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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 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
Description
The development and application of the free-electron X-ray laser (XFEL) to structure and dynamics in biology since its inception in 2009 are reviewed. The research opportunities which result from the ability to outrun most radiation-damage effects are outlined, and some grand challenges are suggested. By avoiding the need to cool samples to minimize damage, the XFEL has permitted atomic resolution imaging of molecular processes on the 100 fs timescale under near-physiological conditions and in the correct thermal bath in which molecular machines operate. Radiation damage, comparisons of XFEL and synchrotron work, single-particle diffraction, fast solution scattering, pump–probe studies on photosensitive proteins, mix-and-inject experiments, caged molecules, pH jump and other reaction-initiation methods, and the study of molecular machines are all discussed. Sample-delivery methods and data-analysis algorithms for the various modes, from serial femtosecond crystallography to fast solution scattering, fluctuation X-ray scattering, mixing jet experiments and single-particle diffraction, are also reviewed.
ContributorsSpence, John (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2017-05-10
Description
Single-particle diffraction from X-ray Free Electron Lasers offers the potential for molecular structure determination without the need for crystallization. In an effort to further develop the technique, we present a dataset of coherent soft X-ray diffraction images of Coliphage PR772 virus, collected at the Atomic Molecular Optics (AMO) beamline with pnCCD detectors in the LAMP instrument at the Linac Coherent Light Source. The diameter of PR772 ranges from 65–70 nm, which is considerably smaller than the previously reported ~600 nm diameter Mimivirus. This reflects continued progress in XFEL-based single-particle imaging towards the single molecular imaging regime. The data set contains significantly more single particle hits than collected in previous experiments, enabling the development of improved statistical analysis, reconstruction algorithms, and quantitative metrics to determine resolution and self-consistency.
ContributorsReddy, Hemanth K. N. (Author) / Yoon, Chun Hong (Author) / Aquila, Andrew (Author) / Awel, Salah (Author) / Ayyer, Kartik (Author) / Barty, Anton (Author) / Berntsen, Peter (Author) / Bielecki, Johan (Author) / Bobkov, Sergey (Author) / Bucher, Maximilian (Author) / Carini, Gabriella A. (Author) / Carron, Sebastian (Author) / Chapman, Henry (Author) / Daurer, Benedikt (Author) / DeMirci, Hasan (Author) / Ekeberg, Tomas (Author) / Fromme, Petra (Author) / Hajdu, Janos (Author) / Hanke, Max Felix (Author) / Hart, Philip (Author) / Hogue, Brenda (Author) / Hasseinizadeh, Ahmad (Author) / Kim, Yoonhee (Author) / Kirian, Richard (Author) / Kurta, Ruslan P. (Author) / Larsson, Daniel S. D. (Author) / Loh, N. Duane (Author) / Maia, Filipe R. N. C. (Author) / Mancuso, Adrian P. (Author) / Muhlig, Kerstin (Author) / Munke, Anna (Author) / Nam, Daewoong (Author) / Nettelblad, Carl (Author) / Ourmazd, Abbas (Author) / Rose, Max (Author) / Schwander, Peter (Author) / Seibert, Marvin (Author) / Sellberg, Jonas A. (Author) / Song, Changyong (Author) / Spence, John (Author) / Svenda, Martin (Author) / van der Schot, Gijs (Author) / Vartanyants, Ivan A. (Author) / Williams, Garth J. (Author) / Xavier, P. Lourdu (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) / School of Life Sciences (Contributor) / Department of Physics (Contributor)
Created2017-06-27
Description
X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.
