Chemists of all fields currently publish about 50 000 crystal structures per year, the vast majority of which are X‐ray structures. We determined two molecular structures by employing electron rather ...than X‐ray diffraction. For this purpose, an EIGER hybrid pixel detector was fitted to a transmission electron microscope, yielding an electron diffractometer. The structure of a new methylene blue derivative was determined at 0.9 Å resolution from a crystal smaller than 1×2 μm2. Several thousand active pharmaceutical ingredients (APIs) are only available as submicrocrystalline powders. To illustrate the potential of electron crystallography for the pharmaceutical industry, we also determined the structure of an API from its pill. We demonstrate that electron crystallography complements X‐ray crystallography and is the technique of choice for all unsolved cases in which submicrometer‐sized crystals were the limiting factor.
Electrons instead of X‐rays: An electron diffractometer was tailored and employed for de novo structure determination from submicrometer‐sized crystals. A new methylene blue derivative was analysed together with a microcrystalline extract of an active pharmaceutical ingredient from a pill. The results obtained on submicrometer‐sized samples complement X‐ray crystallography.
Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural ...biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.
Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency ...is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion
. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies
and peptides
or that address late steps of the viral replication cycle
.
In this review we cover the technical background to negative staining of biomolecules and viruses, and then expand upon the different possibilities and limitations. Topics range from conventional ...air-dry negative staining of samples adsorbed to carbon support films, the variant termed the “negative staining-carbon film” technique and negative staining of samples spread across the holes of holey-carbon support films, to a consideration of dynamic/time-dependent negative staining. For each of these approaches examples of attainable data are given. The cryo-negative staining technique for the specimen preparation of frozen-hydrated/vitrified samples is also presented. A detailed protocol to successfully achieve cryo-negative staining with ammonium molybdate is given, as well as examples of data, which support the claim that cryo-negative staining provides a useful approach for the high-resolution study of macromolecular and viral structure.
Transcription by RNA polymerase II (RNAPII) is a central process in eukaryotic gene regulation. While atomic details exist for the yeast RNAPII, characterization of the human complex lags behind, ...mostly due to the inability to obtain large quantities of purified material. Although the complexes have the same protein composition and high sequence similarity, understanding of transcription and of transcription-coupled DNA repair (TCR) in humans will require the use of human proteins in structural studies. We have used cryo-electron microscopy, image reconstruction, and variance analysis to characterize the structure and dynamics of human RNAPII (hRNAPII). Our studies show that hRNAPII in solution parallels the conformational flexibility of the yeast structures crystallized in different states but also illustrate a more extensive conformational range with potential biological significance. This hRNAPII study will serve as a structural platform to build up higher-order transcription and TCR complexes and to gain information that may be unique to the human RNAPII system.
3D electron diffraction has reached a stage where the structures of chemical compounds can be solved productively. Instrumentation is lagging behind this development, and to date dedicated electron ...diffractometers for data collection based on the rotation method do not exist. Current studies use transmission electron microscopes as a workaround. These are optimized for imaging, which is not optimal for diffraction studies. The beam intensity is very high, it is difficult to create parallel beam illumination and the detectors used for imaging are of only limited use for diffraction studies. In this work, the combination of an EIGER hybrid pixel detector with a transmission electron microscope to construct a productive electron diffractometer is described. The construction not only refers to the combination of hardware but also to the calibration of the system, so that it provides rapid access to the experimental parameters that are necessary for processing diffraction data. Until fully integrated electron diffractometers become available, this describes a setup for productive and efficient operation in chemical crystallography.
Installation of the EIGER X 1M detector onto an electron microscope and system calibration for data collection turns a transmission electron microscope into an electron diffraction instrument. Data can be collected and processed with a throughput that meets the requirements of a modern X‐ray facility. The setup described here offers access to single‐crystal structure determination from microcrystalline powder when large single crystals cannot be produced.
Chemist's caviar: Stimuli‐responsive fusogenic vesicles from a cardanol‐taurine surfactant are reported. The thermo‐responsive behavior of the unsaturations in the alkyl chains, reminiscent of ...homeoviscous alterations, lead to a micelle‐to‐vesicle transformation and to the formation of caviar‐like adhesive vesicles (see photograph).
Single-particle cryogenic electron microscopy (cryo-EM) can now yield near-atomic resolution structures of biological complexes. However, the reference-based alignment algorithms commonly used in ...cryo-EM suffer from reference bias, limiting their applicability (also known as the 'Einstein from random noise' problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous ('four-dimensional') cryo-EM data sets. The methodologies integrated in this pipeline include
camera correction, movie-based full-data-set contrast transfer function determination, movie-alignment algorithms, (Fourier-space) multivariate statistical data compression and unsupervised classification, 'random-startup' three-dimensional reconstructions, four-dimensional structural refinements and Fourier shell correlation criteria for evaluating anisotropic resolution. The procedures exclusively use information emerging from the data set itself, without external 'starting models'. Euler-angle assignments are performed by angular reconstitution rather than by the inherently slower projection-matching approaches. The comprehensive 'ABC-4D' pipeline is based on the two-dimensional reference-free 'alignment by classification' (ABC) approach, where similar images in similar orientations are grouped by unsupervised classification. Some fundamental differences between X-ray crystallography
single-particle cryo-EM data collection and data processing are discussed. The structure of the giant haemoglobin from
at a global resolution of ∼3.8 Å is presented as an example of the use of the ABC-4D procedure.
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