Progress in rational methods of cryoprotection in macromolecular crystallography
Thomas Alcorn,Douglas H. Juers +1 more
TLDR
Measurements of the average thermal contractions of 26 different cryosolutions are presented and discussed in conjunction with other recent advances in the rational design of protocols for cryogenic cooling in macromolecular crystallography.Abstract:
Cryogenic cooling of macromolecular crystals is commonly used for X-ray data collection both to reduce crystal damage from radiation and to gather functional information by cryogenically trapping intermediates. However, the cooling process can damage the crystals. Limiting cooling-induced crystal damage often requires cryoprotection strategies, which can involve substantial screening of solution conditions and cooling protocols. Here, recent developments directed towards rational methods for cryoprotection are described. Crystal damage is described in the context of the temperature response of the crystal as a thermodynamic system. As such, the internal and external parts of the crystal typically have different cryoprotection requirements. A key physical parameter, the thermal contraction, of 26 different cryoprotective solutions was measured between 294 and 72 K. The range of contractions was 2–13%, with the more polar cryosolutions contracting less. The potential uses of these results in the development of cryocooling conditions, as well as recent developments in determining minimum cryosolution soaking times, are discussed.read more
Citations
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Radiation damage in macromolecular crystallography: what is it and why should we care?
TL;DR: The basic causes of the radiation damage inflicted on macromolecular crystals during diffraction experiments are summarized, as well as the current state of research which attempts to understand and to mitigate it.
Journal ArticleDOI
Mapping the conformational landscape of a dynamic enzyme by multitemperature and XFEL crystallography
Daniel A. Keedy,Lillian R. Kenner,Matthew Warkentin,Rahel A. Woldeyes,Jesse B. Hopkins,Michael C. Thompson,Aaron S. Brewster,Andrew H. Van Benschoten,E.L. Baxter,Monarin Uervirojnangkoorn,Monarin Uervirojnangkoorn,Scott E McPhillips,J. Song,Roberto Alonso-Mori,James M. Holton,James M. Holton,James M. Holton,William I. Weis,William I. Weis,Axel T. Brunger,S. Michael Soltis,Henrik T. Lemke,Ana Gonzalez,Nicholas K. Sauter,Aina E. Cohen,Henry van den Bedem,Robert E. Thorne,James S. Fraser +27 more
TL;DR: The diffraction-before-destruction nature of XFEL experiments provides a radiation-damage-free view of the functionally important alternative conformations of CypA, confirming earlier synchrotron-based results and motivating a new generation of temperature- and time-resolved experiments to structurally characterize the dynamic underpinnings of protein function.
Journal ArticleDOI
Image formation modeling in cryo-electron microscopy
Miloš Vulović,Raimond B. G. Ravelli,Lucas J. van Vliet,Abraham J. Koster,Ivan Lazic,Uwe Lücken,Hans Rullgård,Ozan Öktem,Bernd Rieger +8 more
TL;DR: A forward model that accounts for the specimen's scattering properties, microscope optics, and detector response and suggests that beam-induced specimen movements are relevant in the experiments whereas the influence of the solvent amorphousness can be neglected.
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Radiation damage in single-particle cryo-electron microscopy: effects of dose and dose rate
Manikandan Karuppasamy,Fatemeh Karimi Nejadasl,Miloš Vulović,Miloš Vulović,Abraham J. Koster,Raimond B. G. Ravelli +5 more
TL;DR: The effects of dose and dose-rate were investigated for single-particle cryo-electron microscopy using stroboscopic data collection and a dose- rate effect was observed favoring lower flux densities.
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