# Software Tools For Molecular Microscopy/Specific packages

Packages that offer a comprehensive set of tools to permit the analysis of data in a single class of structural problem. For example packages specifically focused on objects with helical, icosahedral, crystalline symmetry, etc.

## Two-dimensional crystals
#### 2dx

    Website: http://2dx.org/ Current version: 3.1.0 Contact: Henning.Stahlberg@unibas.ch A software system designed as a user friendly, platform-independent software package for electron crystallography. 2dx assists in the management of an image-processing project, guides the user through the processing of 2D crystal images, and provides transparence for processing tasks and results. Algorithms are implemented in the form of script templates reminiscent of c-shell scripts. It includes a single-particle Maximum Likelihood module, and 3D merging capability. 2dx builds upon the MRC programs, which were extended by additional functions to interface with the GUI of 2dx, and to implement the optionally automatic processing. Support: Operating systems: Mac OS X, Linux Image format support: MRC, TIFF, CCP4 Cost: Free/Open Source, GPL Primary Publication to Cite: Gipson B, Zeng X, Zhang ZY, Stahlberg H (2007). "2dx--user-friendly image processing for 2D crystals". J. Struct. Biol. 157 (1): 64–72. doi:10.1016/j.jsb.2006.07.020. PMID 17055742. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Additional References: Zeng X, Stahlberg H, Grigorieff N (2007). "A maximum likelihood approach to two-dimensional crystals". J. Struct. Biol. 160 (3): 362–74. doi:10.1016/j.jsb.2007.09.013. PMID 17964808. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)

## Icosahedral viruses
#### AUTO3DEM

    Website: http://cryoem.ucsd.edu/programs.shtm Current version: 2.02 Contact: sinkovit@sdsc.edu An automation system designed to accelerate the computationally intensive process of three-dimensional structure determination from images of vitrified icosahedral virus particles. With minimal user input and intervention, AUTO3DEM manages the flow of data between the major image reconstruction programs, monitors the progress of the computations, and intelligently updates the input parameters as the resolution of the model is improved. Support: Operating systems: Linux, UNIX Image format support: PIF Cost: Free/Open Source, BSD license Primary Publication to Cite: Yan X, Sinkovits RS, Baker TS (2007). "AUTO3DEM--an automated and high throughput program for image reconstruction of icosahedral particles". J. Struct. Biol. 157 (1): 73–82. doi:10.1016/j.jsb.2006.08.007. PMC 1847775. PMID 17029842. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)

## Helices
#### BBHP - Burnham-Brandeis Helical Package

    Website: http://coan.burnham.org/other-projects/ Current version: 20.0 Contact: coan@burnham.org The Brandeis Helical Package has been in use in one form or another for quite some time. Originally developed for VAX/VMS it has been modified, expanded, and ported several times. We first ported the package to Linux in 2006, using the Portland Group compilers. Subsequently we rewrote portions of the fortran code which were not supported by GNU fortran (g77) and now the package compiles and runs with gcc/g77. This has allowed us to support extra modern platforms. The re-christened Burnham-Brandeis Helical Package will now run on 32-bit Linux, 64-bit Linux, Mac OS X (Intel) and even Windows running Cygwin. At the same time, when porting to Linux, we took the opportunity to replace the main GUI portions of the package with updated programs. We had spent some time modifying the Tcl/Tk-based GUI programs Tkir (image viewer) and Tkll (layerline viewer) from SUPRIM to support a plugin architecture, where we could write new plugins to add functionality without further modifications to the core code-base. The first, tkinterp, provides an interface in Tkir for selecting layerlines, replacing the old interp program. The second, brandeisll, is a Tkll plugin to provide support for displaying the Burnham-Brandeis Helical Package "interp" layerline and bessel-function format. We've also included as part of the package, the AVID software for analyzing the variance within the helix. Support: Operating systems: Linux Image format support: MRC Cost: Free Primary Publication to Cite: Owen CH, Morgan DG, DeRosier DJ (1996). "Image analysis of helical objects: the Brandeis Helical Package". J. Struct. Biol. 116: 167–175. PMID 8742740. {{cite journal}}: Cite has empty unknown parameter: |month= (help)CS1 maint: multiple names: authors list (link) Additional References: Rost LE, Hanein D, DeRosier DJ (1998). "Reconstruction of symmetry deviations: a procedure to analyze partially decorated F-actin and other incomplete structures". Ultramicroscopy. 72: 187–197. PMID 9639941. {{cite journal}}: Cite has empty unknown parameter: |month= (help)CS1 maint: multiple names: authors list (link)

