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. 2011 Apr 12;108(15):6127-32.
doi: 10.1073/pnas.1017701108. Epub 2011 Mar 28.

Radiation damage in protein crystals is reduced with a micron-sized X-ray beam

Affiliations

Radiation damage in protein crystals is reduced with a micron-sized X-ray beam

Ruslan Sanishvili et al. Proc Natl Acad Sci U S A. .

Abstract

Radiation damage is a major limitation in crystallography of biological macromolecules, even for cryocooled samples, and is particularly acute in microdiffraction. For the X-ray energies most commonly used for protein crystallography at synchrotron sources, photoelectrons are the predominant source of radiation damage. If the beam size is small relative to the photoelectron path length, then the photoelectron may escape the beam footprint, resulting in less damage in the illuminated volume. Thus, it may be possible to exploit this phenomenon to reduce radiation-induced damage during data measurement for techniques such as diffraction, spectroscopy, and imaging that use X-rays to probe both crystalline and noncrystalline biological samples. In a systematic and direct experimental demonstration of reduced radiation damage in protein crystals with small beams, damage was measured as a function of micron-sized X-ray beams of decreasing dimensions. The damage rate normalized for dose was reduced by a factor of three from the largest (15.6 μm) to the smallest (0.84 μm) X-ray beam used. Radiation-induced damage to protein crystals was also mapped parallel and perpendicular to the polarization direction of an incident 1-μm X-ray beam. Damage was greatest at the beam center and decreased monotonically to zero at a distance of about 4 μm, establishing the range of photoelectrons. The observed damage is less anisotropic than photoelectron emission probability, consistent with photoelectron trajectory simulations. These experimental results provide the basis for data collection protocols to mitigate with micron-sized X-ray beams the effects of radiation damage.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Damage per calculated dose as a function of beam size for 18.5-keV X-rays. (A) Normalized sum of all diffracted intensities as a function of calculated dose for data collected with average beam diameters of 0.84, 1.81, 2.71, 5.35, 8.85, and 15.6 μm. Doses were calculated using RADDOSE (24). The dotted line represents an assumed linear rate of damage for which the intensity decays to 50% at a dose of 4.3 × 107 Gy, as measured with a 100-μm beam (25). (B) Damage rate as a function of beam diameter. The slope of each curve in A was normalized by the value at 15.6 μm. The beam size is the average of the horizontal and vertical beam sizes. The data were collected by rotating the crystal through 1° about the horizontal axis; and therefore a small correction was applied to account for the swept volume (Table S3). Error bars represent ± 1σ of multiple experiments.
Fig. 2.
Fig. 2.
Experimental design to measure the spatial extent of radiation damage. (A) Contiguous-probe protocol. Probe measurements (open circles) were recorded at each position in the pattern before and after each burn dose at the burn position (filled circle). (B) Isolated-probe protocol. The probe measurements (open circles) were not contaminated by neighboring probe measurements because a separate burn position (filled circles) was used for each probe position.
Fig. 3.
Fig. 3.
Spatial extent of radiation damage in lysozyme crystals. Fractional loss of total integrated reflection intensity as a function of distance from the burn position in the horizontal and vertical directions for four probe-burn-probe sequences at 15.1 keV (beam size: 1.16 μm FWHM horizontal; 1.18 μm vertical) and 18.5 keV (0.88 μm horizontal; 0.80 μm vertical). The dose was increased by equal increments for the damage distribution curves in the order black, blue, green, and red. (A) Damage in the horizontal direction at 15.1 keV. (B) Damage in the horizontal direction at 18.5 keV. (C) Damage in the vertical direction at 15.1 keV. (D) Damage in the vertical direction at 18.5 keV. The damage is greatest at the center and decays monotonically to zero (within experimental error) by 4 μm from the beam center for both energies. Error bars represent ± 1σ of multiple experiments.
Fig. 4.
Fig. 4.
Comparison of spatial extent of radiation damage and the beam size at 18.5 keV. The blue and red curves show the damage distribution in the horizontal and vertical directions, respectively. The data from the red curves (maximum dose) in Fig. 3 B and D were averaged about the origin and then normalized at the origin. The widths of the damage distributions are significantly larger than the width of the X-ray beam (green line, average FWHM = 0.84 μm), and are slightly anisotropic.
Fig. 5.
Fig. 5.
Monte Carlo simulation of photoelectron trajectories. (A) 2,000 trajectories for photoelectrons of energy 14.4 keV (Left) and 17.8 keV (Right) in a protein crystal. Trajectories originated at θ = 0° (parallel to the polarization vector, black arrow) and simulate a 1-μm diameter X-ray beam directed into the viewing plane. The 3D mushroom-like distribution is projected onto a horizontal plane and colored from yellow to blue with decreasing photoelectron energy. The spatial extent of the trajectories is considerably shorter for the 14.4-keV electrons than for the 17.8-keV electrons. Only one half of each symmetric distribution is shown. (B) Trajectories of photoelectrons emitted at 0° (Left), 30° (Middle), and 60° (Right) relative to the polarization vector. The number of electrons in the simulation was proportional to the probability of ejection within azimuth angles between 0° and 20° for the 0° beam angle simulation, 20° and 45° for the 30° simulation, and 45° and 75° for the 60° simulation. Actual trajectories are distributed isotropically around the polarization vector for the given azimuth angle range. The strong curvature of the photoelectron trajectories at lower energy toward the end of their travel results in some backscatter (red).

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