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Metal-ligand bond lengths and strengths: are they correlated? Structure determination of the intermediate tin oxide Sn3O4 by precession electron diffraction by White, Thomas A. Sergio and Midgley, Paul A. A crystal chemical approach to superconductivity. Species-specific shells: Chitin synthases and cell mechanics in molluscs by Weiss, Ingrid.
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ufowihij.tk Called MicroED, this technique involves placing the crystal in a transmission electron cryo-microscope, which is a fairly standard piece of equipment in many laboratories. However, Shi et al.
By reducing the electron dose by a factor of , it was possible to collect up to 90 diffraction patterns from the same, very small, three-dimensional crystal, and then—similar to what happens in X-ray crystallography—work backwards to figure out the structure of the protein. Shi et al. This proof-of principle study paves the way for crystallographers to study protein that cannot be studied with existing techniques.
X-ray crystallography depends on large and well-ordered crystals for diffraction studies.
The periodic structure of the crystalline solid acts as a diffraction grating to scatter the X-rays. Therefore, large crystals are required to withstand the high levels of radiation damage received during data collection Henderson, Despite the development of highly sophisticated robotics for crystal growth assays and the implementation of microfocus beamlines Moukhametzianov et al. In an attempt to alleviate this problem, researchers have turned to femtosecond X-ray crystallography Chapman et al.
While this technique shows great promise, the current implementation of the technology requires an extremely large number of crystals millions and access to sources is still in developmental stages. Electron crystallography is a bona fide method for determining protein structure from crystalline material but with important differences. The crystals that are used must be very thin Henderson and Unwin, ; Henderson et al.
Because electrons interact with materials more strongly than X-rays Henderson, , electrons can yield meaningful data from relatively small and thin crystals. This technique has been used successfully to determine the structures of several proteins from thin two-dimensional crystals 2D crystals Wisedchaisri et al. High energy electrons result in a large amount of radiation damage to the sample, leading to loss in resolution and destruction of the crystalline material Glaeser, As each crystal can usually yield only a single diffraction pattern, structure determination is only possible by merging data originating from hundreds of individual crystals.
This diffraction pattern was processed with ImageJ and despeckled for ease of viewing. Moreover, a double tilt cryo holder as well as newly developed goniometer-based grid holders could be used to cover more of the Fourier space. J Gen Physiol — Advances in structural and functional analysis of membrane proteins by electron crystallography. BLN, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article.
For example, electron diffraction data from more than individual crystals were merged to generate a data set for aquaporin-0 at 1. While electron crystallography has been successful with 2D crystals, previous attempts at using electron diffraction for structure determination from protein 3D crystals were not successful. A number of studies detail the difficulties associated with data collection and processing of diffraction data that originates from several hundreds of 3D crystals, limiting the ability to integrate and merge the data in order to determine a structure in such a way Shi et al.
We show here that atomic resolution diffraction data can be collected from crystals with volumes up to six orders of magnitude smaller than those typically used for X-ray crystallography. We developed a strategy for data collection with extremely low electron dose and procedures for indexing and integrating reflections.
We processed the diffraction data and determined the structure of lysozyme at 2. Thus, a high-resolution protein structure can be determined from electron diffraction of three-dimensional protein crystals in an electron microscope. Lysozyme was chosen as a model protein because it is a well-behaved and well-characterized protein that readily forms well-ordered crystals. From the time its structure was first analyzed Blake et al. Figure 1A shows a typical crystallization drop containing microcrystals, which appear as barely visible specks arrows alongside the larger crystals that are typically used for X-ray crystallography.
These specks are up to 6 orders of magnitude smaller in volume than the larger crystals in the drop. The solution containing these microcrystals was applied to an electron microscopy holey-carbon grid with a pipette and plunged into liquid ethane. More than microcrystals were typically observed per grid preparation, and these ranged in size from several microns to sub micron. The crystals typically appeared as electron dense rectangular or triangular forms with very sharp edges. A Light micrograph showing lysozyme microcrystals three examples indicated by arrows in comparison with larger crystals of the size normally used for X-ray crystallography.
B Lysozyme microcrystals visualized in over-focused diffraction mode on the cryo-EM prior to data collection.
Electron diffraction was used to assess the quality of the cryo-preparations. Moreover, we found that for such thin crystals the tilt had no significant adverse affect on the diffraction quality Figure 2D. A Analysis of the effects of crystal thickness on maximum resolution of observed reflections from thick crystals. The analysis shows adverse effects of crystal thickness on the obtainable resolution as large crystals are tilted. B For assessing the quality of our cryo preparations, diffraction data were obtained from lysozyme microcrystals. C An example of lysozyme diffraction data collected at 0.
This diffraction pattern was processed with ImageJ and despeckled for ease of viewing. D Analysis of the effects of crystal thickness on maximum resolution of observed reflections from thin crystals. The small crystal shows a relatively constant maximum resolution that does not appear to be affected by crystal tilt. For 2D electron crystallography, the electron dose that is typically used in diffraction causes significant radiation damage to the sample, leading to a rapid loss in resolution and destruction of the crystal Glaeser, ; Unwin and Henderson, ; Taylor and Glaeser, As a result, each crystal exposed to high dose usually only yields a single diffraction pattern, and structure determination requires the merging of data originating from a large number of individual crystals.
However, 3D crystals can deliver electron diffraction data to atomic resolution with very low doses. Because all the data would originate from a single crystal, indexing, integration and merging should be straightforward and structure determination possible. As a dataset containing multiple exposures from a single crystal is collected, energy transferred by inelastic scattering will damage the crystalline matrix, negatively affecting both the resolution limit and intensities of observed reflections.