The use of cryo-EM and three-dimensional image reconstruction is becoming increasingly common. Our vision for this technique is to provide a straightforward manner in which users can proceed from raw data to a reliable 3D reconstruction through a pipeline that both facilitates management of the processing steps and makes the results at each step more transparent. Tightly integrated with a relational SQL database, Appion is a modular and transparent pipeline that extends existing software applications and procedures. The user manages and controls the software modules via web-based forms, and all results are similarly available using web-based viewers directly linked to the underlying database, enabling even naive users to quickly deduce the quality of their results. The Appion API was designed with the principle that applications should be compatible with a broad range of specimens and that libraries and routines are modular and extensible. Presented here is a description of the design and architecture of the working Appion pipeline prototype and some results of its use.

Solving the structure of macromolecular complexes using transmission electron microscopy can be an arduous task. Many of the steps in this process rely strongly on the aid of pre-existing structural knowledge, and are greatly complicated when this information is unavailable. Here we present two software tools meant to facilitate particle picking, an early stage in the single-particle processing of unknown macromolecules. The first tool, DoG Picker, is an efficient and reasonably general, particle picker based on the Difference of Gaussians (DoG) image transform. It can function alone, as a reference-free particle picker with the unique ability to sort particles based on size, or it can also be used as a way to bootstrap the creation of templates or training datasets for other particle pickers. The second tool is TiltPicker, an interactive graphical interface application designed to streamline the selection of particle pairs from tilted-pair datasets. In many respects, TiltPicker is a reimplementation of the SPIDER WEB tilted-particle picker, but built on modern computer frameworks making it easier to deploy and maintain. The TiltPicker program also includes several useful new features beyond those of its predecessor.

As larger macromolecular structures become available, there is a growing need to understand their ‘internal’ volumes—such as deep clefts, channels and cavities—as these often play critical roles in their function. The 3V web server can automatically extract and comprehensively analyze all the internal volumes from input RNA and protein structures. It rapidly finds internal volumes by taking the difference between two rolling-probe solvent-excluded surfaces, one with as large as possible a probe radius and the other with a solvent radius (typically 1.5 Å for water). The outputs are volumetric representations, both as images and downloadable files, which can be used for further analysis.The 3V server and source code are available from http://3vee.molmovdb.org.