Density and electron density of aqueous cryoprotectant solutions at cryogenic temperatures for optimized cryoprotection and diffraction contrast

Tyree, Timothy J.; Dan, Ritwik; Thorne, Robert

doi:10.1107/s2059798318003078

Cited by 24 publications

(20 citation statements)

References 54 publications

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“…The solutes can include salts present in protein buffers as well as cryoprotectants such as glycerol. However, these reduce the electron-density contrast between the biomolecule and buffer (Tyree et al, 2018), and so reduce the measurement signal to noise. Solutes also include the biomolecules themselves.…”

Section: Reducing Beam-induced Motionmentioning

confidence: 99%

Hypothesis for a mechanism of beam-induced motion in cryo-electron microscopy

Thorne

2020

Int Union Crystallogr J

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Estimates of heat-transfer rates during plunge-cooling and the patterns of ice observed in cryo-EM samples indicate that the grid bars cool much more slowly than do the support foil and sample near the middle of the grid openings. The resulting transient temperature differences generate transient tensile stresses in the support foil. Most of this foil stress develops while the sample is liquid and cooling toward its glass transition T g, and so does not generate tensile sample stress. As the grid bars continue cooling towards the cryogen temperature and contracting, the tensile stress in the foil is released, placing the sample in compressive stress. Radiation-induced creep in the presence of this compressive stress should generate a doming of the sample in the foil openings, as is observed experimentally. Crude estimates of the magnitude of the doming that may be generated by this mechanism are consistent with observation. Several approaches to reducing beam-induced motion are discussed.

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Section: Reducing Beam-induced Motionmentioning

confidence: 99%

Hypothesis for a mechanism of beam-induced motion in cryo-electron microscopy

Thorne

2020

Int Union Crystallogr J

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“…Domain coordinates were those of the WT PheH in the resting conformation (PDB entry 5FGJ) except residues 1-33 and 110 -118, which were modeled as random coils. Missing atoms were added using Modeller (42), and the scattering patterns were simulated using CRYSOL (31) with solvent electron density of 0.35 Å Ϫ3 corresponding to the experimental buffer, which contained 20% glycerol (18). The best-fit ensemble was found using lsqnonneg in MATLAB (MathWorks, Natick, MA).…”

Section: Activation Of Phenylalanine Hydroxylase By a Pku Mutationmentioning

confidence: 99%

The phenylketonuria-associated substitution R68S converts phenylalanine hydroxylase to a constitutively active enzyme but reduces its stability

Khan

Meisburger

Ando

et al. 2019

Journal of Biological Chemistry

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The naturally occurring R68S substitution of phenylalanine hydroxylase (PheH) causes phenylketonuria (PKU). However, the molecular basis for how the R68S variant leads to PKU remains unclear. Kinetic characterization of R68S PheH establishes that the enzyme is fully active in the absence of allosteric binding of phenylalanine, in contrast to the WT enzyme. Analytical ultracentrifugation establishes that the isolated regulatory domain of R68S PheH is predominantly monomeric in the absence of phenylalanine and dimerizes in its presence, similar to the regulatory domain of the WT enzyme. Fluorescence and small-angle X-ray scattering analyses establish that the overall conformation of the resting form of R68S PheH is different from that of the WT enzyme. The data are consistent with the substitution disrupting the interface between the catalytic and regulatory domains of the enzyme, shifting the equilibrium between the resting and activated forms ϳ200-fold, so that the resting form of R68S PheH is ϳ70% in the activated conformation. However, R68S PheH loses activity 2 orders of magnitude more rapidly than the WT enzyme at 37°C and is significantly more sensitive to proteolysis. We propose that, even though this substitution converts the enzyme to a constitutively active enzyme, it results in PKU because of the decrease in protein stability. cro ARTICLE

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“…Between 300 and 240 K liquid water expands by 2% (Hare & Sorensen, 1987) and a 40%(v/v) glycerol solution contracts by 2% (Glycerine Producers Association, 1963). This suggests that solvent must be expelled from or flow into the unit cell to account for the difference between the solvent-cavity and solvent contractions (Juers & Matthews, 2001, 2004Kriminski et al, 2002;Tyree et al, 2018;Juers et al, 2018). As shown in Supplementary Fig.…”

Section: Results and Analysismentioning

confidence: 99%

Solvent flows, conformation changes and lattice reordering in a cold protein crystal

Moreau

Atakisi

Thorne

2019

Acta Cryst Sect D Struct Biol

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When protein crystals are abruptly cooled, the unit-cell, protein and solventcavity volumes all contract, but the volume of bulk-like internal solvent may expand. Outflow of this solvent from the unit cell and its accumulation in defective interior crystal regions has been suggested as one cause of the large increase in crystal mosaicity on cooling. It is shown that when apoferritin crystals are abruptly cooled to temperatures between 220 and 260 K, the unit cell contracts, solvent is pushed out and the mosaicity grows. On temperaturedependent timescales of 10 to 200 s, the unit-cell and solvent-cavity volume then expand, solvent flows back in, and the mosaicity and B factor both drop. Expansion and reordering at fixed low temperature are associated with smallamplitude but large-scale changes in the conformation and packing of apoferritin. These results demonstrate that increases in mosaicity on cooling arise due to solvent flows out of or into the unit cell and to incomplete, arrested relaxation of protein conformation. They indicate a critical role for time in variable-temperature crystallographic studies, and the feasibility of probing interactions and cooperative conformational changes that underlie cold denaturation in the presence of liquid solvent at temperatures down to $200 K.

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Density and electron density of aqueous cryoprotectant solutions at cryogenic temperatures for optimized cryoprotection and diffraction contrast

Cited by 24 publications

References 54 publications

Hypothesis for a mechanism of beam-induced motion in cryo-electron microscopy

Hypothesis for a mechanism of beam-induced motion in cryo-electron microscopy

The phenylketonuria-associated substitution R68S converts phenylalanine hydroxylase to a constitutively active enzyme but reduces its stability

Solvent flows, conformation changes and lattice reordering in a cold protein crystal

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