan; Tolu, İlayda; Olkan, Alpsu; Erdemoğlu, Ece; Yefanov, Oleksandr M.; Dao, E. Han; Hayes, Brandon; Liang, Mengning; Seaberg, Matthew H.; Hunter, Mark S.; Batyuk, Alex; Mariani, Valerio; Su, Zhen; Poitevin, Frederic; Yoon, Chun Hong; Kupitz, Christopher; Sierra, Raymond G.; Snell, Edward H., Koç Üniversitesi İş Bankası Enfeksiyon Hastalıkları Uygulama ve Araştırma Merkezi (EHAM) / Koç University İşbank Center for Infectious Diseases (KU-IS CID), College of Sciences; Graduate School of Sciences and Engineering; School of Nursing, Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Department of Materials Science and Engineering, Demirci, Hasan (ORCID 0000-0002-9135-5397 & YÖK ID 307350); Dağ, Çağdaş; Yığın, Merve; Büyükdağ, Cengizhan; Ertem, Fatma Betül; Yıldırım, Günseli; Destan, Ebru; Güven, Ömür; Ayan, Esra; Yüksel, Büşra; Pekşen, Ayşe Buket; Göçenler, Oktay; Yücel, Ali Doğa; Can, Özgür; Ozabrahamyan, Serena; Shafiei, Alaleh; Akşit, Fulya; Tanısalı, Gökhan; Besler, Sabri Özkan, Durdağı, Serdar; Doğan, Berna; Avşar, Timuçin; Erol, İsmail; Çalış, Şeyma; Orhan, Müge D.; Aksoydan, Busecan; Şahin, Kader; Oktay, Lalehan; Tolu, İlayda; Olkan, Alpsu; Erdemoğlu, Ece; Yefanov, Oleksandr M.; Dao, E. Han; Hayes, Brandon; Liang, Mengning; Seaberg, Matthew H.; Hunter, Mark S.; Batyuk, Alex; Mariani, Valerio; Su, Zhen; Poitevin, Frederic; Yoon, Chun Hong; Kupitz, Christopher; Sierra, Raymond G.; Snell, Edward H., Koç Üniversitesi İş Bankası Enfeksiyon Hastalıkları Uygulama ve Araştırma Merkezi (EHAM) / Koç University İşbank Center for Infectious Diseases (KU-IS CID), College of Sciences; Graduate School of Sciences and Engineering; School of Nursing, and Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering; Department of Materials Science and Engineering

Abstract

The COVID-19 pandemic has resulted in 198 million reported infections and more than 4 million deaths as of July 2021 (covid19.who.int). Research to identify effective therapies for COVID-19 includes: (1) designing a vaccine as future protection; (2) de novo drug discovery; and (3) identifying existing drugs to repurpose them as effective and immediate treatments. To assist in drug repurposing and design, we determine two apo structures of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease at ambient temperature by serial femtosecond X-ray crystallography. We employ detailed molecular simulations of selected known main protease inhibitors with the structures and compare binding modes and energies. The combined structural and molecular modeling studies not only reveal the dynamics of small molecules targeting the main protease but also provide invaluable opportunities for drug repurposing and structure-based drug design strategies against SARS-CoV-2., National Science Foundation (NSF) Science and Technology Centers; BioXFEL; Scientific and Technological Research Council of Turkey (TÜBİTAK); Bahçeşehir University (BAU) Scientific Research Projects (BAP)

Published
2021

48. Case study of high-throughput drug screening and remote data collection for SARS-CoV-2 main protease by using serial femtosecond X-ray crystallography