#### IHRSR

    Website: Contact: egelman@virginia.edu The iterative helical real space reconstruction (IHRSR) algorithm can recontruct helical filaments under conditions when traditional Fourier–Bessel approaches sometimes fail. For example when there is disorder or heterogeneity present, when the specimens diffract weakly, or when Bessel functions overlap. Support: Operating systems: Linux Image format support: SPIDER Cost: Free Primary Publication to Cite: Egelman EH (2007). "The iterative helical real space reconstruction method: surmounting the problems posed by real polymers". J. Struct. Biol. 157 (1): 83–94. doi:10.1016/j.jsb.2006.05.015. PMID 16919474. {{cite journal}}: Unknown parameter |month= ignored (help)

#### Phoelix

    Website: http://ami.scripps.edu/redmine/projects/ami/wiki/Phoelix Current version: 1.3 Contact: amisoftware@scripps.edu A set of procedures and algorithms for helical processing that we refer to as the PHOELIX package. The package was developed to provide a time-efficient and semiautomated method for determining a three-dimensional density map from a specimen with helical symmetry. The procedures which are part of PHOELIXn are drawn from the original MRC helical processing suite with extensions principally developed using the SUPRIM image processing package. The package in its current form has been optimized for the processing of actomyosin filaments but has been modified and applied to other helical structures. Support: Operating systems: Unix Image format support: MRC/suprim Cost: Free/Open Source Primary Publication to Cite: Whittaker M, Carragher BO, Milligan RA (1995). "PHOELIX: a package for semi-automated helical reconstruction". Ultramicroscopy. 58 (3–4): 245–59. PMID 7571117. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Additional References: Carragher B, Whittaker M, Milligan RA (1996). "Helical processing using PHOELIX". J. Struct. Biol. 116 (1): 107–12. doi:10.1006/jsbi.1996.0018. PMID 8742731.{{cite journal}}: CS1 maint: multiple names: authors list (link)

#### Ruby-Helix

    Website: http://structure.m.u-tokyo.ac.jp/English/software/Ruby-Helix-Page/ruby-helix.html Current version: 1.0 Contact: mkikkawa@m.u-tokyo.ac.jp Set of programs for the analysis of “helical” objects with or without a seam. Ruby-Helix is built on top of the Ruby programming language and is the first implementation of asymmetric helical reconstruction for practical image analysis. It also allows easier and semi-automated analysis, performing iterative unbending and accurate determination of the repeat length. Support: Operating systems: Fedora, Mac OS X Image format support: MRC Cost: Free/Open Source, GPL Primary Publication to Cite: Metlagel Z, Kikkawa YS, Kikkawa M (2007). "Ruby-Helix: an implementation of helical image processing based on object-oriented scripting language". J. Struct. Biol. 157 (1): 95–105. doi:10.1016/j.jsb.2006.07.015. PMID 16996276. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Additional References: Kikkawa M (2004). "A new theory and algorithm for reconstructing helical structures with a seam". J. Mol. Biol. 343 (4): 943–55. doi:10.1016/j.jmb.2004.08.051. PMID 15476812. {{cite journal}}: Unknown parameter |month= ignored (help)

#### Stokes Lab Procedures

    Website: http://skirball.med.nyu.edu/research/sb/stokeslab/image-analysis.html Contact: stokes@saturn.med.nyu.edu Image Processing Software for Helical Crystals: outlines, procedures and hints for using the MRC helical processing software which has since been modified by several groups including Chikashi Toyoshima, Nigel Unwin, David DeRosier and David Stokes. Support: Operating systems: Unix Image format support: MRC Cost: Free/Open Source Primary Publication to Cite: Unpublished

## Single particles
#### Frealign

    Website: http://emlab.rose2.brandeis.edu/ Current version: 8.08 Contact: niko @brandeis.edu Frealign provides algorithms optimized for the efficient refinement of three-dimensional reconstructions and correction for the contrast transfer function of the microscope in the determination of macromolecular structures by single particle electron microscopy. Support: Operating systems: Linux, IRIX, OSF, Mac OS X Image format support: MRC, Spider, IMAGIC Cost: Free/Open Source, GPL Primary Publication to Cite: Grigorieff N (2007). "FREALIGN: high-resolution refinement of single particle structures". J. Struct. Biol. 157 (1): 117–25. doi:10.1016/j.jsb.2006.05.004. PMID 16828314. {{cite journal}}: Unknown parameter |month= ignored (help)

#### PFT3DR

    Website: http://www.chem.byu.edu/node/807 Current version: 2 Contact: David_Belnap at byu.edu PFT3DR is package for determining orientations and origins of imaged particles and computing a three-dimensional reconstruction from the images and their assigned orientations and origins. The programs are enhanced versions of the PFT algorithm developed by Baker and Cheng (2000) and the Fourier Bessel reconstruction algorithm of Crowther et al. (1970). The original adaptations of the PFT3DR programs were specific to icosahedral viruses, but were enhanced to support nearly all symmetries found in biological particles. Other enhancements are described on the PFT3DR web site. Support: Operating systems: Unix (Linux, Mac OS X, etc.), VMS Image format support: same as Bsoft Cost: Free/Open Source Primary Publication to Cite: Baker TS, Cheng RH (1996). "A model-based approach for determining orientations of biological macromolecules imaged by cryoelectron microscopy". J. Struct. Biol. 116 (1): 120–30. doi:10.1006/jsbi.1996.0020. PMID 8742733. Additional References: Crowther RA, Amos LA, Finch JT, De Rosier DJ, Klug A (1970). "Three dimensional reconstructions of spherical viruses by fourier synthesis from electron micrographs". Nature. 226 (5244): 421–5. PMID 4314822. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Fuller SD, Butcher SJ, Cheng RH, Baker TS (1996). "Three-dimensional reconstruction of icosahedral particles--the uncommon line". J. Struct. Biol. 116 (1): 48–55. doi:10.1006/jsbi.1996.0009. PMID 8742722.{{cite journal}}: CS1 maint: multiple names: authors list (link) Bubeck D, Filman DJ, Cheng N, Steven AC, Hogle JM, Belnap DM (2005). "The structure of the poliovirus 135S cell entry intermediate at 10-angstrom resolution reveals the location of an externalized polypeptide that binds to membranes". J. Virol. 79 (12): 7745–55. doi:10.1128/JVI.79.12.7745-7755.2005. PMC 1143686. PMID 15919927. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Sanz-García, E, Stewart, AB, Belnap DM (2010). "The random-model method enables ab initio three-dimensional reconstruction of asymmetric particles and determination of particle symmetry". J. Struct. Biol. 171 (2): 216–222. doi:10.1016/j.jsb.2010.03.017. PMC 2885456. PMID 20353825.{{cite journal}}: CS1 maint: multiple names: authors list (link)

#### Search/Refine/Build

    Website: https://sites.google.com/site/rubinsteingroup/ Current version: 1.01 Contact: john.rubinstein at utoronto.ca Search_Fspace, Refine_Fspace, and Build_Fspace, are relatively simple programs for calculating and refining 3D maps. Many of the approaches used are similar to approaches applied by Frealign. The website provided also has a number of other useful utilities for working on micrographs, stacks of MRC images, and parameter files. Support: Operating systems: Linux Image format support: MRC Cost: Free/Open Source, GPL Primary Publication to Cite: Lau, W.C.Y, Rubinstein, J.L. (2012). "Subnanometre-resolution structure of the intact Thermus thermophilus H+-driven ATP synthase". Nature. 481: 214–218. PMID 22753497. {{cite journal}}: Cite has empty unknown parameter: |1= (help)CS1 maint: multiple names: authors list (link) Additional References: Benlekbir S, Bueler SA, Rubinstein JL (2012). "Structure of the vacuolar-type ATPase from Saccharomyces cerevisiae at 11-Å resolution". Nature Structural and Molecular Biology. 19 pages=1356-52. PMID 23142977. {{cite journal}}: Cite has empty unknown parameter: |1= (help); Missing pipe in: |volume= (help)CS1 maint: multiple names: authors list (link)

## Tomography
#### ASTRA Toolbox

    Website: http://www.visielab.ua.ac.be/software Contact: astra@ua.ac.be The ASTRA Toolbox is an open platform for 3D image reconstruction in tomography. The ASTRA Toolbox provides an extensive set of fast (GPU accelerated) and flexible building blocks that can be used in advanced reconstruction algorithms. Support: Operating systems: Linux, Microsoft Windows Image format support: EM,MRC,Matlab Cost: Free Primary Publication to Cite: Palenstijn WJ, Batenburg KJ, Sijbers J (2011). "Performance improvements for iterative electron tomography reconstruction using graphics processing units (GPUs)". J. Struct. Biol. 176 (2): 250–253. doi:10.1016/j.jsb.2011.07.017. PMID 21840398. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Additional References: Palenstijn WJ, Batenburg KJ, Sijbers J (2013). "The ASTRA Tomography Toolbox)" (PDF). 13th International Conference on Computational and Mathematical Methods in Science and Engineering.{{cite journal}}: CS1 maint: multiple names: authors list (link)

