Chapter 19.8. Use of SPIDER and SPIRE in image reconstruction

SPIDER (System for Processing Image Data in Electron microscopy and Related fields) was one of the earliest imageprocessing packages for single-particle reconstruction and electron tomography. It was first released in 1978, and has been continually improved and updated since then (Frank et al., 1981, 1996; Baxter et al., 2007; Shaikh et al., 2008; Yang et al., 2007). It is currently available as free open-source code under Gnu Public License (GPL). In its most common usage, SPIDER allows a researcher to create a three-dimensional (3D) reconstruction of a macromolecule from a collection of transmission electron micrographs, interpreted as projections showing the molecule in different orientations. Using cryo-electron microscopy these methods, pioneered by Joachim Frank at the Wadsworth Center in Albany (see Frank, 2006), have recently allowed researchers to determine the 3D structures of several macromolecular complexes at near-atomic resolution (~3.8–4.5 Å; Zhou, 2008). These methods provide a way of studying the structure of such complexes in a more natural environment than is possible using X-ray crystallography and of studying conformational changes of molecular machines as they perform their work (Saibil, 2000). One way in which SPIDER differs from most other software for single-particle reconstruction [Imagic (van Heel et al., 1996; Chapter 19.9), Frealign (Grigorieff, 2007), EMAN2 (Tang et al., 2007; Chapter 19.10), SPARX (Hohn et al., 2007) and Xmipp (Sorzano et al., 2004)] is that it provides alternative methods for many procedures and thus can serve as a developmental platform for testing different approaches to single-particle reconstruction. For instance, alternative alignment operations and statistical analysis operations can be combined inside scripts to investigate different approaches to overcoming molecular heterogeneity. The prime reasons for the continued success of SPIDER are its validated reputation for providing the highest-resolution reconstructions, its ease of installation and its comprehensive documentation, which is constantly being updated (Shaik et al., 2008). SPIDER’s procedure language makes it easy to use for controlling the flow of a complex train of operations and addressing problems related to heterogeneity of particles. With the Quick Start Guide and tutorials as an introduction, a new user can quickly acquire competence in the procedure language with only a few hours’ introduction. The SPIDER system consists of six major components: