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Journal logoSTRUCTURAL
BIOLOGY
ISSN: 2059-7983

Recent developments in the CCP-EM software suite

aScientific Computing Department, Science and Technology Facilities Council, Research Complex at Harwell, Didcot OX11 0FA, England

(Received 14 May 2017; accepted 26 May 2017; online 31 May 2017)

As part of its remit to provide computational support to the cryo-EM community, the Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM) has produced a software framework which enables easy access to a range of programs and utilities. The resulting software suite incorporates contributions from different collaborators by encapsulating them in Python task wrappers, which are then made accessible via a user-friendly graphical user interface as well as a command-line interface suitable for scripting. The framework includes tools for project and data management. An overview of the design of the framework is given, together with a survey of the functionality at different levels. The current CCP-EM suite has particular strength in the building and refinement of atomic models into cryo-EM reconstructions, which is described in detail.

1. Introduction

The Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM) was initiated in 2012 to support the computational needs of the macromolecular electron cryo-microscopy (cryo-EM) community. To this end, it aims to support both software developers and users in a manner analogous to the way in which the long-running Collaborative Computational Project, Number 4 (CCP4; Winn et al., 2011[Winn, M. D. et al. (2011). Acta Cryst. D67, 235-242.]) has supported the macromolecular crystallography (MX) community. CCP-EM is mandated to provide user training and developer support and to establish a coherent community for the exchange of best practices and novel ideas.

The creation of the CCP-EM project has been described previously (Wood et al., 2015[Wood, C., Burnley, T., Patwardhan, A., Scheres, S., Topf, M., Roseman, A. & Winn, M. (2015). Acta Cryst. D71, 123-126.]). In this contribution, we specifically address the CCP-EM software suite: a multi-platform suite of tools that, in time, aims to cover all aspects of cryo-EM data processing from image manipulation to the building of atomic models, and to cover multiple techniques such as single-particle reconstruction (SPR), tomography and diffraction. Packaging tools together allows better management of structural biology projects, as well as better distribution and testing of software, to the benefit of both users and developers. Here, we describe the development of the CCP-EM software suite and its initial functionality.

The CCP-EM software suite was conceived as a generic framework that could support a wide variety of functionalities, whether written by ourselves or provided by external programs. As a collaborative project, the ability to incorporate programs from external partners is a high priority. There are clear conceptual similarities to other frameworks such as Appion (Lander et al., 2009[Lander, G. C., Stagg, S. M., Voss, N. R., Cheng, A., Fellmann, D., Pulokas, J., Yoshioka, C., Irving, C., Mulder, A., Lau, P.-W., Lyumkis, D., Potter, C. S. & Carragher, B. (2009). J. Struct. Biol. 166, 95-102.]), Scipion (de la Rosa-Trevín et al., 2016[Rosa-Trevín, J. M. de la et al. (2016). J. Struct. Biol. 195, 93-99.]) and Focus (Biyani et al., 2017[Biyani, N., Righetto, R. D., McLeod, R., Caujolle-Bert, D., Castano-Diez, D., Goldie, K. N. & Stahlberg, H. (2017). J. Struct. Biol. 198, 124-133.]). One unique feature of the CCP-EM framework is its close connection to the highly successful CCP4 suite for macromolecular crystallography, which arises out of historic links. This connection allows the reuse of software-engineering technologies deployed previously for CCP4, and the easy incorporation of crystallographic programs for the interpretation of high-resolution reconstructions. Nevertheless, the CCP-EM suite is distinct from CCP4 and is firmly directed towards the cryo-EM community.

A public beta release of the CCP-EM suite was made in 2016, and has been used since then in several CCP-EM training courses. The initial focus has been on fitting, building and refining of atomic models into high- or medium-resolution single-particle reconstructions. This focus partly reflects our historical links to the macromolecular crystallography community, but is also timely given the recent `resolution revolution' (Kühlbrandt, 2014[Kühlbrandt, W. (2014). Science, 343, 1443-1444.]). The CCP-EM suite aims to assist microscopists who are perhaps obtaining high-resolution structures for the first time, and may be unfamiliar with topics such as reciprocal-space refinement or the use of structural restraints. Equally, for the many crystallographers who are moving into the cryo-EM field, the suite aims to help them adapt familiar tools to new data sets. In all cases, the CCP-EM suite provides convenient pipelining and data-management tools, which are becoming essential as cryo-EM moves to become a high-throughput and widespread technique (Stuart et al., 2016[Stuart, D. I., Subramaniam, S. & Abrescia, N. G. A. (2016). Nature Methods, 13, 607-608.]).

In the following section, we describe the generic software framework and the design decisions that have been made. We cover the underlying software libraries which support applications visible to the general user, as well as facilitating further development. We then go on to give an overview of the current functionality, including small utilities and major programs. We finish with a quick discussion of future plans.