#### EM3D

    Website: http://em3d.stanford.edu/index.html Current version: 2.0 Contact: grantser@bio.tamu.edu EM3D is a software application designed to analyze and visualize electron microscope (EM) tomography data. It is especially for cellular and molecular biologists. From a tilt series of 2D electron micrographs taken at many tilt angles with respect to the electron beam, this program can then perform auto-align and quickly render those data into a lucid 3D model, which allows you to perform object rotating for viewing. In addition, EM3D also provides analysis tools for quantify structural information from the models, including their moments, proximity relationships, and spatial reliability. And all of these functions can be executed with a very intuited graphic user interface. EM3D is available free-of-charge Mac OS X for PowerPC or Intel, and Windows. EM3D was developed in the laboratory of Dr. U. J. McMahan, Professor of Neurobiology and of Structural Biology at Stanford University School of Medicine and continues to be developed at the Dept. of Biology, Texas A&M University. Support: Operating systems: Windows, Mac OS X for PowerPC or Intel Image format support: Cost: Free for academic use Primary Publication to Cite: Ress D, Harlow ML, Schwarz M, Marshall RM, McMahan UJ (1999). "Automatic acquisition of fiducial markers and alignment of images in tilt series for electron tomography". J Electron Microsc (Tokyo). 48 (3): 277–87. PMID 10425746EM3D's scheme for the automatic alignment of tilt-images is described.{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: postscript (link) Additional References: Ress DB, Harlow ML, Marshall RM, McMahan UJ (2004). "Methods for generating high-resolution structural models from electron microscope tomography data". Structure. 12 (10): 1763–74. doi:10.1016/j.str.2004.07.022. PMID 15458626EM3D approach to model generation is detailed. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) CS1 maint: postscript (link) Harlow ML, Ress D, Stoschek A, Marshall RM, McMahan UJ (2001). "The architecture of active zone material at the frog's neuromuscular junction". Nature. 409 (6819): 479–84. doi:10.1038/35054000. PMID 11206537EM3D is first used for exposing cellular architecture at macromolecular resolution. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) CS1 maint: postscript (link) Petersen JD, Chen X, Vinade L, Dosemeci A, Lisman JE, Reese TS (2003). "Distribution of postsynaptic density (PSD)-95 and Ca2+/calmodulin-dependent protein kinase II at the PSD". J. Neurosci. 23 (35): 11270–8. PMID 14657186EM3D is used to examine the architecture of the postsynaptic density at brain synapses. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) CS1 maint: postscript (link) Ress, D., Harlow, M.L., Marshall, R.A., and McMahan, U.J. (2003). "Optimized Method for Isodensity Surface Models Obtained with Electron Microscope Tomography Data". Proceedings of the 25th Annual International Conference of IEEE. pp. 774–777EM3D's method for creating optimal isodensity surface models is described.{{cite book}}: CS1 maint: multiple names: authors list (link) CS1 maint: postscript (link)

#### IMOD

    Website: http://bio3d.colorado.edu/imod/ Current version: 4.1 Contact: mast at colorado dot edu IMOD is a set of image processing, modeling and display programs used for tomographic reconstruction and for 3D reconstruction of EM serial sections and optical sections. The package contains tools for assembling and aligning data within multiple types and sizes of image stacks, viewing 3-D data from any orientation, and modeling and display of the image files. It includes a complete graphical user interface for generating tomograms, combining tomograms from tilt series taken around two axes, and stacking tomograms from serial sections. Support: Operating systems: Linux, Windows, Mac OS X Image format support: MRC, TIFF Cost: Free/Open Source, GPL Primary Publication to Cite: Kremer JR, Mastronarde DN, McIntosh JR (1996). "Computer visualization of three-dimensional image data using IMOD". J. Struct. Biol. 116 (1): 71–6. doi:10.1006/jsbi.1996.0013. PMID 8742726.{{cite journal}}: CS1 maint: multiple names: authors list (link) Additional References: Mastronarde DN (2008). "Correction for non-perpendicularity of beam and tilt axis in tomographic reconstructions with the IMOD package". J Microsc. 230 (Pt 2): 212–7. doi:10.1111/j.1365-2818.2008.01977.x. PMID 18445149. {{cite journal}}: Unknown parameter |month= ignored (help) Mastronarde DN (1997). "Dual-axis tomography: an approach with alignment methods that preserve resolution". J. Struct. Biol. 120 (3): 343–52. doi:10.1006/jsbi.1997.3919. PMID 9441937. {{cite journal}}: Unknown parameter |month= ignored (help)

