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

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

PLoS ONE ◽

10.1371/journal.pone.0239702 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0239702

Author(s):

Timothy R. Stachowski ◽

Mary E. Snell ◽

Edward H. Snell

Keyword(s):

Radiation Damage ◽

Disulfide Bond ◽

Large Scale ◽

Specific Interaction ◽

X Rays ◽

Low Resolution ◽

Damage Site ◽

Site Specific ◽

X Ray ◽

X Ray Crystallography

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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Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913022117 ◽

2013 ◽

Vol 69 (12) ◽

pp. 2381-2394 ◽

Cited By ~ 39

Author(s):

Kristin A. Sutton ◽

Paul J. Black ◽

Kermit R. Mercer ◽

Elspeth F. Garman ◽

Robin L. Owen ◽

...

Keyword(s):

Radiation Damage ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Bond Cleavage ◽

Absorbed Dose ◽

Product Formation ◽

Visible Absorption ◽

Structural Investigations ◽

X Ray ◽

Uv Visible

Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV–visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5–0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.

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X-ray radiation damage to biological macromolecules: further insights

Journal of Synchrotron Radiation ◽

10.1107/s160057751602018x ◽

2017 ◽

Vol 24 (1) ◽

pp. 1-6 ◽

Cited By ~ 21

Author(s):

Elspeth F. Garman ◽

Martin Weik

Keyword(s):

Radiation Damage ◽

Radiation Chemistry ◽

Mitigation Strategies ◽

Biological Macromolecules ◽

Conformational Heterogeneity ◽

X Ray Diffraction ◽

X Ray ◽

Tyrosine Residues ◽

Experimental Phasing ◽

Collection Strategies

Despite significant progress made over more than 15 years of research, structural biologists are still grappling with the issue of radiation damage suffered by macromolecular crystals which is induced by the resultant radiation chemistry occurring during X-ray diffraction experiments. Further insights into these effects and the possible mitigation strategies for use in both diffraction and SAXS experiments are given in eight papers in this volume. In particular, damage during experimental phasing is addressed, scavengers for SAXS experiments are investigated, microcrystals are imaged, data collection strategies are optimized, specific damage to tyrosine residues is reexamined, and room temperature conformational heterogeneity as a function of dose is explored. The brief summary below puts these papers into perspective relative to other ongoing radiation damage research on macromolecules.

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Studies of metaphase chromosomes in the scanning transmission x-ray microscope: Image fidelity, radiation damage and specimen preparation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100129826 ◽

1992 ◽

Vol 50 (2) ◽

pp. 1038-1039

Author(s):

Shawn Williams ◽

Xiaodong Zhang ◽

Susan Lamm ◽

Jack Van’t Hof

Keyword(s):

Visible Light ◽

Radiation Damage ◽

Cross Section ◽

Imaging Techniques ◽

Dose Range ◽

Specimen Preparation ◽

X Rays ◽

Metaphase Chromosomes ◽

X Ray ◽

Scanning Transmission

The Scanning Transmission X-ray Microscope (STXM) is well suited for investigating metaphase chromosome structure. The absorption cross-section of soft x-rays having energies between the carbon and oxygen K edges (284 - 531 eV) is 6 - 9.5 times greater for organic specimens than for water, which permits one to examine unstained, wet biological specimens with resolution superior to that attainable using visible light. The attenuation length of the x-rays is suitable for imaging micron thick specimens without sectioning. This large difference in cross-section yields good specimen contrast, so that fewer soft x-rays than electrons are required to image wet biological specimens at a given resolution. But most imaging techniques delivering better resolution than visible light produce radiation damage. Soft x-rays are known to be very effective in damaging biological specimens. The STXM is constructed to minimize specimen dose, but it is important to measure the actual damage induced as a function of dose in order to determine the dose range within which radiation damage does not compromise image quality.

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Characterization of defects in tabular silver halide grains

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100178367 ◽

1990 ◽

Vol 48 (4) ◽

pp. 1046-1047

Author(s):

C. Goessens ◽

D. Schryvers ◽

J. Van Landuyt ◽

A. Verbeeck ◽

R. De Keyzer

Keyword(s):

Radiation Damage ◽

Silver Halide ◽

Chemical Characteristics ◽

X Ray ◽

Free Region ◽

Central Defect ◽

Crystallographic Defects ◽

Physical And Chemical ◽

Two Sides

Silver halide grains (AgX, X=Cl,Br,I) are commonly recognized as important entities in photographic applications. Depending on the preparation specifications one can grow cubic, octahedral, tabular a.o. morphologies, each with its own physical and chemical characteristics. In the present study crystallographic defects introduced by the mixing of 5-20% iodide in a growing AgBr tabular grain are investigated. X-ray diffractometry reveals the existence of a homogeneous Ag(Br1-xIx) region, expected to be formed around the AgBr kernel. In fig. 1 a two-beam BF image, taken at T≈100 K to diminish radiation damage, of a triangular tabular grain is presented, clearly showing defect contrast fringes along four of the six directions; the remaining two sides show similar contrast under relevant diffraction conditions. The width of the central defect free region corresponds with the pure AgBr kernel grown before the mixing with I. The thickness of a given grain lies between 0.15 and 0.3 μm: as indicated in fig. 2 triangular (resp. hexagonal) grains exhibit an uneven (resp. even) number of twin interfaces (i.e., between + and - twin variants) parallel with the (111) surfaces. The thickness of the grains and the existence of the twin variants was confirmed from CTEM images of perpendicular cuts.

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Factors in Photographic Emulsions for Low Dose Electron Crystallography

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s143192760000101x ◽

1980 ◽

Vol 38 ◽

pp. 230-231

Author(s):

T.W. Jeng ◽

W. Chiu

Keyword(s):

Radiation Damage ◽

Low Dose ◽

Damage Effect ◽

Photographic Emulsion ◽

Electron Crystallography ◽

Electron Microscopic ◽

X Ray ◽

Signal Contrast ◽

Diffraction Patterns ◽

Photographic Emulsions

With the advances in preparing biological materials in a thin and highly ordered form, and in maintaining them hydrated under vacuum, electron crystallography has become an important tool for biological structure investigation at high resolution (1,2). However, the electron radiation damage would limit the capability of recording reflections with low intensities in an electron diffraction pattern. It has been demonstrated that the use of a low temperature stage can reduce the radiation damage effect and that one can expose the specimen with a higher dose in order to increase the signal contrast (3). A further improvement can be made by selecting a proper photographic emulsion. The primary factors in evaluating the suitability of photographic emulsion for recording low dose diffraction patterns are speed, fog level, electron response at low electron exposure, linearity, and usable range of exposure. We have compared these factors with three photographic emulsions including Kodak electron microscopic plate (EMP), Industrex AA x-ray film (AA x-ray) and Kodak nuclear track film (NTB3).

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