The precise structural solution of crystals on a mesostructural scale is challenging due to the difficulties in obtaining electron diffraction and the complicated relationship between the crystal structure factors (CSFs) and the conventional underfocus phase-contrast transmission electron microscopy (TEM) images due to the large unit cell and the complex structures. Here, we present the structural investigation of mesostructured crystals via the combination of electron crystallographic Fourier synthesis and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) that only relies on the mass-thickness contrast. The three-dimensional electrostatic potential is reconstructed from the amplitudes and phases extracted from the Fourier transforms of the corresponding HAADF-STEM images and merged into a set of CSFs. This method is verified on silica scaffolds following a shifted double-diamond surface network with space group I41/amd. The results indicate that electron crystallography reconstruction by HAADF-STEM images is more suitable and accurate in determining the structure in comparison with conventional TEM electron crystallography reconstruction. This approach transfers the contrast of mesostructured crystals to images more accurately and the relationship between the Fourier transforms of HAADF-STEM images and the CSFs is more intuitive. It shows great advantages for the structural solution of crystals on the mesostructural scale.

A scanning precession electron diffraction system has been integrated with a direct electron detector to allow the collection of improved quality diffraction patterns. This has been used on a two-phase α–β titanium alloy (Timetal® 575) for phase and orientation mapping using an existing pattern-matching algorithm and has been compared to the commonly used detector system, which consisted of a high-speed video-camera imaging the small phosphor focusing screen. Noise is appreciably lower with the direct electron detector, and this is especially noticeable further from the diffraction pattern center where the real electron scattering is reduced and both diffraction spots and inelastic scattering between spots are weaker. The results for orientation mapping are a significant improvement in phase and orientation indexing reliability, especially of fine nanoscale laths of α-Ti, where the weak diffracted signal is rather lost in the noise for the optically coupled camera. This was done at a dose of ~19 e−/Å2, and there is clearly a prospect for reducing the current further while still producing indexable patterns. This opens the way for precession diffraction phase and orientation mapping of radiation-sensitive crystalline materials.

Charoite, ideally (K,Sr,Ba,Mn)15–16(Ca,Na)32[(Si70(O,OH)180)](OH,F)4·nH20, is a rock-forming mineral from the Murun massif in Yakutia, Sakha Republic, Siberia, Russia, where it occurs in a unique alkaline intrusion. Charoite occurs as four different polytypes, which are commonly intergrown in nanocrystallme fibres. We report the structure of charoite-96 (a = 32.11(6), b = 19.77(4), c = 7.23(1) Å, β = 95.85(9)°, V = 4565(24) Å3, space group P21/m), which was solved ab initio by direct methods on the basis of 2676 unique electron diffraction reflections collected by automated diffraction tomography and refined to R1/wR2 = 0.34/0.37. The structure of charoite-96 is related to that of the charoite-90, which was also solved recently. Both structures are composed of three different types of dreier silicate chains running along [001] and separated by ribbons of edge-sharing Ca- and Na-centred octahedra. In the structure of charoite-96, adjacent blocks formed by three different silicate chains and stacked along the x axis, are shifted by a translation of 1/2 c. The shifts involve a hybrid dreier quadruple chain, [Si17O43]18– and a double dreier chain, [Si6O17]10–. In charoite-90 adjacent blocks are stacked without shifts.

Single-particle reconstruction (SPR) and electron crystallography (EC), two major applications in electron microscopy, can be used to determine the structure of membrane proteins. The three-dimensional (3D) map is obtained from separated particles in conventional SPR, but from periodic unit cells in EC. Here, we report a refined SPR procedure for processing 2D crystal images. The method is applied to 2D crystals of melibiose permease, a secondary transporter in Escherichia coli. The current procedure is improved from our previously published one in several aspects. The “gold standard Fourier shell correlation” resolution of our final reconstruction reaches 13 Å, which is significantly better than the previously obtained 17 Å resolution. The choices of different refinement parameters for reconstruction are discussed. Our refined SPR procedure could be applied to determine the structure of other membrane proteins in small or locally distorted 2D crystals, which are not ideal for EC.

Specimen charging may be one of the most significant factors that contribute to the high variability and generally low quality of images in cryo-electron microscopy. Understanding the nature of specimen charging can help in devising methods to reduce or even avoid its effects and thus improve the rate of data collection as well as the quality of the data. We describe a series of experiments that help to characterize the charging phenomenon, which has been termed the Berriman effect. The pattern of buildup and disappearance of the charge pattern has led to several suggestions for how to alleviate the effect. Experiments are described that demonstrate the feasibility of such charge mitigation.

A new assay using low-dose electron diffraction to measure the protection of protein structure against damage from drying is described. When thin single crystals of catalase are dried within water alone, low-dose electron diffraction yields no Bragg spots. Drying within an experimental aqueous solution that permits detection of diffraction spots thereby indicates a positive result, and the extent of these Bragg reflections into the high angle range gives a quantitative measure of the degree of protection. Bragg spots out to 3.7–3.9 Å are recorded for drying within 100 mM solutions of the known structure-preserving sugars, sucrose, tannin, and trehalose. The ability of trehalose to maintain native protein structure during drying starts between 10 and 25 mM, and changes only slightly at concentrations above this threshold; with drying in 150-mM trehalose, catalase crystals yield diffraction spots out to 3.7 Å. Drying within the organic nonsugar polymer polyvinylpyrrolidone gives Bragg spots to 4.0 Å. This new assay should be useful to measure the unexamined structure-preserving capabilities of modified sugars, other nonsugars, and mixtures to identify which protective matrix maintains native protein structure to the greatest extent during drying; electron crystallography using that optimal matrix should yield protein structure at improved levels of high resolution.

Many of the techniques that have been developed in X-ray crystallography are being applied in electron crystallographic studies of proteins. Electron crystallography has the advantage of measuring structure factor phases directly from high resolution images with an accuracy substantially higher than is common in X-ray crystallography. However, electron diffraction amplitudes are often not as precise as those obtained in X-ray work. We discuss here some approaches to maximizing the reliability of the diffraction amplitudes through choice of exposure and data processing schemes. With accurate measurement of diffraction data, Fourier difference methods can be used in electron crystallographic studies of small, localized changes of proteins that exist in two-dimensional crystals. The mathematical basis for the power of these methods in detecting small changes is reviewed. We then discuss several issues related to optimizing the quality of the diffraction data and derive an expression for the best exposure for recording diffraction patterns. An application of Fourier difference maps in localizing drug binding sites on the protein tubulin is discussed.

An efficient method of merging images from electron crystallographic data has been developed that is several orders of magnitude faster than the method of phase shift origin search. The results of this new cross-correlation method are compared with the phase residual in terms of speed, consistency with the phase residual method, and the ease of incorporating symmetry constraints along with the image-to-image merging. The program is further accelerated by a more efficient sorting procedure.