#### protomo

    Website: http://www.electrontomography.org Current version: 1.1 Contact: protomo@electrontomography.org Protomo is a suite of programs and shell scripts primarily developed for electron tomography. It offers routines for preprocessing micrographs, CTF-correction of images of untilted and tilted specimens, marker-free alignment of tilt series, and 3D reconstruction, besides some general image processing functionality. Support: Operating systems: Linux Image format support: Most formats Cost: Free Primary Publication to Cite: Winkler H (2007). "3D reconstruction and processing of volumetric data in cryo-electron tomography". J. Struct. Biol. 157 (1): 126–37. doi:10.1016/j.jsb.2006.07.014. PMID 16973379. {{cite journal}}: Unknown parameter |month= ignored (help) Additional References: Winkler H, Taylor KA (2006). "Accurate marker-free alignment with simultaneous geometry determination and reconstruction of tilt series in electron tomography". Ultramicroscopy. 106 (3): 240–54. doi:10.1016/j.ultramic.2005.07.007. PMID 16137829. {{cite journal}}: Unknown parameter |month= ignored (help) Winkler H, Taylor KA (2003). "Focus gradient correction applied to tilt series image data used in electron tomography". J. Struct. Biol. 143 (1): 24–32. PMID 12892723. {{cite journal}}: Unknown parameter |month= ignored (help) Taylor KA, Tang J, Cheng Y, Winkler H (1997). "The use of electron tomography for structural analysis of disordered protein arrays". J. Struct. Biol. 120 (3): 372–86. doi:10.1006/jsbi.1997.3932. PMID 9441940. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)

#### PyTom

    Website: http://www.biochem.mpg.de/en/rg/foerster/Content_Software/PyTom/index.html Current version: 1.0.1 Contact: foerster@biochem.mpg.de, hrabe@biochem.mpg.de, ychen@biochem.mpg.de PyTom is a python-based package for tomogram analysis. It supports tasks such as particle localization and identification by template matching, subtomogram averaging, and subtomogram classification. The package is open source, does not rely on any commercial software, and is platform-independent. The implementation in python should allow easy scripting for specific tasks. Support: Operating systems: Linux, Mac OS, Windows Image format support: .em, .spi, .mrc Cost: Free, GPL / BSD Written In: Python Primary Publication to Cite: Hrabe T, Chen Y, Pfeffer S, Cuellar LK, Mangold AV, Förster F (2012). "PyTom: a python-based toolbox for localization of macromolecules in cryo-electron tomograms and subtomogram analysis". J Struct Biology. 178 (2): 178–88. doi:10.1016/j.jsb.2011.12.003.{{cite journal}}: CS1 maint: multiple names: authors list (link) Additional References: Chen Y, Hrabe T, Pfeffer S, Pauly O, Mateus D, Navab N, Förster F (2012). "Detection and identification of macromolecular complexes in cryo-electron tomograms using support vector machines". Biomedical Imaging (ISBI), 2012 9th IEEE International Symposium on: 1373–76. doi:10.1109/ISBI.2012.6235823.{{cite journal}}: CS1 maint: multiple names: authors list (link)

#### RAPTOR

    Website: http://www-vlsi.stanford.edu/TEM/software.htm Current version: 2.1 Contact: famat at stanford dot edu Robust Alignment and Projection Estimation for Tomographic Reconstruction (RAPTOR) is a free available software to align raw stacks obtained from electron microscopes for tomographic purposes. It is intended to automatically obtain a full-precision alignment comparable to the one obtained with extended manual intervention. Fiducial particles are needed in the image for the alignment. It has been designed to be compatible with IMOD software, so results can be checked with the usual IMOD tools for alignment and reconstruction. Support: Operating systems: Linux Image format support: MRC Cost: Free Primary Publication to Cite: Amat F, Moussavi F, Comolli LR, Elidan G, Downing KH, Horowitz M (2008). "Markov random field based automatic image alignment for electron tomography". J. Struct. Biol. 161 (3): 260–75. doi:10.1016/j.jsb.2007.07.007. PMID 17855124. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)

#### SerialEM

    Website: http://bio3d.colorado.edu/SerialEM/ Current version: 2.8 Contact: mast at colorado dot edu SerialEM is a program to acquire tilt series for electron tomography on Tecnai and newer JEOL microscopes. It uses an approach based on prediction of specimen position during the tilt series from the position at previous tilts. With this method, it achieves both the robustness of the older approach to tilt series acquisition (track and focus at every tilt) and the speed of the newer precalibration approach. It provides a complete interface for camera control and image acquisition, viewing, and saving. It includes a low dose mode for tracking and focusing away from the area of interest, energy filter control, acquisition of tilt series using a montage of overlapping frames, and a navigator module for mapping the grid and returning to selected locations. It supports Gatan, Tietz, FEI, AMT, and some Direct Electron CCD cameras. Support: Operating systems: Microsoft Windows Image format support: MRC Cost: Free for academic use Primary Publication to Cite: Mastronarde DN (2005). "Automated electron microscope tomography using robust prediction of specimen movements". J. Struct. Biol. 152 (1): 36–51. doi:10.1016/j.jsb.2005.07.007. PMID 16182563. {{cite journal}}: Unknown parameter |month= ignored (help)

