Radiation damage in single-particle cryo-electron microscopy : effects of dose and dose rate

Radiation damage is an important resolution limiting factor both in macromolecular X-ray crystallography and cryo-electron microscopy. Systematic studies in macromolecular X-ray crystallography greatly benefited from the use of dose, expressed as energy deposited per mass unit, which is derived from...

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Veröffentlicht in:Journal of synchrotron radiation. - 1994. - 18(2011), Pt 3 vom: 21. Mai, Seite 398-412
1. Verfasser: Karuppasamy, Manikandan (VerfasserIn)
Weitere Verfasser: Karimi Nejadasl, Fatemeh, Vulovic, Milos, Koster, Abraham J, Ravelli, Raimond B G
Format: Online-Aufsatz
Sprache:English
Veröffentlicht: 2011
Zugriff auf das übergeordnete Werk:Journal of synchrotron radiation
Schlagworte:Journal Article Research Support, Non-U.S. Gov't
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520 |a Radiation damage is an important resolution limiting factor both in macromolecular X-ray crystallography and cryo-electron microscopy. Systematic studies in macromolecular X-ray crystallography greatly benefited from the use of dose, expressed as energy deposited per mass unit, which is derived from parameters including incident flux, beam energy, beam size, sample composition and sample size. In here, the use of dose is reintroduced for electron microscopy, accounting for the electron energy, incident flux and measured sample thickness and composition. Knowledge of the amount of energy deposited allowed us to compare doses with experimental limits in macromolecular X-ray crystallography, to obtain an upper estimate of radical concentrations that build up in the vitreous sample, and to translate heat-transfer simulations carried out for macromolecular X-ray crystallography to cryo-electron microscopy. Stroboscopic exposure series of 50-250 images were collected for different incident flux densities and integration times from Lumbricus terrestris extracellular hemoglobin. The images within each series were computationally aligned and analyzed with similarity metrics such as Fourier ring correlation, Fourier ring phase residual and figure of merit. Prior to gas bubble formation, the images become linearly brighter with dose, at a rate of approximately 0.1% per 10 MGy. The gradual decomposition of a vitrified hemoglobin sample could be visualized at a series of doses up to 5500 MGy, by which dose the sample was sublimed. Comparison of equal-dose series collected with different incident flux densities showed a dose-rate effect favoring lower flux densities. Heat simulations predict that sample heating will only become an issue for very large dose rates (50 e(-)Å(-2) s(-1) or higher) combined with poor thermal contact between the grid and cryo-holder. Secondary radiolytic effects are likely to play a role in dose-rate effects. Stroboscopic data collection combined with an improved understanding of the effects of dose and dose rate will aid single-particle cryo-electron microscopists to have better control of the outcome of their experiments 
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700 1 |a Karimi Nejadasl, Fatemeh  |e verfasserin  |4 aut 
700 1 |a Vulovic, Milos  |e verfasserin  |4 aut 
700 1 |a Koster, Abraham J  |e verfasserin  |4 aut 
700 1 |a Ravelli, Raimond B G  |e verfasserin  |4 aut 
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