The advances in electron cryo-microscopy have enabled high-resolution structural studies of vitrified macromolecular complexes in situ by cryo-electron tomography (cryo-ET). Since utilization of cryo-ET is generally limited to the specimens with thickness < 500 nm, a complex sample preparation protocol to study larger samples such as single eukaryotic cells by cryo-ET was developed and optimized over the last decade. The workflow is based on the preparation of a thin cellular lamella by cryo-focused ion beam milling (cryo-FIBM) from the vitrified cells. The sample preparation protocol is a multi-step process which includes utilization of several high-end instruments and comprises sample manipulation prone to sample deterioration. Here, we present a workflow for preparation of three different model specimens that was optimized to provide high-quality lamellae for cryo-ET or electron diffraction tomography with high reproducibility. Preparation of lamellae from large adherent mammalian cells, small suspension eukaryotic cell line, and protein crystals of intermediate size is described which represents examples of the most frequently studied samples used for cryo-FIBM in life sciences.

• MeSH
• Cells ultrastructure   MeSH
• Cryoelectron Microscopy methods   MeSH
• Ions MeSH
• Macromolecular Substances ultrastructure   MeSH
• Molecular Biology methods   MeSH
• Specimen Handling methods   MeSH
• Proteins ultrastructure   MeSH
• Workflow MeSH
• Reproducibility of Results MeSH
• Saccharomyces cerevisiae ultrastructure   MeSH
• Electron Microscope Tomography methods   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Cryo-electron microscopy has established as a mature structural biology technique to elucidate the three-dimensional structure of biological macromolecules. The Coulomb potential of the sample is imaged by an electron beam, and fast semi-conductor detectors produce movies of the sample under study. These movies have to be further processed by a whole pipeline of image-processing algorithms that produce the final structure of the macromolecule. In this chapter, we illustrate this whole processing pipeline putting in value the strength of "meta algorithms," which are the combination of several algorithms, each one with different mathematical rationale, in order to distinguish correctly from incorrectly estimated parameters. We show how this strategy leads to superior performance of the whole pipeline as well as more confident assessments about the reconstructed structures. The "meta algorithms" strategy is common to many fields and, in particular, it has provided excellent results in bioinformatics. We illustrate this combination using the workflow engine, Scipion.

• MeSH
• Algorithms * MeSH
• Cryoelectron Microscopy methods   MeSH
• Macromolecular Substances ultrastructure   MeSH
• Molecular Biology methods   MeSH
• Image Processing, Computer-Assisted methods   MeSH
• Workflow MeSH
• Computational Biology MeSH
• Single Molecule Imaging methods   MeSH
• Imaging, Three-Dimensional methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

3D macromolecular structural data is growing ever more complex and plentiful in the wake of substantive advances in experimental and computational structure determination methods including macromolecular crystallography, cryo-electron microscopy, and integrative methods. Efficient means of working with 3D macromolecular structural data for archiving, analyses, and visualization are central to facilitating interoperability and reusability in compliance with the FAIR Principles. We address two challenges posed by growth in data size and complexity. First, data size is reduced by bespoke compression techniques. Second, complexity is managed through improved software tooling and fully leveraging available data dictionary schemas. To this end, we introduce BinaryCIF, a serialization of Crystallographic Information File (CIF) format files that maintains full compatibility to related data schemas, such as PDBx/mmCIF, while reducing file sizes by more than a factor of two versus gzip compressed CIF files. Moreover, for the largest structures, BinaryCIF provides even better compression-factor ten and four versus CIF files and gzipped CIF files, respectively. Herein, we describe CIFTools, a set of libraries in Java and TypeScript for generic and typed handling of CIF and BinaryCIF files. Together, BinaryCIF and CIFTools enable lightweight, efficient, and extensible handling of 3D macromolecular structural data.

• MeSH
• Databases, Chemical MeSH
• Data Compression methods   MeSH
• Crystallography methods   MeSH
• Macromolecular Substances chemistry   ultrastructure   MeSH
• Models, Molecular * MeSH
• Software * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH