Explore the vast range of titles available.

The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.

Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data. This article reports sub- and near-atomic structures of triclinic lysozyme and serine protease proteinase K, respectively, providing first demonstrations of ab initio phasing using electron counted MicroED data to solve macromolecular structures.

High-resolution information is important for accurate structure modeling but is challenging to attain in macromolecular crystallography due to the rapid fading of diffracted intensities at increasing resolution. While direct electron detection essentially eliminates the read-out noise during MicroED data collection, other sources of noise remain and limit the measurement of faint high-resolution reflections. Inelastic scattering significantly contributes to noise, raising background levels and broadening diffraction peaks. We demonstrate a substantial improvement in signal-to-noise ratio by using energy filtering to remove inelastically scattered electrons. This strategy results in sub-atomic resolution MicroED data from proteinase K crystals, enabling the visualization of detailed structural features. Interestingly, reducing the noise further reveals diffuse scattering that may hold additional structural information. Our findings suggest that combining energy filtering and direct detection provides more accurate measurements at higher resolution, facilitating precise model refinement and improved insights into protein structure and function. High-resolution data are crucial for accurate structural modeling. Here, authors enhance MicroED data quality using energy filtering, achieving sub-atomic resolution protein data and uncovering diffuse scattering, offering detailed insights into protein structure and function.

MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013 ). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.

Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae. Here, authors demonstrate a method for milling vitrified biological material. Using correlative fluorescence and electron microscopy images, MicroED data is collected for the adenosine receptor.

hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19–29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15–25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19–29 S20G may serve as a model for the toxic spine of hIAPP. In Type-II Diabetes, an individual’s cells fail to respond correctly to the hormone insulin, leaving them unable to counteract high levels of sugar in the blood. Another hormone, human islet amyloid polypeptide (hIAPP), works with insulin to regulate blood sugar levels. hIAPP is an amyloid protein, which means that it can lose its normal structure and form fibrils. Fibrils are difficult for cells to break down and are often associated with disease. Indeed, fibrils of hIAPP often form in the pancreas as part of Type-II Diabetes. Some studies have shown that hIAPP fibrils are toxic to pancreatic cells and worsen the symptoms of Type-II Diabetes. Others suggest that it is the process of fibril formation that is toxic, not the fibrils themselves. Although the structures of the fibrils have been described, whether these structures cause cell toxicity has not been investigated. Krotee et al. have now explored the structures of two overlapping segments of hIAPP using a new cryo electron microscopy method called MicroED that is ideal for studying such segments. One segment, called 19-29 S20G, forms a standard amyloid fibril structure that is similar to the structure of full-length hIAPP fibrils. Adding these segments to human cells causes similar levels of toxicity as the full-length hIAPP fibrils. The second segment, called 15-25 WT, forms a non-toxic structure that is less stable than standard amyloid fibrils. The results presented by Krotee et al. support the view that standard amyloid fibril structures are toxic to cells and suggest that 19-29 S20G may be a good model to use when studying how full-length hIAPP fibrils behave. The structure of 19-29 S20G may also be useful as a template for designing molecules that block amyloid fibril growth. If amyloid fibrils cause cell toxicity in the pancreas, then these molecules could be used to treat Type-II Diabetes.

There is considerable potential for X-ray free electron lasers (XFELs) to enable determination of macromolecular crystal structures that are difficult to solve using current synchrotron sources. Prior XFEL studies often involved the collection of thousands to millions of diffraction images, in part due to limitations of data processing methods. We implemented a data processing system based on classical post-refinement techniques, adapted to specific properties of XFEL diffraction data. When applied to XFEL data from three different proteins collected using various sample delivery systems and XFEL beam parameters, our method improved the quality of the diffraction data as well as the resulting refined atomic models and electron density maps. Moreover, the number of observations for a reflection necessary to assemble an accurate data set could be reduced to a few observations. These developments will help expand the applicability of XFEL crystallography to challenging biological systems, including cases where sample is limited. Large biological molecules (or macromolecules) have intricate three-dimensional structures. X-ray crystallography is a technique that is commonly used to determine these structures and involves directing a beam of X-rays at a crystal that was grown from the macromolecule of interest. The macromolecules in the crystal scatter the X-rays to produce a diffraction pattern, and the crystal is rotated to provide further diffraction images. It is then possible to work backwards from these images and elucidate the structure of the macromolecule in three dimensions. X-ray beams are powerful enough to damage crystals, and scientists are developing new approaches to overcome this problem. One recent development uses ‘X-ray free electron lasers’ to circumvent the damage caused to crystals. However, early applications of this approach required many crystals and thousands to millions of diffraction patterns to be collected—largely because methods to process the diffraction data were far from optimal. Uervirojnangkoorn et al. have now developed a new data-processing procedure that is specifically designed for diffraction data obtained using X-ray free electron lasers. This method was applied to diffraction data collected from crystals of three different macromolecules (which in this case were three different proteins). For all three, the new method required many fewer diffraction images to determine the structure, and in one case revealed more details about the structure than the existing methods. This new method is now expected to allow a wider range of macromolecules to be studied using crystallography with X-ray free electron lasers, including cases where very few crystals are available.

MicroED is a cryo-EM technique for collecting electron diffraction data from microcrystals and nanocrystals. This protocol from Gonen and colleagues describes how to prepare the protein crystal samples, how to set up the electron microscope for MicroED, and diffraction data collection. The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.