Abstract
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.
Highlights
Single-particle cryo-electron microscopy (SP cryo-EM) is a unique technique widely used to elucidate the three-dimensional structures of macromolecules of molecular mass greater than a few hundred kDa (Saibil, 2000; Frank, 2009; Jonic & Venien-Bryan, 2009; Orlova & Saibil, 2010)
Radiation damage should not be treated as a binary nuisance, neither in macromolecular X-ray crystallography (MX) nor in SP cryo-EM
We introduced a new metric, analogous to MX, for ascertaining phase qualities, namely the average cosine of phase errors (FOM)
Summary
Single-particle cryo-electron microscopy (SP cryo-EM) is a unique technique widely used to elucidate the three-dimensional structures of macromolecules of molecular mass greater than a few hundred kDa (Saibil, 2000; Frank, 2009; Jonic & Venien-Bryan, 2009; Orlova & Saibil, 2010). A three-dimensional electron-density map of molecules to a resolution of $ 10 A (1 nm) can be obtained from these projection images (Frank, 2009; Wendler & Saibil, 2010). 18, 398–412 radiation damage resolution by fitting the atomic models of some of the components available from X-ray diffraction studies into the reconstructed EM map of the entire complex (for example, Zhou, 2008; Bhushan et al, 2010; Sindelar & Downing, 2010; Baker et al, 2010; Fujii et al, 2010). A full-atom model of a non-enveloped aquareovirus at 3.3 Awas recently obtained by SP reconstruction in which side-chain densities for non-Gly amino acids were clearly visible (Zhang et al, 2010). Technological improvements in electron optics, sample preparation, and data collection and processing have enabled these recent advances
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