#### TOM Toolbox

    Website: http://www.biochem.mpg.de/tom/ Contact: tom@biochem.mpg.de The Tomography Toolbox is a collection of functions that extend the capability of the MATLAB (+ Image Procesing Toolbox) numeric computing environment. The toolbox supports a wide range of functions for tomography. Automated data acquisition procedures have changed the perspectives of electron tomography (ET) in a profound manner. Elaborate data acquisition schemes with autotuning functions minimize exposure of the specimen to the electron beam and sophisticated image analysis routines retrieve a maximum of information from noisy data sets. ‘TOM software toolbox’ integrates established algorithms and new concepts tailored to the special needs of low dose ET. It provides a user-friendly unified platform for all processing steps: acquisition, alignment, reconstruction, and analysis. Designed as a collection of computational procedures it is a complete software solution within a highly flexible framework. TOM represents a new way of working with the electron microscope and can serve as the basis for future high-throughput applications. Support: Operating systems: Linux, Microsoft Windows Image format support: EM,MRC,Spider Cost: Free Primary Publication to Cite: Nickell S, Förster F, Linaroudis A; et al. (2005). "TOM software toolbox: acquisition and analysis for electron tomography". J. Struct. Biol. 149 (3): 227–34. doi:10.1016/j.jsb.2004.10.006. PMID 15721576. {{cite journal}}: Explicit use of et al. in: |author= (help); Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)

#### TomoJ

    Website: http://u759.curie.fr/en/download/softwares/TomoJ Current version: 2.15 Contact: tomoj@curie.fr TomoJ is a Java plug-in for ImageJ to perform tomographic reconstruction using ART, SIRT and WBP algorithms. It includes free reference alignment procedures in addition to standard cross-correlation methods in a friendly user interface. Support: Operating systems: All, same as ImageJ program Image format support: Cost: Free/Open Source, CeCILL Licence Written In: Java Primary Publication to Cite: Messaoudii C, Boudier T, Sanchez Sorzano CO, Marco S (2007). "TomoJ: tomography software for three-dimensional reconstruction in transmission electron microscopy". BMC Bioinformatics. 8: 288. doi:10.1186/1471-2105-8-288. PMC 1976622. PMID 17683598.{{cite journal}}: CS1 maint: multiple names: authors list (link) Additional References: Sorzano CO, Messaoudi C, Eibauer M; et al. (2009). "Marker-free image registration of electron tomography tilt-series". BMC Bioinformatics. 10: 124. doi:10.1186/1471-2105-10-124. PMC 2694187. PMID 19397789. {{cite journal}}: Explicit use of et al. in: |author= (help)CS1 maint: multiple names: authors list (link)

#### TxBR

    Website: https://confluence.crbs.ucsd.edu/display/ncmir/TxBR Current version: 3.0 Contact: sph at ncmir dot ucsd dot edu Tomographic reconstruction from large-format electron microscope data requires special procedures to handle geometric distortions arising from electron optics as opposed to light-ray optics. In particular, electrons travel in curvilinear paths through the sample, and defocus and other aberrations can have significant effects. TxBR handles geometric nonlinearities associated with charged-particle optics, distortion due to sample warping, and transform sets associated with non-standard modes of data collection. In addition, TxBR makes provision for marker-free alignment and specialized montaging for tomography of serial sections. Support: Operating systems: Linux, Windows (≥ XP), Mac OS X (PowerPC and Intel) Image format support: MRC Cost: Free Primary Publication to Cite: S. Phan, A. Lawrence (2008). "Tomography of Large Format Electron Microscope Tilt Series: Image Alignment and Volume Reconstruction". Congress on Image and Signal Processing, Vol. 2. pp. 176–182. Additional References: Lawrence A, Bouwer JC, Perkins G, Ellisman MH (2006). "Transform-based backprojection for volume reconstruction of large format electron microscope tilt series". J. Struct. Biol. 154 (2): 144–67. doi:10.1016/j.jsb.2005.12.012. PMID 16542854. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)