ContributorsFrank, Matthias (Author) / Carlson, David B. (Author) / Hunter, Mark S. (Author) / Williams, Garth J. (Author) / Messerschmidt, Marc (Author) / Zatsepin, Nadia (Author) / Barty, Anton (Author) / Benner, W. Henry (Author) / Chu, Kaiqin (Author) / Graf, Alexander T. (Author) / Hau-Riege, Stefan P. (Author) / Kirian, Richard A. (Author) / Padeste, Celestino (Author) / Pardini, Tommaso (Author) / Pedrini, Bill (Author) / Segelke, Brent (Author) / Seibert, M. Marvin (Author) / Spence, John (Author) / Tsai, Ching-Ju (Author) / Lane, Stephen M. (Author) / Li, Xiao-Dan (Author) / Schertler, Gebhard (Author) / Boutet, Sebastien (Author) / Coleman, Matthew (Author) / Evans, James E. (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2014-02-28
Description
The invention of the laser in the 1950 s for visible light and microwaves, and the slow but steady recognition of its manifold uses, is a truly remarkable story in the history of science. But the severe λ[superscript 3] dependence of the ratio of stimulated (mostly coherent) to spontaneous (incoherent) emission meant that efforts to build an X-ray laser seemed hopeless for decades. As so often happens in the history of science, the breakthrough eventually occurred at the interface of several fields – synchrotron science (and especially their insertion devices), laser physics, and work on microwave tubes for radar, emerging from the second world war. Synchrotrons themselves were an outgrowth of the particle accelerators of nuclear physics, whose X-ray radiation was considered a nuisance. All of this culminated recently in the construction of the first hard-X-ray laser, the US Department of Energy's Linac Coherent Light Source (LCLS), at their SLAC laboratory near Stanford. The first X-ray lasing occurred in that two-mile long tunnel on April 21, 2009, at about 2 kV, in an all-or-nothing moment of intense excitement, as theoretical predictions proved spot-on. The new laser principle needed for hard-X-ray lasing, the free-electron laser (FEL), was first demonstrated in the infra-red region at Stanford in 1975 in John Madey's group, following earlier theoretical work by Motz and Phillips on microwave tubes. Other FELs soon followed, in the microwave and visible region, leading to the LCLS. The XFEL method provides brief pulses of X-ray laser radiation by the SASE (self-amplified spontaneous emission) process, using a resonant undulator driven by a LINAC electron accelerator. Each LCLS pulse, of 10 fs duration (repeated 120 times a second) contains about 10[superscript 12] hard-X-ray photons, about the same number that a synchrotron might generate in a second.
ContributorsSpence, John (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2014-04-30
Description
Diamond is considered as an ideal material for high field and high power devices due to its high breakdown field, high lightly doped carrier mobility, and high thermal conductivity. The modeling and simulation of diamond devices are therefore important to predict the performances of diamond based devices. In this context, we use Silvaco[superscript ®] Atlas, a drift-diffusion based commercial software, to model diamond based power devices. The models used in Atlas were modified to account for both variable range and nearest neighbor hopping transport in the impurity bands associated with high activation energies for boron doped and phosphorus doped diamond. The models were fit to experimentally reported resistivity data over a wide range of doping concentrations and temperatures. We compare to recent data on depleted diamond Schottky PIN diodes demonstrating low turn-on voltages and high reverse breakdown voltages, which could be useful for high power rectifying applications due to the low turn-on voltage enabling high forward current densities. Three dimensional simulations of the depleted Schottky PIN diamond devices were performed and the results are verified with experimental data at different operating temperatures.
ContributorsHathwar, Raghuraj (Author) / Dutta, Maitreya (Author) / Koeck, Franz (Author) / Nemanich, Robert (Author) / Chowdhury, Srabanti (Author) / Goodnick, Stephen (Author) / Ira A. Fulton Schools of Engineering (Contributor) / School of Electrical, Computer and Energy Engineering (Contributor) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2016-06-08
Description
Thermionic energy conversion, a process that allows direct transformation of thermal to electrical energy, presents a means of efficient electrical power generation as the hot and cold side of the corresponding heat engine are separated by a vacuum gap. Conversion efficiencies approaching those of the Carnot cycle are possible if material parameters of the active elements at the converter, i.e., electron emitter or cathode and collector or anode, are optimized for operation in the desired temperature range.
These parameters can be defined through the law of Richardson–Dushman that quantifies the ability of a material to release an electron current at a certain temperature as a function of the emission barrier or work function and the emission or Richardson constant. Engineering materials to defined parameter values presents the key challenge in constructing practical thermionic converters. The elevated temperature regime of operation presents a constraint that eliminates most semiconductors and identifies diamond, a wide band-gap semiconductor, as a suitable thermionic material through its unique material properties. For its surface, a configuration can be established, the negative electron affinity, that shifts the vacuum level below the conduction band minimum eliminating the surface barrier for electron emission.
In addition, its ability to accept impurities as donor states allows materials engineering to control the work function and the emission constant. Single-crystal diamond electrodes with nitrogen levels at 1.7 eV and phosphorus levels at 0.6 eV were prepared by plasma-enhanced chemical vapor deposition where the work function was controlled from 2.88 to 0.67 eV, one of the lowest thermionic work functions reported. This work function range was achieved through control of the doping concentration where a relation to the amount of band bending emerged. Upward band bending that contributed to the work function was attributed to surface states where lower doped homoepitaxial films exhibited a surface state density of ∼3 × 10[superscript 11] cm[superscript −2]. With these optimized doped diamond electrodes, highly efficient thermionic converters are feasible with a Schottky barrier at the diamond collector contact mitigated through operation at elevated temperatures.
