Matching Items (38)
Description
Serial femtosecond crystallography requires reliable and efficient delivery of fresh crystals across the beam of an X-ray free-electron laser over the course of an experiment. We introduce a double-flow focusing nozzle to meet this challenge, with significantly reduced sample consumption, while improving jet stability over previous generations of nozzles. We demonstrate its use to determine the first room-temperature structure of RNA polymerase II at high resolution, revealing new structural details. Moreover, the double flow-focusing nozzles were successfully tested with three other protein samples and the first room temperature structure of an extradiol ring-cleaving dioxygenase was solved by utilizing the improved operation and characteristics of these devices.
ContributorsOberthuer, Dominik (Author) / Knoska, Juraj (Author) / Wiedorn, Max O. (Author) / Beyerlein, Kenneth R. (Author) / Bushnell, David A. (Author) / Kovaleva, Elena G. (Author) / Heymann, Michael (Author) / Gumprecht, Lars (Author) / Kirian, Richard (Author) / Barty, Anton (Author) / Mariani, Valerio (Author) / Tolstikova, Aleksandra (Author) / Adriano, Luigi (Author) / Awel, Salah (Author) / Barthelmess, Miriam (Author) / Dorner, Katerina (Author) / Xavier, P. Lourdu (Author) / Yefanov, Oleksandr (Author) / James, Daniel (Author) / Nelson, Garrett (Author) / Wang, Dingjie (Author) / Calvey, George (Author) / Chen, Yujie (Author) / Schmidt, Andrea (Author) / Szczepek, Michael (Author) / Frielingsdorf, Stefan (Author) / Lenz, Oliver (Author) / Snell, Edward (Author) / Robinson, Philip J. (Author) / Sarler, Bozidar (Author) / Belsak, Grega (Author) / Macek, Marjan (Author) / Wilde, Fabian (Author) / Aquila, Andrew (Author) / Boutet, Sebastien (Author) / Liang, Mengning (Author) / Hunter, Mark S. (Author) / Scheerer, Patrick (Author) / Lipscomb, John D. (Author) / Weierstall, Uwe (Author) / Kornberg, Roger D. (Author) / Spence, John (Author) / Pollack, Lois (Author) / Chapman, Henry N. (Author) / Bajt, Sasa (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2017-03-16
Description
X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.
ContributorsLi, Xuanxuan (Author) / Chiu, Chun-Ya (Author) / Wang, Hsiang-Ju (Author) / Kassemeyer, Stephan (Author) / Botha, Sabine (Author) / Shoeman, Robert L. (Author) / Lawrence, Robert (Author) / Kupitz, Christopher (Author) / Kirian, Richard (Author) / James, Daniel (Author) / Wang, Dingjie (Author) / Nelson, Garrett (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Hartman, Elisabeth (Author) / Jafarpour, Aliakbar (Author) / Foucar, Lutz M. (Author) / Barty, Anton (Author) / Chapman, Henry (Author) / Liang, Mengning (Author) / Menzel, Andreas (Author) / Wang, Fenglin (Author) / Basu, Shibom (Author) / Fromme, Raimund (Author) / Doak, R. Bruce (Author) / Fromme, Petra (Author) / Weierstall, Uwe (Author) / Huang, Michael H. (Author) / Spence, John (Author) / Schlichting, Ilme (Author) / Hogue, Brenda (Author) / Liu, Haiguang (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Department of Physics (Contributor) / School of Life Sciences (Contributor)
Created2017-04-11
Description
Multiple co-occurring environmental changes are affecting soil nitrogen cycling processes, which are mainly mediated by microbes. While it is likely that various nitrogen-cycling functional groups will respond differently to such environmental changes, very little is known about their relative responsiveness. Here we conducted four long-term experiments in a steppe ecosystem by removing plant functional groups, mowing, adding nitrogen, adding phosphorus, watering, warming, and manipulating some of their combinations. We quantified the abundance of seven nitrogen-cycling genes, including those for fixation (nifH), mineralization (chiA), nitrification (amoA of ammonia-oxidizing bacteria (AOB) or archaea (AOA)), and denitrification (nirS, nirK and nosZ). First, for each gene, we compared its sensitivities to different environmental changes and found that the abundances of various genes were sensitive to distinct and different factors. Overall, the abundances of nearly all genes were sensitive to nitrogen enrichment. In addition, the abundances of the chiA and nosZ genes were sensitive to plant functional group removal, the AOB-amoA gene abundance to phosphorus enrichment when nitrogen was added simultaneously, and the nirS and nirK gene abundances responded to watering. Second, for each single- or multi-factorial environmental change, we compared the sensitivities of the abundances of different genes and found that different environmental changes primarily affected different gene abundances. Overall, AOB-amoA gene abundance was most responsive, followed by the two denitrifying genes nosZ and nirS, while the other genes were less sensitive. These results provide, for the first time, systematic insights into how the abundance of each type of nitrogen-cycling gene and the equilibrium state of all these nitrogen-cycling gene abundances would shift under each single- or multi-factorial global change.
