Matching Items (14)
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Description
Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the

Phytochromes are a family of photoreceptors that control light responses of plants, fungi and bacteria. A sequence of structural changes, which is not yet fully understood, leads to activation of an output domain. Time-resolved serial femtosecond crystallography (SFX) can potentially shine light on these conformational changes. Here we report the room temperature crystal structure of the chromophore-binding domains of the Deinococcus radiodurans phytochrome at 2.1 Å resolution. The structure was obtained by serial femtosecond X-ray crystallography from microcrystals at an X-ray free electron laser. We find overall good agreement compared to a crystal structure at 1.35 Å resolution derived from conventional crystallography at cryogenic temperatures, which we also report here. The thioether linkage between chromophore and protein is subject to positional ambiguity at the synchrotron, but is fully resolved with SFX. The study paves the way for time-resolved structural investigations of the phytochrome photocycle with time-resolved SFX.
ContributorsEdlund, Petra (Author) / Takala, Heikki (Author) / Claesson, Elin (Author) / Henry, Leocadie (Author) / Dods, Robert (Author) / Lehtivuori, Heli (Author) / Panman, Matthijs (Author) / Pande, Kanupriya (Author) / White, Thomas (Author) / Nakane, Takanori (Author) / Berntsson, Oskar (Author) / Gustavsson, Emil (Author) / Bath, Petra (Author) / Modi, Vaibhav (Author) / Roy Chowdhury, Shatabdi (Author) / Zook, James (Author) / Berntsen, Peter (Author) / Pandey, Suraj (Author) / Poudyal, Ishwor (Author) / Tenboer, Jason (Author) / Kupitz, Christopher (Author) / Barty, Anton (Author) / Fromme, Petra (Author) / Koralek, Jake D. (Author) / Tanaka, Tomoyuki (Author) / Spence, John (Author) / Liang, Mengning (Author) / Hunter, Mark S. (Author) / Boutet, Sebastien (Author) / Nango, Eriko (Author) / Moffat, Keith (Author) / Groenhof, Gerrit (Author) / Ihalainen, Janne (Author) / Stojkovic, Emina A. (Author) / Schmidt, Marius (Author) / Westenhoff, Sebastian (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2016-10-19
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Description
Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement.

Serial femtosecond crystallography (SFX) using X-ray free-electron laser sources is an emerging method with considerable potential for time-resolved pump-probe experiments. Here we present a lipidic cubic phase SFX structure of the light-driven proton pump bacteriorhodopsin (bR) to 2.3 Å resolution and a method to investigate protein dynamics with modest sample requirement. Time-resolved SFX (TR-SFX) with a pump-probe delay of 1 ms yields difference Fourier maps compatible with the dark to M state transition of bR. Importantly, the method is very sample efficient and reduces sample consumption to about 1 mg per collected time point. Accumulation of M intermediate within the crystal lattice is confirmed by time-resolved visible absorption spectroscopy. This study provides an important step towards characterizing the complete photocycle dynamics of retinal proteins and demonstrates the feasibility of a sample efficient viscous medium jet for TR-SFX.
ContributorsNogly, Przemyslaw (Author) / Panneels, Valerie (Author) / Nelson, Garrett (Author) / Gati, Cornelius (Author) / Kimura, Tetsunari (Author) / Milne, Christopher (Author) / Milathianaki, Despina (Author) / Kubo, Minoru (Author) / Wu, Wenting (Author) / Conrad, Chelsie (Author) / Coe, Jesse (Author) / Bean, Richard (Author) / Zhao, Yun (Author) / Bath, Petra (Author) / Dods, Robert (Author) / Harimoorthy, Rajiv (Author) / Beyerlein, Kenneth R. (Author) / Rheinberger, Jan (Author) / James, Daniel (Author) / Deponte, Daniel (Author) / Li, Chufeng (Author) / Sala, Leonardo (Author) / Williams, Garth J. (Author) / Hunter, Mark S. (Author) / Koglin, Jason E. (Author) / Berntsen, Peter (Author) / Nango, Eriko (Author) / Iwata, So (Author) / Chapman, Henry N. (Author) / Fromme, Petra (Author) / Frank, Matthias (Author) / Abela, Rafael (Author) / Boutet, Sebastien (Author) / Barty, Anton (Author) / White, Thomas A. (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Neutze, Richard (Author) / Schertler, Gebhard (Author) / Standfuss, Jorg (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / School of Molecular Sciences (Contributor)
Created2016-08-22
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Description
In materials science, developing GeSn alloys is major current research interest concerning the production of efficient Group-IV photonics. These alloys are particularly interesting because the development of next-generation semiconductors for ultrafast (terahertz) optoelectronic communication devices could be accomplished through integrating these novel alloys with industry-standard silicon technology. Unfortunately, incorporating a

