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- Creators: Spence, John
- Member of: Programs and Communities
- Status: Published
Description
Briefly explains how lack of monetary savings serves as a barrier to accessing to finance capital for women of color seeking to launch their own tech startup.
ContributorsBrodock, Kate (Author) / Women of Color in Computing Research Collaborative (Contributor)
Created2021 (year uncertain)
Description
Intense femtosecond x-ray pulses from free-electron laser sources allow the imaging of individual particles in a single shot. Early experiments at the Linac Coherent Light Source (LCLS) have led to rapid progress in the field and, so far, coherent diffractive images have been recorded from biological specimens, aerosols, and quantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLS held a workshop to discuss the scientific and technical challenges for reaching the ultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap toward reaching atomic resolution, 3D imaging at free-electron laser sources.
ContributorsAquila, A. (Author) / Barty, A. (Author) / Bostedt, C. (Author) / Boutet, S. (Author) / Carini, G. (Author) / dePonte, D. (Author) / Drell, P. (Author) / Doniach, S. (Author) / Downing, K. H. (Author) / Earnest, T. (Author) / Elmlund, H. (Author) / Elser, V. (Author) / Guhr, M. (Author) / Hajdu, J. (Author) / Hastings, J. (Author) / Hau-Riege, S. P. (Author) / Huang, Z. (Author) / Lattman, E. E. (Author) / Maia, F. R. N. C. (Author) / Marchesini, S. (Author) / Ourmazd, A. (Author) / Pellegrini, C. (Author) / Santra, R. (Author) / Schlichting, I. (Author) / Schroer, C. (Author) / Spence, John (Author) / Vartanyants, I. A. (Author) / Wakatsuki, S. (Author) / Weis, W. I. (Author) / Williams, G. J. (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2015-04-21
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, 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
Description
Toward the end of his career, Zewail developed strong interest in fast electron spectroscopy and imaging, a field to which he made important contributions toward his aim of making molecular movies free of radiation damage. We therefore compare here the atomistic mechanisms leading to destruction of protein samples in diffract-and-destroy experiments for the cases of high-energy electron beam irradiation and X-ray laser pulses. The damage processes and their time-scales are compared and relevant elastic, inelastic, and photoelectron cross sections are given. Inelastic mean-free paths for ejected electrons at very low energies in insulators are compared with the bioparticle size. The dose rate and structural damage rate for electrons are found to be much lower, allowing longer pulses, reduced beam current, and Coulomb interactions for the formation of smaller probes. High-angle electron scattering from the nucleus, which has no parallel in the X-ray case, tracks the slowly moving nuclei during the explosion, just as the gain of the XFEL (X-ray free-electron laser) has no parallel in the electron case. Despite reduced damage and much larger elastic scattering cross sections in the electron case, leading to not dissimilar elastic scattering rates (when account is taken of the greatly increased incident XFEL fluence), progress for single-particle electron diffraction is seen to depend on the effort to reduce emittance growth due to Coulomb interactions, and so allow formation of intense sub-micron beams no larger than a virus.
ContributorsSpence, John (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2017-06-01
Description
Current hard X-ray free-electron laser (XFEL) sources can deliver doses to biological macromolecules well exceeding 1 GGy, in timescales of a few tens of femtoseconds. During the pulse, photoionization can reach the point of saturation in which certain atomic species in the sample lose most of their electrons. This electronic radiation damage causes the atomic scattering factors to change, affecting, in particular, the heavy atoms, due to their higher photoabsorption cross sections. Here, it is shown that experimental serial femtosecond crystallography data collected with an extremely bright XFEL source exhibit a reduction of the effective scattering power of the sulfur atoms in a native protein. Quantitative methods are developed to retrieve information on the effective ionization of the damaged atomic species from experimental data, and the implications of utilizing new phasing methods which can take advantage of this localized radiation damage are discussed.
ContributorsGalli, L. (Author) / Son, S.-K. (Author) / Klinge, M. (Author) / Bajt, S. (Author) / Barty, A. (Author) / Bean, R. (Author) / Betzel, C. (Author) / Beyerlein, K. R. (Author) / Caleman, C. (Author) / Doak, R. B. (Author) / Duszenko, M. (Author) / Fleckenstein, H. (Author) / Gati, C. (Author) / Hunt, B. (Author) / Kirian, R. A. (Author) / Liang, M. (Author) / Nanao, M. H. (Author) / Nass, K. (Author) / Oberthur, D. (Author) / Redecke, L. (Author) / Shoeman, R. (Author) / Stellato, F. (Author) / Yoon, C. H. (Author) / White, T. A. (Author) / Yefanov, O. (Author) / Spence, John (Author) / Chapman, H. N. (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2015-04-29
Description
CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.
ContributorsLee, Ho-Hsien (Author) / Cherni, Irene (Author) / Yu, HongQi (Author) / Fromme, Raimund (Author) / Doran, Jeffrey (Author) / Grotjohann, Ingo (Author) / Mittman, Michele (Author) / Basu, Shibom (Author) / Deb, Arpan (Author) / Dorner, Katerina (Author) / Aquila, Andrew (Author) / Barty, Anton (Author) / Boutet, Sebastien (Author) / Chapman, Henry N. (Author) / Doak, R. Bruce (Author) / Hunter, Mark (Author) / James, Daniel (Author) / Kirian, Richard (Author) / Kupitz, Christopher (Author) / Lawrence, Robert (Author) / Liu, Haiguang (Author) / Nass, Karol (Author) / Schlichting, Ilme (Author) / Schmidt, Kevin (Author) / Seibert, M. Marvin (Author) / Shoeman, Robert L. (Author) / Spence, John (Author) / Stellato, Francesco (Author) / Weierstall, Uwe (Author) / Williams, Garth J. (Author) / Yoon, Chun Hong (Author) / Wang, Dingjie (Author) / Zatsepin, Nadia (Author) / Hogue, Brenda (Author) / Matoba, Nobuyuki (Author) / Fromme, Petra (Author) / Mor, Tsafrir (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / Department of Chemistry and Biochemistry (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Biodesign Institute (Contributor) / Infectious Diseases and Vaccinology (Contributor) / Department of Physics (Contributor)
Created2014-08-20
Description
Crystallographic auto-indexing algorithms provide crystal orientations and unit-cell parameters and assign Miller indices based on the geometric relations between the Bragg peaks observed in diffraction patterns. However, if the Bravais symmetry is higher than the space-group symmetry, there will be multiple indexing options that are geometrically equivalent, and hence many ways to merge diffraction intensities from protein nanocrystals. Structure factor magnitudes from full reflections are required to resolve this ambiguity but only partial reflections are available from each XFEL shot, which must be merged to obtain full reflections from these `stills'. To resolve this chicken-and-egg problem, an expectation maximization algorithm is described that iteratively constructs a model from the intensities recorded in the diffraction patterns as the indexing ambiguity is being resolved. The reconstructed model is then used to guide the resolution of the indexing ambiguity as feedback for the next iteration. Using both simulated and experimental data collected at an X-ray laser for photosystem I in the P63 space group (which supports a merohedral twinning indexing ambiguity), the method is validated.
ContributorsLiu, Haiguang (Author) / Spence, John (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2014-09-23
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