#### UCSF Tomography

    Website: http://www.msg.ucsf.edu/em/EMNEW2/tomography_page.html Current version: v7.7.4E4 Contact: agard@msg.ucsf.edu UCSF Tomography is an integrated software suite that provides full automation from target finding, sequential tomographic data collection, to real-time reconstruction for both single and dual axes as well as automated acquisition of random conical data sets. This software was implemented based upon a novel approach in which the compustage tilting is modeled as geometric rotation. The spatial movement of the sample as a result of stage tilting can be predicted based upon previously collected tomographic images. Therefore, there is no need to collect tracking and focusing images during the entire tomographic data collection. A significant dose saving can thus be achieved and is critical in collecting cryo tilt series. Real-time reconstruction is achieved by calculating a weighted back-projection on a small Linux cluster (five dual-processor computer nodes) concurrently with the UCSF tomography data collection running on the microscope’s computer, and using the fiducial-marker free alignment data generated during the data collection process. The real-time reconstructed 3D volume provides users with immediate feedback to fully asses all aspects of the experiment ranging from sample choice, ice thickness, experimental parameters to the quality of specimen preparation. To facilitate dual-axis tomographic data collection, a hierarchical scheme for target finding and relocation after specimen rotation was developed and integrated with the predictive data collection and real-time reconstruction, allowing full automation from target finding to data collection and to reconstruction of 3D volumes with little user intervention. An on-site scheme was developed for random conical data collection where tracking and focusing are performed at the same location as the final conical tilt images. Lower magnifications combined with short exposure are used to substantially reduce dose and to allow larger tilt steps. The system also includes a feature for montaging untilted images to ensure that all of the particles in the tilted image may be used in the reconstruction. Support: Operating systems: Windows 2000, XP Image format support: MRC Cost: Free for academic use Primary Publication to Cite: Zheng SQ, Keszthelyi B, Branlund E; et al. (2007). "UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction". J. Struct. Biol. 157 (1): 138–47. doi:10.1016/j.jsb.2006.06.005. PMID 16904341. {{cite journal}}: Explicit use of et al. in: |author= (help); Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Additional References: Zheng QS, Braunfeld MB, Sedat JW, Agard DA (2004). "An improved strategy for automated electron microscopic tomography". J. Struct. Biol. 147 (2): 91–101. doi:10.1016/j.jsb.2004.02.005. PMID 15193638. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Zheng SQ, Kollman JM, Braunfeld MB, Sedat JW, Agard DA (2007). "Automated acquisition of electron microscopic random conical tilt sets". J. Struct. Biol. 157 (1): 148–55. doi:10.1016/j.jsb.2006.10.026. PMC 2556511. PMID 17169745. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Zheng SQ, Matsuda A, Braunfeld MB, Sedat JW, Agard DA (2009). "Dual-axis target mapping and automated sequential acquisition of dual-axis EM tomographic data". J. Struct. Biol. 168 (2): 323–31. doi:10.1016/j.jsb.2009.06.010. PMC 2776717. PMID 19545637. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)

#### FEI Xplore3D™ Tomography Suite

    Website: http://www.fei.com/LifeSciences/ Contact: robert dot snyder at fei dot com Xplore3D provides a complete solution for 3D tomographic acquisition, reconstruction, and visualization into one integrated package. The advanced capabilities include batch acquisition, dual-axis tomography, low-dose imaging, STEM tomography, energy filtering, and hardware acceleration for reconstruction. Batch scheduling permits unattended overnight data acquisition from multiple grid positions. Accurate, interactive alignment routines can use cross-correlation (no markers needed), bead tracking, or general feature tracking. Advanced reconstruction techniques include weighted back-projection, ART, and SIRT. Xplore3D includes a post-alignment and reconstruction module, Inspect3D. Support: Operating systems: Windows XP, Image format support: most image formats Cost: Price available upon request, Perpetual right-to-use license Primary Publication to Cite: R.H.M. Schoenmakers, R.A. Perquin, T.F. Fliervoet, W. Voorhout, H. Schirmacher (2005). "New software for high resolution, high throughput electron tomography". Microscopy and Analysis. 19 (4): 5–6. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Additional References: Li Peng, Sergey Ryazantsev, Ren Sun, Z. Hong Zhou (2010). "Three-Dimensional Visualization of Gammaherpesvirus Life Cycle in Host Cells by Electron Tomography". Structure. 18 (1): 47–58. doi:10.1016/j.str.2009.10.017. PMID 20152152. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Linda F. van Driel, Jack A. Valentijn, Karine M. Valentijn, Roman I. Koning, Abraham J. Koster (2009). "Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells". European Journal of Cell Biology. 88 (11): 669–684. doi:10.1016/j.ejcb.2009.07.002. PMID 19726102. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Laura van Niftrik, Willie J.C. Geerts, Elly G. van Donselaar, Bruno M. Humbel, Alevtyna Yakushevska, Arie J. Verkleij, Mike S.M. Jetten, Marc Strous (2008). "Combined structural and chemical analysis of the anammoxosome: A membrane-bounded intracytoplasmic compartment in anammox bacteria". Journal of Structural Biology. 161 (3): 401–410. doi:10.1016/j.jsb.2007.05.005. PMID 17604181. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link)