In materials science, developing GeSn alloys is major current research interest concerning the production of efficient Group-IV photonics. These alloys are particularly interesting because the development of next-generation semiconductors for ultrafast (terahertz) optoelectronic communication devices could be accomplished through integrating these novel alloys with industry-standard silicon technology. Unfortunately, incorporating a maximal amount of Sn into a Ge lattice has been difficult to achieve experimentally. At ambient conditions, pure Ge and Sn adopt cubic (α) and tetragonal (β) structures, respectively, however, to date the relative stability and structure of α and β phase GeSn alloys versus percent composition Sn has not been thoroughly studied. In this research project, computational tools were used to perform state-of-the-art predictive quantum simulations to study the structural, bonding and energetic trends in GeSn alloys in detail over a range of experimentally accessible compositions. Since recent X-Ray and vibrational studies have raised some controversy about the nanostructure of GeSn alloys, the investigation was conducted with ordered, random and clustered alloy models.
By means of optimized geometry analysis, pure Ge and Sn were found to adopt the alpha and beta structures, respectively, as observed experimentally. For all theoretical alloys, the corresponding αphase structure was found to have the lowest energy, for Sn percent compositions up to 90%. However at 50% Sn, the correspondingβ alloy energies are predicted to be only ~70 meV higher. The formation energy of α-phase alloys was found to be positive for all compositions, whereas only two beta formation energies were negative. Bond length distributions were analyzed and dependence on Sn incorporation was found, perhaps surprisingly, not to be directly correlated with cell volume. It is anticipated that the data collected in this project may help to elucidate observed complex vibrational properties in these systems.
ContributorsLiberman-Martin, Zoe Elise (Author) / Chizmeshya, Andrew (Thesis director) / Sayres, Scott (Committee member) / Wolf, George (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description
Carbon allotropes are the basis for many exciting advancements in technology. While sp² and sp³ hybridizations are well understood, the sp¹ hybridized carbon has been elusive. However, with recent advances made using a pulsed laser ablation in liquid technique, sp¹ hybridized carbon allotropes have been created. The fabricated carbon chain

Carbon allotropes are the basis for many exciting advancements in technology. While sp² and sp³ hybridizations are well understood, the sp¹ hybridized carbon has been elusive. However, with recent advances made using a pulsed laser ablation in liquid technique, sp¹ hybridized carbon allotropes have been created. The fabricated carbon chain is composed of sp¹ and sp³ hybridized bonds, but it also incorporates nanoparticles such as gold or possibly silver to stabilize the chain. The polyyne generated in this process is called pseudocarbyne due to its striking resemblance to the theoretical carbyne. The formation of these carbon chains is yet to be fully understood, but significant progress has been made in determining the temperature of the plasma in which the pseudocarbyne is formed. When a 532 nm pulsed laser with a pulsed energy of 250 mJ and pulse length of 10ns is used to ablate a gold target, a peak temperature of 13400 K is measured. When measured using Laser-Induced Breakdown spectroscopy (LIBS) the average temperature of the neutral carbon plasma over one second was 4590±172 K. This temperature strongly suggests that the current theoretical model used to describe the temperature at which pseudocarbyne generates is accurate.
ContributorsWala, Ryland Gerald (Co-author) / Wala, Ryland (Co-author) / Sayres, Scott (Thesis director) / Steimle, Timothy (Committee member) / Drucker, Jeffery (Committee member) / Historical, Philosophical & Religious Studies (Contributor) / Dean, W.P. Carey School of Business (Contributor) / Department of Physics (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
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Description
Dielectrophoretic trapping is a separatory/analytical method that is capable of achieving high levels of analyte differentiation using a combination of electroosmotic flow, electrophoresis, and dielectrophoresis. The form of dielectrophoretic device used in these trials was of a gradient insulator-based design that induced the non-uniform electric fields necessary for dielectrophoretic trapping