Author

Güven, Ömür; Gül, Mehmet; Ayan, Esra; Johnson, Jerome Austin; Çakılkaya, Barış; Usta, Gözde Karakadıoğlu; Ertem, Fatma Betül; Tokay, Nurettin; Yüksel, Büşra; Göcenler, Oktay; Büyükdağ, Cengizhan; Demirci, Hasan (ORCID 0000-0002-9135-5397 & YÖK ID 307350), Botha, Sabine; Ketawala, Gihan; Su, Zhen; Hayes, Brandon; Poitevin, Frederic; Batyuk, Alexander; Yoon, Chun Hong; Kupitz, Christopher; Durdağı, Serdar; Sierra, Raymond G., Koç Üniversitesi İş Bankası Enfeksiyon Hastalıkları Uygulama ve Araştırma Merkezi (EHAM) / Koç University İşbank Center for Infectious Diseases (KU-IS CID), Graduate School of Sciences and Engineering; College of Sciences, Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering, Güven, Ömür; Gül, Mehmet; Ayan, Esra; Johnson, Jerome Austin; Çakılkaya, Barış; Usta, Gözde Karakadıoğlu; Ertem, Fatma Betül; Tokay, Nurettin; Yüksel, Büşra; Göcenler, Oktay; Büyükdağ, Cengizhan; Demirci, Hasan (ORCID 0000-0002-9135-5397 & YÖK ID 307350), Botha, Sabine; Ketawala, Gihan; Su, Zhen; Hayes, Brandon; Poitevin, Frederic; Batyuk, Alexander; Yoon, Chun Hong; Kupitz, Christopher; Durdağı, Serdar; Sierra, Raymond G., Koç Üniversitesi İş Bankası Enfeksiyon Hastalıkları Uygulama ve Araştırma Merkezi (EHAM) / Koç University İşbank Center for Infectious Diseases (KU-IS CID), Graduate School of Sciences and Engineering; College of Sciences, and Department of Molecular Biology and Genetics; Department of Chemical and Biological Engineering

Abstract

Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing; therefore, confirmation of in silico drug screening studies have been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with nine drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol., National Science Foundation (NSF) Science and Technology Centers Grant; Biology with X-ray Lasers (BioXFEL); Scientific and Technological Research Council of Turkey (TÜBİTAK); 2232 International Fellowship for Outstanding Researchers Program; 2244 Industrial PhD Fellowship Program; 1001 Scientific and Technological Research Projects Funding Program

Published
2021

49. Direct Observation of the Mechanism of Antibiotic Resistance by Mix-and-Inject at the European XFEL

Author

Pandey, Suraj, Bean, Richard, Hunter, Mark, Abdellatif, Mohamed H., Bajt, Saša, Bondar, Valerii, Echelmeier, Austin, Doppler, Diandra, Emons, Moritz, Frank, Matthias, Fromme, Raimund, Gevorkov, Yaroslav, Sato, Tokushi, Giovanetti, Gabriele, Jiang, Man, Kim, Daihyun, Kim, Yoonhee, Kirkwood, Henry, Klimovskaia, Anna, Knoska, Juraj, Koua, Faisal H. M., Letrun, Romain, Lisova, Stella, Poudyal, Ishwor, Maia, Luis, Mazalova, Victoria, Meza, Domingo, Michelat, Thomas, Ourmazd, Abbas, Palmer, Guido, Ramilli, Marco, Schubert, Robin, Schwander, Peter, Silenzi, Alessandro, Bielecki, Johan, Sztuk-Dambietz, Jolanta, Tolstikova, Alexandra, Chapman, Henry N., Ros, Alexandra, Barty, Anton, Fromme, Petra, Mancuso, Adrian P., Schmidt, Marius, Cruz Villarreal, Jorvani, Yefanov, Oleksandr, Mariani, Valerio, White, Thomas A., and Kupitz, Christopher

Subjects
Enzyme substrate complex, Millisecond, Materials science, Bacterial antibiotic resistance, Chemical physics, Atomic resolution, Direct observation, Substrate (chemistry), Time resolution, ddc:610, Ultrashort pulse
Abstract

In this study, we follow the diffusion and buildup of occupancy of the substrate ceftriaxone inM. tuberculosisβ-lactamase BlaC microcrystals by structural analysis of the enzyme substrate complex at single millisecond time resolution. We also show the binding and the reaction of an inhibitor, sulbactam, on a slower millisecond time scale. We use the ‘mix-and-inject’ technique to initiate these reactions by diffusion, and determine the resulting structures by serial crystallography using ultrafast, intense X-ray pulses from the European XFEL (EuXFEL) arriving at MHz repetition rates. Here, we show how to use the EuXFEL pulse structure to dramatically increase the size of the data set and thereby the quality and time resolution of “molecular movies” which unravel ligand binding and enzymatically catalyzed reactions. This shows the great potential for the EuXFEL as a tool for biomedically relevant research, particularly, as shown here, for investigating bacterial antibiotic resistance.One Sentence SummaryDirect observation of fast ligand binding in a biomedically relevant enzyme at near atomic resolution with MHz X-ray pulses at the European XFEL.

Published
2020

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