Characterization of Conditions Required for X-Ray Diffraction Experiments with Protein Microcrystals

Glaeser, Robert M.; Facciotti, Marc T.; Walian, Peter J.; Rouhani, Shahab; Holton, James M.; MacDowell, Alastair A.; Celestre, Richard; Cambie, Daniela; Padmore, H. A.

doi:10.1016/s0006-3495(00)76854-8

Cited by 36 publications

(37 citation statements)

References 9 publications

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“…The ®rst approach emphasizes the study of diffraction data in reciprocal space; the second emphasizes structural damage in real space. Our study (Teng & Moffat, 2000) and that of Glaeser et al (2000) clearly belong to the ®rst approach; those of Burmeister (2000), and Weik et al (2000) belong mostly to the second. In reciprocal space, the reduction in diffraction quality caused by X-ray radiation damage is evident in the decay of diffraction intensities and in the increase of unit-cell volume, overall B-factor and merged R-factor.…”

Section: Introductionmentioning

confidence: 46%

“…In our previous study (Teng & Moffat, 2000), a striking quantitative feature was observed, that the effect of radiation damage on several Table 1 Comparison of the upper limit for X-ray radiation damage and the lifetime of a 100 mm protein crystal (Teng & Moffat, 2000;Glaeser et al, 2000;Burmeister, 2000). parameters characteristic of the diffraction data quality was linearly dependent on the absorbed dose, up to a threshold value of approximately 1 Â 10 7 Gy.…”

Section: Resultsmentioning

confidence: 99%

“…A better understanding of the factors that give rise to chemical damage through the production and diffusion of radicals in protein crystals and in frozen solution is essential. Five studies on X-ray radiation damage from third-generation synchrotron sources were published in the year 2000 alone (Burmeister, 2000;Glaeser et al, 2000;Teng & Moffat, 2000;Weik et al, 2000), in which a wide variety of protein crystals were investigated at different beamlines. These studies took two different yet complementary approaches.…”

Section: Introductionmentioning

confidence: 99%

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Radiation damage of protein crystals at cryogenic temperatures between 40 K and 150 K

Teng

Moffat

2002

J Synchrotron Radiat

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X-ray radiation damage of lysozyme single crystals by an intense monochromatic beam from the Advanced Photon Source is studied at cryogenic temperatures between 40 K and 150 K. The results con®rm that primary radiation damage is both linearly dependent on the X-ray dose and independent of temperature. The upper limit for the primary radiation damage observed in our previous study [Teng & Moffat (2000), J. Synchrotron Rad. 7, 313±317] holds over the wider temperature range of this study. The X-ray diffraction quality of the data acquired at 40 K is superior to those at 100 K, apparently due to temperature dependence of secondary and tertiary radiation damage and to reduced thermal motion.

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Section: Introductionmentioning

confidence: 46%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Radiation damage of protein crystals at cryogenic temperatures between 40 K and 150 K

Teng

Moffat

2002

J Synchrotron Radiat

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“…1B) is direct evidence that radiation damage within the diffracting volume can be reduced by reducing the size of the X-ray beam, as proposed by Nave and Hill (18). Several observations of greater than anticipated robustness of very small (<10 μm) crystals (26,27) or of larger crystals probed with very small (<10 μm) beams (3,28,29) have been attributed to photoelectron escape from the illuminated volume and provided indirect evidence of this phenomenon. In our direct measurements, the sample survivability was increased by a factor of three over the range of beam sizes studied with 18.5-keV incident X-rays.…”

Section: Discussionmentioning

confidence: 79%

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

Sanishvili

Yoder

Pothineni

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

124

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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.microcrystallography | synchrotron radiation T he brilliance of synchrotron radiation from undulator devices on third-generation sources has been an enormous boon to crystallography of biological macromolecules. The high flux density and low divergence of undulator beams led to a rapid decrease in the minimum crystal size and minimum beam size that can yield usable diffraction data (1-4). However, the resulting decrease in diffracting volume necessitates an increase in X-ray exposure per unit sample volume, increasing radiation damage and severely compromising the substantial benefits of brilliant undulator sources. Thus, there is considerable interest in understanding the mechanism and spatial extent of X-rayinduced damage to crystals of biological macromolecules.Diffraction experiments are typically performed at cryotemperatures (approximately 100 K) to prevent the diffusion of free radicals, which are a major source of damage in crystals exposed to X-rays at higher temperatures (5), but cryocooling does not eliminate X-ray damage. Many experimental approaches to circumventing the effects of radiation damage have been investigated (6-10) but have not yet yielded a breakthrough result. Zero-dose diffraction intensities have been extrapolated from measured values by mathematical modeling (7-9). The effects of radiation damage have also been exploited for crystallograp...

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“…The air paths before and after the sample, up until the X-ray beamstop, or the cryo-gas stream, also contribute significantly to the background scatter. A reduction in air scatter has been demonstrated for microcrystals on improvements to the collimation and by replacing nitrogen with helium in the cold gas stream (Glaeser et al, 2000). Further enhancements have also been gained through close alignment of the apertures and beamstop to within just a few millimetres of the sample position (Madsen et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

In vacuoX-ray data collection from graphene-wrapped protein crystals

Warren¹,

Crawshaw²,

Trincão³

et al. 2015

Acta Cryst Sect A

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The measurement of diffraction data from macromolecular crystal samples held in vacuo holds the promise of a very low X-ray background and zero absorption of incident and scattered beams, leading to better data and the potential for accessing very long X-ray wavelengths (>3 Å ) for native sulfur phasing. Maintaining the hydration of protein crystals under vacuum is achieved by the use of liquid jets, as with serial data collection at free-electron lasers, or is sidestepped by cryocooling the samples, as implemented at new synchrotron beamlines. Graphene has been shown to protect crystals from dehydration by creating an extremely thin layer that is impermeable to any exchanges with the environment. Furthermore, owing to its hydrophobicity, most of the aqueous solution surrounding the crystal is excluded during sample preparation, thus eliminating most of the background caused by liquid. Here, it is shown that high-quality data can be recorded at room temperature from graphene-wrapped protein crystals in a rough vacuum. Furthermore, it was observed that graphene protects crystals exposed to different relative humidities and a chemically harsh environment.

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Characterization of Conditions Required for X-Ray Diffraction Experiments with Protein Microcrystals

Cited by 36 publications

References 9 publications

Radiation damage of protein crystals at cryogenic temperatures between 40 K and 150 K

Radiation damage of protein crystals at cryogenic temperatures between 40 K and 150 K

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

In vacuoX-ray data collection from graphene-wrapped protein crystals

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