Dielectrophoretic trapping is a separatory/analytical method that is capable of achieving high levels of analyte differentiation using a combination of electroosmotic flow, electrophoresis, and dielectrophoresis. The form of dielectrophoretic device used in these trials was of a gradient insulator-based design that induced the non-uniform electric fields necessary for dielectrophoretic trapping to occur. Development of such microfluidic devices began in the early 2000s and has produced several successful trials and refinements since then. Improvements have led to the ability of these devices to separate analytes to extremely high degrees of resolution as was demonstrated by the simultaneous separation of antibiotic resistant and antibiotic susceptible strains of bacteria in other experiments. The majority of analytes examined with these microfluidic devices have been biological in nature and on the scale of micrometers in size. The objective of this experiment was to test the lower limit of the device's resolution by attempting to use dielectrophoresis to trap gold nanoparticles via the balancing point between electrophoretic and dielectrophoretic mobilities. Trials successfully captured 10 nm fluorophore tagged gold nanoparticles at a mobility ratio of 6.16 x 1011 V2/m3, 60 nm citrate-capped gold nanoparticles at approximately 3.61 x 1010 V2/m3, and bare 10 nm gold nanoparticle aggregates at both 1.63 x 1010 V2/m3 and 1.68 x 1010 V2/m3. The corresponding voltages that were applied to achieve trapping were -1500 V, -2000 V, and -1500 V respectively. These findings were promising but reproducibility of the results was very low, largely due to matters of contaminants entering the devices and preventing the even, continuous flow of the analyte solution. Refinement of the analytical process should be pursued.
ContributorsRamirez, Alexis Jordan (Author) / Hayes, Mark (Thesis director) / Sayres, Scott (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
Description

The photodissociation of 1-bromobutane is explored using pump-probe spectroscopy and time-of-flight mass spectrometry. Fragments of bromobutane are constructed computationally and theoretical energies are calculated using Gaussian 16 software. It is determined that the dissociation of bromine from the parent molecule is the most observed fragmentation pathway arising from the excitation

The photodissociation of 1-bromobutane is explored using pump-probe spectroscopy and time-of-flight mass spectrometry. Fragments of bromobutane are constructed computationally and theoretical energies are calculated using Gaussian 16 software. It is determined that the dissociation of bromine from the parent molecule is the most observed fragmentation pathway arising from the excitation of the ground state parent molecule to a dissociative A state using two 400 nm, 3.1 eV pump photons. The dissociation energy of this pathway is 2.91 eV, leaving 3.3 eV of energy that is redistributed into the product fragments as vibrational energy. C4H9 has the highest relative intensity in the mass spectrum with a relative intensity of 1.00. It is followed by C2H5 and C2H4 at relative intensities of 0.73 and 0.29 respectively. Because of the negative correlation between C4H9 and these two fragments at positive time delays, it is concluded that most of these smaller molecules are formed from the further dissociation of the fragment C4H9 rather than any alternative pathways from the parent molecule. Thermodynamic analysis of these pathways has displayed the power of thermodynamic prediction as well as its limitations as it fails to consider kinetic limitations in dissociation reactions.

ContributorsGosman, Robert (Author) / Sayres, Scott (Thesis director) / Chizmeshya, Andrew (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor) / Department of Physics (Contributor)
Created2023-05
Description

Dielectrophoresis is an analytical technique which involves electroosmotic flow, electrophoresis, and dielectrophoretic force. These factors, when in correct proportions for a given analyte, allow for dielectrophoretic trapping, otherwise known as dielectrophoretic capture. Non-uniform electric fields are required for this phenomenon, and the device in this trial used to induce such

