Automated tracing of filaments in 3D electron tomography reconstructions using Sculptor and Situs

Abstract

The molecular graphics program Sculptor and the command-line suite Situs are software packages for the integration of biophysical data across spatial resolution scales. Herein, we provide an overview of recently developed tools relevant to cryo-electron tomography (cryo-ET), with an emphasis on functionality supported by Situs 2.7 and Sculptor 2.1. We describe a work flow for automatically segmenting filaments in cryo-ET maps including denoising, local normalization, feature detection, and tracing. Tomograms of cellular actin networks exhibit both cross-linked and bundled filament densities. Such filamentous regions in cryo-ET data sets can then be segmented using a stochastic template-based search, VolTrac. The approach combines a genetic algorithm and a bidirectional expansion with a tabu search strategy to localize and characterize filamentous regions. The automated filament segmentation by VolTrac compares well to a manual one performed by expert users, and it allows an efficient and reproducible analysis of large data sets. The software is free, open source, and can be used on Linux, Macintosh or Windows computers.

Numerical geometry of map and model assessment

“…The approach also led to an implementation of symmetry constraints during the refinement (Wriggers, 2012), and it opened the door to a new high-dimensional conformational search (López-Blanco and Chacón, 2013) of multiple fragments, which were optimized in parallel using genetic algorithms in combination with tabu search techniques (Rusu and Birmanns, 2010). We have recently adopted this approach, termed voltrac , to trace a-helices in high-resolution EM maps (Rusu and Wriggers, 2012) and to segment filaments in tomograms (Rusu et al, 2012). …”

Quantifying Variability of Manual Annotation in Cryo-Electron Tomograms

“…Ice embedding was developed to keep biological specimens in a frozen-hydrated state for electron microscopy (Taylor & Glaeser, 1974; Lepault et al, 1983). Recent advances in sample preparation methods, as well as new imaging technologies, have made the once impossible task of imaging intact mammalian cells by cryo-electron tomography (cryoET) a reality (Koning et al, 2008; Moussavi et al, 2010; Rigort et al, 2012; Rusu et al, 2012; Shahmoradian et al, 2014; Asano et al, 2015). However, both the imaging technique (cryoET) and the native environment of cells pose difficulties for the visualization and interpretation of tomographic data sets.…”

“…It is critical to understand the variability inherent in manual segmentation in order to determine the role it should play in structural biology research. Numerous studies have used manual annotations to make comparisons with various automated segmentation algorithms (Garduño et al, 2008; Nguyen & Ji, 2008; Rigort et al, 2012; Rusu et al, 2012). Many groups have taken this a step further by evaluating the use of manual annotations as a good “ground truth”.…”

Developing a denoising filter for electron microscopy and tomography data in the cloud

“…The HIV-1 map is a frequently used test system for denoising (van der Heide et al 2007; Fernandez 2009; Wei and Yin 2010); therefore, our results can be compared to those in the literature. Our DPSV filter is quite effective, even after one application with relatively short paths P =2 (see also Rusu et al 2012 for a comparison with Gaussian filtering). The filtered cross-sections (Fig.…”