Biology subjects

Han, B.-G.

Publications and source records attributed to Han, B.-G..

4 recordsLinked to original sources

bioRxiv2021

SIMPLE ASSAY FOR ADSORPTION OF PROTEINS TO THE AIR-WATER INTERFACE

A rapid assay is described, based upon the Marangoni effect, which detects the formation of a denatured-protein film at the air-water interface (AWI) of aqueous samples. This assay requires no more than a 20 L aliquot of sample, at a protein concentration of no more than1 mg/ml, and it can be performed with any buffer that is used to prepare grids for electron cryo-microscopy (cryo-EM). In addition, this assay provides an easy way to estimate the rate at which a given protein forms such a film at the AWI. Use of this assay is suggested as a way to pre-screen the effect of various additives and chemical modifications that one might use to optimize the preparation of grids, although the final proof of optimization still requires further screening of grids in the electron microscope. In those cases when the assay establishes that a given protein does form a sacrificial, denatured-protein monolayer, it is suggested that subsequent optimization strategies might focus on discovering how to improve the adsorption of native proteins onto that monolayer, rather than to prevent its formation. A second alternative might be to bind such proteins to the surface of rationally designed affinity grids, in order to prevent their diffusion to, and unwanted interaction with, the AWI.

biophysics↗

bioRxiv2020

Defocus-dependent Thon-ring fading

The brightness of modern Schottky field-emission guns can produce electron beams that have very high spatial coherence, especially for the weak-illumination conditions that are used for single-particle electron cryo-microscopy in structural biology. Even so, many users have observed defocus-dependent Thon-ring fading that has led them to restrict their data collection strategy to imaging with relatively small defocus values. In this paper, we reproduce the observation of defocus-dependent Thon-ring fading and produce a quantitative analysis and clear explanation of its causes. We demonstrate that a major cause is the delocalization of high-resolution Fourier components outside the field of view of the camera. We also show that it is important to make a correction for linear magnification anisotropy, even if it is quite small, before circular averaging of the Thon rings, as is also true before merging data from particles in many orientations. Under the conditions used in this paper, which are typical of those used in single-particle electron cryomicroscopy, fading of the Thon rings due to source coherence is negligible. The principal conclusion is that much higher values of defocus can be used than is currently thought to be possible. This increased understanding should give electron microscopists the confidence to use higher amounts of defocus to allow, for example, better visibility of their particles and Ewald sphere correction.

biophysics↗

bioRxiv2019

Micro-scale fluid behavior during cryo-EM sample blotting

Blotting has been the standard technique for preparing aqueous samples for single-particle electron cryo-microscopy (cryo-EM) for over three decades. This technique removes excess solution from a TEM grid by pressing absorbent filter paper against the specimen prior to vitrification. However, this standard technique produces vitreous ice with inconsistent thickness from specimen to specimen and from region to region within the same specimen, the reasons for which are not understood. Here, high-speed interference-contrast microscopy is used to demonstrate that the irregular pattern of fibers in the filter paper imposes tortuous, highly variable boundaries during removal of excess liquid from a flat, hydrophilic surface. As a result, aqueous films of nonuniform thickness are formed while the filter paper is pressed against the substrate. This pattern of nonuniform liquid thickness changes again after the filter paper is pulled away, but the thickness still does not become completely uniform. We suggest that similar topological features of the liquid film are produced during the standard technique used to blot EM grids and that these manifest in nonuniform ice after vitrification. These observations suggest that alternative thinning techniques, which do not rely on direct contact between the filter paper and the grid, may result in more repeatable and uniform sample thicknesses.\n\nSTATEMENT OF SIGNIFICANCEMultiple imaging techniques are used to observe dynamic, micro-scale events as excess water is removed from a substrate by blotting with filter paper. As a result, new insight is gained about why the thickness values of remaining sample material are so variable across a single EM grid, as well as from one grid to the next. In addition, quantitative estimates are made of the shear forces to which macromolecular complexes can be exposed during blotting. The fact that sample thicknesses and flow rates are seen to be inherently under poor control during blotting suggests that other methods of removing excess water may be better suited for consistently achieving large sample areas that are suitable for use in electron cryo-microscopy.

biophysics↗

bioRxiv2016

Monolayer-crystal streptavidin support films provide an internal standard of cryo-EM image quality

Analysis of images of biotinylated Escherichia coli 70S ribosome particles, bound to streptavidin affinity grids, demonstrates that the image-quality of particles can be predicted by the image-quality of the monolayer crystalline support film. The quality of the Thon rings is also a good predictor of the image-quality of particles, but only when images of the streptavidin crystals extend to relatively high resolution. When the estimated resolution of streptavidin was 5 [A] or worse, for example, the ribosomal density map obtained from 22,697 particles went to only 9.5 [A], while the resolution of the map reached 4.0 [A] for the same number of particles, when the estimated resolution of streptavidin crystal was 4 [A] or better. It thus is easy to tell which images in a data set ought to be retained for further work, based on the highest resolution seen for Bragg peaks in the computed Fourier transforms of the streptavidin component. The refined density map obtained from 57,826 particles obtained in this way extended to 3.6 [A], a marked improvement over the value of 3.9 [A] obtained previously from a subset of 52,433 particles obtained from the same initial data set of 101,213 particles after 3-D classification. These results are consistent with the hypothesis that interaction with the air-water interface can damage particles when the sample becomes too thin. Streptavidin monolayer crystals appear to provide a good indication of when that is the case.

biophysics↗