Dielectrophoresis is an analytical technique which involves electroosmotic flow, electrophoresis, and dielectrophoretic force. These factors, when in correct proportions for a given analyte, allow for dielectrophoretic trapping, otherwise known as dielectrophoretic capture. Non-uniform electric fields are required for this phenomenon, and the device in this trial used to induce such an electric field was a gradient insulator-based design. Similar devices have been previously used to separate or identify a wide variety of analytes within solution. Much of the previous work has been focused on the differences in dielectrophoretic trapping between strains of bacteria, whereas this experiment focused on the differentiation of phenotypes within a single bacterial strain, Staphylococcus aureus isolate 35984. A control sample was tested, as well as a sample heated at 70oC for 15 minutes to induce phenotypic changes. The control sample was found to exhibit dielectrophoretic capture at a given gate at a potential of 800V and higher, whereas the heated sample was not observed to capture at any potential in this experiment, which reached a maximum of 1200V. Notably, neither of the samples were found to capture at or below 600V. The results of this experiment were encouraging, though it is worth noting that several experimental trials failed to produce any noteworthy results. As such, the procedure of this experiment should be refined to increase reproducibility of results.

ContributorsLehfeldt, Jase (Author) / Hayes, Mark (Thesis director) / Sayres, Scott (Committee member) / Williams, Peter (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
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Description
Time-resolved serial femtosecond crystallography is an emerging method that allows for structural discovery to be performed on biomacromolecules during their dynamic trajectory through a reaction pathway after activation. This is performed by triggering a reaction on an ensemble of molecules in nano- or microcrystals and then using femtosecond X-ray

Time-resolved serial femtosecond crystallography is an emerging method that allows for structural discovery to be performed on biomacromolecules during their dynamic trajectory through a reaction pathway after activation. This is performed by triggering a reaction on an ensemble of molecules in nano- or microcrystals and then using femtosecond X-ray laser pulses produced by an X-ray free electron laser to collect near-instantaneous data on the crystal. A full data set can be collected by merging a sufficient number of these patterns together and multiple data sets can be collected at different points along the reaction pathway by manipulating the delay time between reaction initiation and the probing X-rays. In this way, these ‘snapshot’ structures can be viewed in series to make a molecular movie, allowing for atomic visualization of a molecule in action and, thereby, a structural basis for the mechanism and function of a given biomacromolecule.

This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained.
ContributorsCoe, Jesse (Author) / Fromme, Petra (Thesis advisor) / Sayres, Scott (Thesis advisor) / Mujica, Vladimiro (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2018
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Description
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

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[subscript 2A] adenosine receptor (A[subscript 2A]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 A[subscript 2A]AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A[subscript 2A]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.
ContributorsMartin Garcia, Jose Manuel (Author) / Conrad, Chelsie (Author) / Nelson, Garrett (Author) / Stander, Natasha (Author) / Zatsepin, Nadia (Author) / Zook, James (Author) / Zhu, Lan (Author) / Geiger, James (Author) / Chun, Eugene (Author) / Kissick, David (Author) / Hilgart, Mark C. (Author) / Ogata, Craig (Author) / Ishchenko, Andrii (Author) / Nagaratnam, Nirupa (Author) / Roy Chowdhury, Shatabdi (Author) / Coe, Jesse (Author) / Subramanian, Ganesh (Author) / Schaffer, Alexander (Author) / James, Daniel (Author) / Ketwala, Gihan (Author) / Venugopalan, Nagarajan (Author) / Xu, Shenglan (Author) / Corcoran, Stephen (Author) / Ferguson, Dale (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Cherezov, Vadim (Author) / Fromme, Petra (Author) / Fischetti, Robert F. (Author) / Liu, Wei (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2017-05-24
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Description
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,

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 ∼4 Å 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.
ContributorsAbdallah, Bahige (Author) / Zatsepin, Nadia (Author) / Roy Chowdhury, Shatabdi (Author) / Coe, Jesse (Author) / Conrad, Chelsie (Author) / Dorner, Katerina (Author) / Sierra, Raymond G. (Author) / Stevenson, Hilary P. (Author) / Camacho Alanis, Fernanda (Author) / Grant, Thomas D. (Author) / Nelson, Garrett (Author) / James, Daniel (Author) / Calero, Guillermo (Author) / Wachter, Rebekka (Author) / Spence, John (Author) / Weierstall, Uwe (Author) / Fromme, Petra (Author) / Ros, Alexandra (Author) / Department of Chemistry and Biochemistry (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2015-08-19