ContributorsKoeck, Franz (Author) / Nemanich, Robert (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2017-12-06
Description
X-ray free-electron lasers (XFELs) provide new opportunities for structure determination of biomolecules, viruses and nanomaterials. With unprecedented peak brilliance and ultra-short pulse duration, XFELs can tolerate higher X-ray doses by exploiting the femtosecond-scale exposure time, and can thus go beyond the resolution limits achieved with conventional X-ray diffraction imaging techniques. Using XFELs, it is possible to collect scattering information from single particles at high resolution, however particle heterogeneity and unknown orientations complicate data merging in three-dimensional space. Using the Linac Coherent Light Source (LCLS), synthetic inorganic nanocrystals with a core–shell architecture were used as a model system for proof-of-principle coherent diffractive single-particle imaging experiments. To deal with the heterogeneity of the core–shell particles, new computational methods have been developed to extract the particle size and orientation from the scattering data to assist data merging. The size distribution agrees with that obtained by electron microscopy and the merged data support a model with a core–shell architecture.
ContributorsLi, Xuanxuan (Author) / Spence, John (Author) / Hogue, Brenda (Author) / Liu, Haiguang (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor)
Created2017-08-27
Description
A major conceptual step forward in understanding the logical architecture of living systems was advanced by von Neumann with his universal constructor, a physical device capable of self-reproduction. A necessary condition for a universal constructor to exist is that the laws of physics permit physical universality, such that any transformation (consistent with the laws of physics and availability of resources) can be caused to occur. While physical universality has been demonstrated in simple cellular automata models, so far these have not displayed a requisite feature of life—namely open-ended evolution—the explanation of which was also a prime motivator in von Neumann’s formulation of a universal constructor. Current examples of physical universality rely on reversible dynamical laws, whereas it is well-known that living processes are dissipative. Here we show that physical universality and open-ended dynamics should both be possible in irreversible dynamical systems if one entertains the possibility of state-dependent laws. We demonstrate with simple toy models how the accessibility of state space can yield open-ended trajectories, defined as trajectories that do not repeat within the expected Poincaré recurrence time and are not reproducible by an isolated system. We discuss implications for physical universality, or an approximation to it, as a foundational framework for developing a physics for life.
ContributorsAdams, Alyssa (Author) / Berner, Angelica (Author) / Davies, Paul (Author) / Walker, Sara (Author) / College of Liberal Arts and Sciences (Contributor) / BEYOND: Center for Fundamental Concepts in Science (Contributor) / Department of Physics (Contributor) / School of Earth and Space Exploration (Contributor) / ASU-SFI Center for Biosocial Complex Systems (Contributor)
Created2017-09-01
Description
Background
Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.
Methodology
We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.
Principal Findings
We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.
Conclusions
Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.
Grading schemes for breast cancer diagnosis are predominantly based on pathologists' qualitative assessment of altered nuclear structure from 2D brightfield microscopy images. However, cells are three-dimensional (3D) objects with features that are inherently 3D and thus poorly characterized in 2D. Our goal is to quantitatively characterize nuclear structure in 3D, assess its variation with malignancy, and investigate whether such variation correlates with standard nuclear grading criteria.
Methodology
We applied micro-optical computed tomographic imaging and automated 3D nuclear morphometry to quantify and compare morphological variations between human cell lines derived from normal, benign fibrocystic or malignant breast epithelium. To reproduce the appearance and contrast in clinical cytopathology images, we stained cells with hematoxylin and eosin and obtained 3D images of 150 individual stained cells of each cell type at sub-micron, isotropic resolution. Applying volumetric image analyses, we computed 42 3D morphological and textural descriptors of cellular and nuclear structure.
Principal Findings
We observed four distinct nuclear shape categories, the predominant being a mushroom cap shape. Cell and nuclear volumes increased from normal to fibrocystic to metastatic type, but there was little difference in the volume ratio of nucleus to cytoplasm (N/C ratio) between the lines. Abnormal cell nuclei had more nucleoli, markedly higher density and clumpier chromatin organization compared to normal. Nuclei of non-tumorigenic, fibrocystic cells exhibited larger textural variations than metastatic cell nuclei. At p<0.0025 by ANOVA and Kruskal-Wallis tests, 90% of our computed descriptors statistically differentiated control from abnormal cell populations, but only 69% of these features statistically differentiated the fibrocystic from the metastatic cell populations.
Conclusions
Our results provide a new perspective on nuclear structure variations associated with malignancy and point to the value of automated quantitative 3D nuclear morphometry as an objective tool to enable development of sensitive and specific nuclear grade classification in breast cancer diagnosis.
ContributorsNandakumar, Vivek (Author) / Kelbauskas, Laimonas (Author) / Hernandez, Kathryn (Author) / Lintecum, Kelly (Author) / Senechal, Patti (Author) / Bussey, Kimberly (Author) / Davies, Paul (Author) / Johnson, Roger (Author) / Meldrum, Deirdre (Author) / Ira A. Fulton Schools of Engineering (Contributor) / School of Electrical, Computer and Energy Engineering (Contributor) / Biodesign Institute (Contributor) / Center for Biosignatures Discovery Automation (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Department of Physics (Contributor)
Created2012-01-05