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1. A SAXS-based approach to rationally evaluate radical scavengers – toward eliminating radiation damage in solution and crystallographic studies

   https://doi.org/10.1107/s1600577521004045  

   Stachowski, Timothy R; Snell, Mary E; Snell, Edward H (September 2021, Journal of Synchrotron Radiation)

   X-ray-based techniques are a powerful tool in structural biology but the radiation-induced chemistry that results can be detrimental and may mask an accurate structural understanding. In the crystallographic case, cryocooling has been employed as a successful mitigation strategy but also has its limitations including the trapping of non-biological structural states. Crystallographic and solution studies performed at physiological temperatures can reveal otherwise hidden but relevant conformations, but are limited by their increased susceptibility to radiation damage. In this case, chemical additives that scavenge the species generated by radiation can mitigate damage but are not always successful and the mechanisms are often unclear. Using a protein designed to undergo a large-scale structural change from breakage of a disulfide bond, radiation damage can be monitored with small-angle X-ray scattering. Using this, we have quantitatively evaluated how three scavengers commonly used in crystallographic experiments – sodium nitrate, cysteine, and ascorbic acid – perform in solution at 10°C. Sodium nitrate was the most effective scavenger and completely inhibited fragmentation of the disulfide bond at a lower concentration (500 µM) compared with cysteine (∼5 mM) while ascorbic acid performed best at 5 mMbut could only reduce fragmentation by ∼75% after a total accumulated dose of 792 Gy. The relative effectiveness of each scavenger matches their reported affinities for solvated electrons. Saturating concentrations of each scavenger shifted fragmentation from first order to a zeroth-order process, perhaps indicating the direct contribution of photoabsorption. The SAXS-based method can detect damage at X-ray doses far lower than those accessible crystallographically, thereby providing a detailed picture of scavenger processes. The solution results are also in close agreement with what is known about scavenger performance and mechanism in a crystallographic setting and suggest that a link can be made between the damage phenomenon in the two scenarios. Therefore, our engineered approach might provide a platform for more systematic and comprehensive screening of radioprotectants that can directly inform mitigation strategies for both solution and crystallographic experiments, while also clarifying fundamental radiation damage mechanisms. 

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2. SAXS studies of X-ray induced disulfide bond damage: Engineering high-resolution insight from a low-resolution technique

   https://doi.org/10.1371/journal.pone.0239702  

   Stachowski, Timothy R; Snell, Mary E; Snell, Edward H (November 2020, PLOS ONE)

   Boggon, Titus J (Ed.)

   A significant problem in biological X-ray crystallography is the radiation chemistry caused by the incident X-ray beam. This produces both global and site-specific damage. Site specific damage can misdirect the biological interpretation of the structural models produced. Cryo-cooling crystals has been successful in mitigating damage but not eliminating it altogether; however, cryo-cooling can be difficult in some cases and has also been shown to limit functionally relevant protein conformations. The doses used for X-ray crystallography are typically in the kilo-gray to mega-gray range. While disulfide bonds are among the most significantly affected species in proteins in the crystalline state at both cryogenic and higher temperatures, there is limited information on their response to low X-ray doses in solution, the details of which might inform biomedical applications of X-rays. In this work we engineered a protein that dimerizes through a susceptible disulfide bond to relate the radiation damage processes seen in cryo-cooled crystals to those closer to physiologic conditions. This approach enables a low-resolution technique, small angle X-ray scattering (SAXS), to detect and monitor a residue specific process. A dose dependent fragmentation of the engineered protein was seen that can be explained by a dimer to monomer transition through disulfide bond cleavage. This supports the crystallographically derived mechanism and demonstrates that results obtained crystallographically can be usefully extrapolated to physiologic conditions. Fragmentation was influenced by pH and the conformation of the dimer, providing information on mechanism and pointing to future routes for investigation and potential mitigation. The novel engineered protein approach to generate a large-scale change through a site-specific interaction represents a promising tool for advancing radiation damage studies under solution conditions. 

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3. XFELs for structure and dynamics in biology

   https://doi.org/10.1107/S2052252517005760  

   Spence, J. C. (July 2017, IUCrJ)

   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. 

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4. Predicting data quality in biological X-ray solution scattering

   https://doi.org/10.1107/S2059798318005004  

   Wang, Chenzheng; Lin, Yuexia; Bougie, Devin; Gillilan, Richard E. (August 2018, Acta Crystallographica Section D Structural Biology)

   null (Ed.)

   Biological small-angle X-ray solution scattering (BioSAXS) is now widely used to gain information on biomolecules in the solution state. Often, however, it is not obvious in advance whether a particular sample will scatter strongly enough to give useful data to draw conclusions under practically achievable solution conditions. Conformational changes that appear to be large may not always produce scattering curves that are distinguishable from each other at realistic concentrations and exposure times. Emerging technologies such as time-resolved SAXS (TR-SAXS) pose additional challenges owing to small beams and short sample path lengths. Beamline optics vary in brilliance and degree of background scatter, and major upgrades and improvements to sources promise to expand the reach of these methods. Computations are developed to estimate BioSAXS sample intensity at a more detailed level than previous approaches, taking into account flux, energy, sample thickness, window material, instrumental background, detector efficiency, solution conditions and other parameters. The results are validated with calibrated experiments using standard proteins on four different beamlines with various fluxes, energies and configurations. The ability of BioSAXS to statistically distinguish a variety of conformational movements under continuous-flow time-resolved conditions is then computed on a set of matched structure pairs drawn from the Database of Macromolecular Motions (http://molmovdb.org). The feasibility of experiments is ranked according to sample consumption, a quantity that varies by over two orders of magnitude for the set of structures. In addition to photon flux, the calculations suggest that window scattering and choice of wavelength are also important factors given the short sample path lengths common in such setups. 

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5. Chaotic advection mixer for capturing transient states of diverse biological macromolecular systems with time-resolved small-angle X-ray scattering

   https://doi.org/10.1107/S2052252523003482  

   Zielinski, Kara A.; Katz, Andrea M.; Calvey, George D.; Pabit, Suzette A.; Milano, Shawn K.; Aplin, Cody; San Emeterio, Josue; Cerione, Richard A.; Pollack, Lois (May 2023, IUCrJ)

   Advances in time-resolved structural techniques, mainly in macromolecular crystallography and small-angle X-ray scattering (SAXS), allow for a detailed view of the dynamics of biological macromolecules and reactions between binding partners. Of particular promise, are mix-and-inject techniques, which offer a wide range of experimental possibility as microfluidic mixers are used to rapidly combine two species just prior to data collection. Most mix-and-inject approaches rely on diffusive mixers, which have been effectively used within crystallography and SAXS for a variety of systems, but their success is dependent on a specific set of conditions to facilitate fast diffusion for mixing. The use of a new chaotic advection mixer designed for microfluidic applications helps to further broaden the types of systems compatible with time-resolved mixing experiments. The chaotic advection mixer can create ultra-thin, alternating layers of liquid, enabling faster diffusion so that even more slowly diffusing molecules, like proteins or nucleic acids, can achieve fast mixing on timescales relevant to biological reactions. This mixer was first used in UV–vis absorbance and SAXS experiments with systems of a variety of molecular weights, and thus diffusion speeds. Careful effort was also dedicated to making a loop-loading sample-delivery system that consumes as little sample as possible, enabling the study of precious, laboratory-purified samples. The combination of the versatile mixer with low sample consumption opens the door to many new applications for mix-and-inject studies. 

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