ContributorsZhang, Ximei (Author) / Liu, Wei (Author) / Schloter, Michael (Author) / Zhang, Guangming (Author) / Chen, Quansheng (Author) / Jianhui, Huang (Author) / Li, Linghao (Author) / Elser, James (Author) / Han, Xingguo (Author) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor)
Created2013-10-04
Description
Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes.
ContributorsZhou, X. Edward (Author) / Gao, Xiang (Author) / Barty, Anton (Author) / Kang, Yanyong (Author) / He, Yuanzheng (Author) / Liu, Wei (Author) / Ishchenko, Andrii (Author) / White, Thomas A. (Author) / Yefanov, Oleksandr (Author) / Han, Gye Won (Author) / Xu, Qingping (Author) / de Waal, Parker W. (Author) / Suino-Powell, Kelly M. (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Wang, Meitian (Author) / Li, Dianfan (Author) / Caffrey, Martin (Author) / Chapman, Henry N. (Author) / Spence, John (Author) / Fromme, Petra (Author) / Weierstall, Uwe (Author) / Stevens, Raymond C. (Author) / Cherezov, Vadim (Author) / Melcher, Karsten (Author) / Xu, H. Eric (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2016-04-12
Description
Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.
ContributorsLi, Dianfan (Author) / Stansfeld, Phillip J. (Author) / Sansom, Mark S. P. (Author) / Keogh, Aaron (Author) / Vogeley, Lutz (Author) / Howe, Nicole (Author) / Lyons, Joseph A. (Author) / Aragao, David (Author) / Fromme, Petra (Author) / Fromme, Raimund (Author) / Basu, Shibom (Author) / Grotjohann, Ingo (Author) / Kupitz, Christopher (Author) / Rendek, Kimberley (Author) / Weierstall, Uwe (Author) / Zatsepin, Nadia (Author) / Cherezov, Vadim (Author) / Liu, Wei (Author) / Bandaru, Sateesh (Author) / English, Niall J. (Author) / Gati, Cornelius (Author) / Barty, Anton (Author) / Yefanov, Oleksandr (Author) / Chapman, Henry N. (Author) / Diederichs, Kay (Author) / Messerschmidt, Marc (Author) / Boutet, Sebastien (Author) / Williams, Garth J. (Author) / Seibert, M. Marvin (Author) / Caffrey, Martin (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2015-12-17
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. 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
Description
Porphyromonas gingivalis (P. gingivalis) is an oral pathogen known for causing periodontal diseases like periodontitis and alveolar bone loss. In this study, we investigate the molecular mechanisms of P. gingivalis with focus of the molecular cloning of the two DNA strains of the bacteria PGN_1740 and PGN_0012 in the Ampr pTCow. PGN_1740 is an RNA polymerase ECF-type sigma factor used for transcription. PGN_0012 is a two-component system regulator gene that is important in signal transduction. We demonstrated the cloning mechanism through transformation and confirmed the results through gel electrophoresis and using a positive transformant as a control. The process of cloning the DNA inserts into the bacteria followed a polymerase chain reaction for the amplification of the DNA fragments, digestion of the plasmid and DNA fragments with the restriction endonucleases (BamHI and HindIII), ligation and finally heat shock transformation are presented in this thesis. The effectiveness of these procedures was observed through agarose gel electrophoresis and ethanol precipitation for the purification of the PCR products. In this investigation, we discuss molecular and biological characterization of the P. gingivalis bacteria in regard to cloning and ampicillin resistance.