## Subvolume averaging
#### Dynamo

    Website: http://www.dynamo-em.org Current version: 1.1.136 Contact: daniel.castano@unibas.ch Dynamo is a software package for subtomogram averaging and classification of Cryo-EM data. It is intended to provide users with big flexibility when adding new algorithms into standard procedures and adapting them for different computing environments as desktops, multicore machines, (multi)GPUs or clusters of CPUs. It includes several tools for tomogram visualization and particle picking in several geometries (as filamentes, vesicles, membranes) Support: Operating systems: Linux, Mac OS, Windows Image format support: .em, .spi, .mrc Cost: Free for academic use, GPL / BSD Primary Publication to Cite: Castaño-Díez D, Kudryashev M, Arheit M, Stahlberg H (2012). "Dynamo: A flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments". J Struct Biology (3). doi:http://dx.doi.org/10.1016/j.jsb.2011.12.017. {{cite journal}}: Check |doi= value (help); External link in |doi= (help)CS1 maint: multiple names: authors list (link)

#### Jsubtomo

    Website: http://www.opic.ox.ac.uk/jsubtomo Current version: 1.2 Contact: juha at strubi dot ox dot ac dot uk Jsubtomo is an open source software package based on Bsoft for averaging of tomographic sub-volumes. It includes tools for locating sub-volumes in tomograms using constrained cross-correlation, missing wedge-weighted averaging and refinement. Initial estimate of particle orientation can be easily included to constrain refinement. Python scripts are available for parallel processing and iterative refinement. Support: Operating systems: Linux, Mac OS, Windows Image format support: Most, including .map, .mrc, .em, .spi, .img, .pif Cost: Free Written In: C Primary Publication to Cite: Huiskonen JT, Hepojoki J, Laurinmäki P, Vaheri A, Lankinen H, Butcher SJ, Grünewald K. (2010). "Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses". J Virology. 84 (10): 4889–97. doi:10.1128/JVI.00057-10.{{cite journal}}: CS1 maint: multiple names: authors list (link)

#### PEET

    Website: http://bio3d.colorado.edu/PEET Current version: 1.8.0 Contact: john.heumann@colorado.edu PEET is an open-source package for subvolume alignment, averaging, and classification. PEET is most easily used in conjunction with IMOD. Support: Operating systems: Linux, Mac OS-X, Windows Image format support: .mrc Cost: Free, GPL Written In: Matlab, C, C++ Primary Publication to Cite: Nicastro D, Schwartz C, Pierson J, Gaudette R, Porter M, McIntosh J.R. (2006). "The molecular architecture of axonemes revealed by cryoelectron tomography". Science. 313 (5789): 944–948. doi:10.1126/science.1128618.{{cite journal}}: CS1 maint: multiple names: authors list (link)

## Mass measurements
#### MASDET

    Website: http://campus.uni-muenster.de/masdet.html Current version: 1.13 Contact: krzyzane at uni-muenster dot de A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy. MASDET offers several subroutines for the main mass analysis steps, namely image display and selection of regions of interest (ROI), mass evaluation and data analysis. Two program-modes constitute the core procedures for mass determination: (i) the program-mode AREA determines the mass-per-area of sheet-like structures (e.g., protein S-layers, thin organic and inorganic films) and the mass-per-box for globular structures (e.g., single macromolecules, globular assemblies, organic/inorganic nanoparticles), and (ii) the program-mode FILAMENT determines the mass-per-length for filamentous structures (e.g., intermediate filaments, DNA-protein complexes, tobacco mosaic virus, nanowires) in individual ROIs. The statistical analysis of the mass data in individual ROIs contains graphical data display (histograms and XY-plots) and allows up to 10 Gaussian curves to be fitted. The dark-field micrographs can also be recalculated to a mass-thickness profile and/or mass-thickness map using data from Monte Carlo simulations. Support: Operating systems: Windows (both standalone program and p-files run from MATLAB); Mac OS X, Linux (p-files to be run from MATLAB) Image format support: Basel IMAG, Muenster TIFF (other formats will be implemented by us on request) Cost: Free for academic use Primary Publication to Cite: Krzyzanek V, Müller SA, Engel A, Reichelt R (2009). "MASDET--A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy". J. Struct. Biol. 165 (2): 78–87. doi:10.1016/j.jsb.2008.10.006. PMID 19041401. {{cite journal}}: Unknown parameter |month= ignored (help)CS1 maint: multiple names: authors list (link) Additional References: Krzyzanek V, Reichelt R (2003). "MONCA: A New MATLAB Package for Monte Carlo Simulation of Electron Scattering in Thin Specimens in the Energy Range 10 - 200 keV". Microsc. Microanal. 9 (Suppl. 3): 110–111. doi:10.1017/S1431927603014065. {{cite journal}}: Cite has empty unknown parameter: |month= (help) Reichelt R, Engel A (1984). "Monte Carlo calculations of elastic and inelastic electron scattering in biological and plastic materials". Ultramicrosc. 13 (3): 279–293. doi:10.1016/0304-3991(84)90206-7. {{cite journal}}: Cite has empty unknown parameter: |month= (help)