Models of insulin secretory vesicles from pancreatic beta cells have been created using the cellPACK suite of tools to research, curate, construct and visualise the current state of knowledge. The model integrates experimental information from proteomics, structural biology, cryoelectron microscopy and X-ray tomography, and is used to generate models of mature and immature vesicles. A new method was developed to generate a confidence score that reconciles inconsistencies between three available proteomes using expert annotations of cellular localisation. The models are used to simulate soft X-ray tomograms, allowing quantification of features that are observed in experimental tomograms, and in turn, allowing interpretation of X-ray tomograms at the molecular level.

Cryo-electron tomography (cryo-ET) has the potential to revolutionize our understanding of the building blocks of life since it provides the unique opportunity to study molecules and membrane architectures in the context of cellular interaction. In particular, the combination of fluorescence imaging with focused ion beam (FIB) milling allows the targeting of specific structures in thick cellular samples by preparing thin lamellae that contain a specific fluorescence marker. This technique has conventionally been time-consuming, as it requires sample transfer to multiple microscopes and presents several technical challenges that currently limit its success. Here we describe METEOR, a FIB-integrated microscopy solution that streamlines the correlative cryo-ET workflow. It protects the sample from ice contamination by minimizing handling steps, thus increasing the likelihood of high-quality data. It also allows for monitoring of the milling procedure to ensure the molecule of interest is captured and can then be imaged by cryo-ET.

Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron cryo-microscopy (cryo-EM) as well as to improvements in sample preparation. The present analysis provides an overview of the extent and modes of usage of the various types of surfactants for cryo-EM studies. Digitonin, dodecylmaltoside, protein-based nanodiscs, lauryl maltoside-neopentyl glycol, glyco-diosgenin, and amphipols (APols) are the most popular surfactants at the vitrification step. Surfactant exchange is frequently used between MP purification and grid preparation, requiring extensive optimization each time the study of a new MP is undertaken. The variety of both the surfactants and experimental approaches used over the past few years bears witness to the need to continue developing innovative surfactants and optimizing conditions for sample preparation. The possibilities offered by novel APols for EM applications are discussed.

In this review, we describe how the interplay among science, technology and community interests contributed to the evolution of four structural biology data resources. We present the method by which data deposited by scientists are prepared for worldwide distribution, and argue that data archiving in a trusted repository must be an integral part of any scientific investigation.

All common negative stains are salts of heavy metals. To remedy several technical defects inherent in the use of heavy metal compounds, this study investigates whether salts of the light metals sodium, magnesium, and aluminum can function as negative stains. Screening criteria require aqueous solubility at pH 7.0, formation of a smooth amorphous layer upon drying, and transmission electron microscope imaging of the 87-Å (8.7-nm) lattice periodicity in thin catalase crystals. Six of 23 salts evaluated pass all three screens; detection of the protein shell in ferritin macromolecules indicates that light metal salts also provide negative staining of single particle specimens. Appositional contrast is less than that given by heavy metal negative stains; image density can be raised by increasing electron phase contrast and by selecting salts with phosphate or sulfate anions, thereby adding strong scattering from P or S atoms. Low-dose electron diffraction of catalase crystals negatively stained with 200 mM magnesium sulfate shows Bragg spots extending out to 4.4 Å. Future experimental use of sodium phosphate buffer and magnesium sulfate for negative staining is anticipated, particularly in designing new cocktail (multicomponent) negative stains able to support and protect protein structure to higher resolution levels than are currently achieved.