ContributorsOkeyo, Diana (Author) / Shi, Yixin (Thesis director) / Liu, Wei (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The heliobacteria, a family of anoxygenic phototrophs, are significant to photosynthesis evolution research, as they possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria may also grow chemotrophically via pyruvate metabolism in the absence of light. In Heliobacterium modesticaldum, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for cytochrome c553 other than the photosynthetic reaction center. Therefore, it was hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. Under this hypothesis, a mutant of H. modesticaldum lacking the cytochrome bc complex was predicted to be viable, but non-phototrophic. In this project, a two-step method for CRISPR-based genome editing was used in H. modesticaldum to delete the genes encoding the cytochrome bc complex. Genotypic analysis verified the deletion of the petC, B, D, and A genes encoding the catalytic components of complex. Spectroscopic studies revealed that re-reduction of cytochrome c553 after flash-induced photo-oxidation was ~130 to 190 times slower in the ∆petCBDA mutant compared to wildtype, phenotypically confirming the removal of the cytochrome bc complex. The resulting ∆petCBDA mutant was unable to grow phototrophically, instead relying on pyruvate metabolism to grow chemotrophically as does wildtype in the dark.
ContributorsLeung, Sabrina (Author) / Redding, Kevin (Thesis director) / Liu, Wei (Committee member) / Vermaas, Wim (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
The human gastrin receptor (CCKBR or CCK2R) is a class A G protein-coupled receptor (GPCR) found throughout the central nervous system, stomach, and a variety of cancer cells. CCK2R is implicated in the regulation of biological processes, including anxiety, satiety, arousal, analgesia, psychosis, and cancer cell growth and proliferation. While CCK2R is an attractive drug target, few drugs have managed to effectively target the receptor, and none have been brought to market. Contributory to this is the lack of high-resolution crystal structure capable of elucidating the binding regions of CCK2R to streamlining drug screening. While GPCRs are not amenable to traditional structural analysis methodologies, the advent of lipidic cubic phase (LCP) crystallography and serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs), has extended the applicability of X-ray crystallography to these integral membrane proteins. LCP-SFX depends on optimizing the protein of interest for extraction, purification, and crystallization. Here we report our findings regarding the optimization of CCK2R suggesting the synergistic relationship between N-terminal truncations and the insertion of a fusion protein along ICL3, in addition to a 30-residue truncation of the C-terminus. Samples were expressed in Sf9 insect cells using a Bac-to-Bac baculovirus expression system, extracted using n-Dodecyl-β-D-Maltoside detergent, and purified via TALON immobilized metal-ion affinity chromatography. The constructs were characterized via SDS-PAGE, Western blot, and size exclusion chromatography. These findings demonstrate the improvements to CCK2R’s crystallographic amenability upon these modifications, however significant improvements must be made prior to crystallization trials. Future work will involve screening C-terminal truncations, thermostabilizing point mutations, and co-crystallizing ligands. Ideally this investigation will serve as a model for future CCK2R structural analysis and contribute to a heightened interest in CCK2R as a therapeutic target.
ContributorsStevens, Alexander Wade (Author) / Liu, Wei (Thesis director) / Chiu, Po-Lin (Committee member) / Mills, Jeremy (Committee member) / School of Human Evolution & Social Change (Contributor) / School of Molecular Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The understanding of normal human physiology and disease pathogenesis shows great promise for progress with increasing ability to profile genomic loci and transcripts in single cells in situ. Using biorthogonal cleavable fluorescent oligonucleotides, a highly multiplexed single-cell in situ RNA and DNA analysis is reported. In this report, azide-based cleavable linker connects oligonucleotides to fluorophores to show nucleic acids through in situ hybridization. Post-imaging, the fluorophores are effectively cleaved off in half an hour without loss of RNA or DNA integrity. Through multiple cycles of hybridization, imaging, and cleavage this approach proves to quantify thousands of different RNA species or genomic loci because of single-molecule sensitivity in single cells in situ. Different nucleic acids can be imaged by shown by multi-color staining in each hybridization cycle, and that multiple hybridization cycles can be run on the same specimen. It is shown that in situ analysis of DNA, RNA and protein can be accomplished using both cleavable fluorescent antibodies and oligonucleotides. The highly multiplexed imaging platforms will have the potential for wide applications in both systems biology and biomedical research. Thus, proving to be cost effective and time effective.
ContributorsSamuel, Adam David (Author) / Guo, Jia (Thesis director) / Liu, Wei (Committee member) / Wang, Xu (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05