2. Software framework

The CCP-EM software framework is primarily written in Python. Python is an interpreted language that is widely used in the scientific community; examples in structural biology include CCP4 (Winn et al., 2011[Winn, M. D. et al. (2011). Acta Cryst. D67, 235-242.]), PHENIX (Adams et al., 2010[Adams, P. D. et al. (2010). Acta Cryst. D66, 213-221.]), PyMOL (Schrödinger), Scipion (de la Rosa-Trevín et al., 2016[Rosa-Trevín, J. M. de la et al. (2016). J. Struct. Biol. 195, 93-99.]) and DIALS (Waterman et al., 2016[Waterman, D. G., Winter, G., Gildea, R. J., Parkhurst, J. M., Brewster, A. S., Sauter, N. K. & Evans, G. (2016). Acta Cryst. D72, 558-575.]). Its convenience and shallow learning curve have aided its popularity, and furthermore it is cross-platform as it does not require compilation.

2.1. Libraries and utilities

The CCP-EM software framework has a modular organization which can be roughly divided into three layers (Fig. 1[link]). The top-level GUI (graphical user interface) layer is written using the PyQt toolkit. This provides a simple graphical interface to the associated programs. Distinct from this is the mid-level management layer, which is written in pure Python. This provides a bridge between the GUI layer and the third layer: the set of functional programs. These programs originate from collaborating developers and are written in a wide variety of languages (including C, C++, Fortran and Python) with distinct control methods and input conventions.

[Figure 1]
Figure 1
Architecture of the CCP-EM software suite. The task wrappers and core libraries shown in green are written in pure Python, whereas the GUI layer is written in PyQt4. The GUI thread is independent of the job processes; task progress is monitored by a job-launch module and is recorded in an SQLite database. JSON files serve as intermediaries allowing the task to be controlled `headless' without the GUI layer.

The second, management layer provides Python task wrappers for each of the functional programs and gives a common interface style accessible via the GUI or the CLI (command-line interface). Command arguments are in the JSON (JavaScript Object Notation) format. This is a lightweight metadata format which is commonly used as it is more human-readable than other markup formats (for example XML) and is fully supported in the Python standard library (Fig. 2[link] shows an example JSON input file). Each specific task is derived from from two principal base classes. A CCPEMTask class provides the pure Python wrapper to the application, defining in a generic way the parameters appropriate to the task, which are then translated as inputs to the various APIs of the underlying applications. Workflows can be constructed using these wrappers, allowing tasks to be linked together and/or run in parallel. The second base class, CCPEMWindow, contains the PyQt4 functionality that provides the GUI window. Each CCPEMWindow holds an instance of the relevant CCPEMTask to allow access to the defined input arguments and trigger the activation of a job. A simple PyQt image viewer has been developed to visualize the contents of MRC-format image stacks.

[Figure 2]
Figure 2
JSON files are used as a convenient, human-readable store of parameters and provide a consistent input for CCP-EM-supported applications. In this example, input parameters for a MOLREP job are shown, including the use of a spherically averaged phase translation function and searching for two copies of the search model in the EM volume.

The software framework has a suite of unit tests to ensure the reliability and reusability of the codebase, and the support programs also have a series of implementation tests to allow autonomous testing of the suite before distribution. It should be noted that the suite has a number of third-party dependencies. These have been selected with care, and are mainstream and well maintained. Every effort has been made to ensure the modularity of the framework such that in the event of a dependency becoming unavailable or unsuitable it could be substituted with an equivalent, either sourced from another third party or developed in-house.

2.2. Python MRC file library

The MRC file format is one of the principal formats for cryo-EM data, and is used in common programs such as RELION (Scheres et al., 2008[Scheres, S. H. W., Núñez-Ramírez, R., Sorzano, C. O. S., Carazo, J. M. & Marabini, R. (2008). Nature Protoc. 3, 977-990.]) and for the deposition of experimental volumes in the EMDB (Tagari et al., 2002[Tagari, M., Newman, R., Chagoyen, M., Carazo, J. M. & Henrick, K. (2002). Trends Biochem. Sci. 27, 589.]; Lawson et al., 2016[Lawson, C. L., Patwardhan, A., Baker, M. L., Hryc, C., Garcia, E. S., Hudson, B. P., Lagerstedt, I., Ludtke, S. J., Pintilie, G., Sala, R., Westbrook, J. D., Berman, H. M., Kleywegt, G. J. & Chiu, W. (2016). Nucleic Acids Res. 44, D396-D403.]). Closely related to the CCP4 map format, it can be used to store individual micrographs, stacks of two-dimensional images, three-dimensional volumes and stacks of three-dimensional volumes. Several variants of the MRC format had emerged, but recently the developers of several major EM software packages agreed a standardized definition, known as the MRC2014 format (Cheng et al., 2015[Cheng, A., Henderson, R., Mastronarde, D., Ludtke, S. J., Schoenmakers, R. H., Short, J., Marabini, R., Dallakyan, S., Agard, D. & Winn, M. D. (2015). J. Struct. Biol. 192, 146-150.]), together with a process for agreeing future revisions. In order to allow developers to use MRC files as easily and flexibly as possible, we have written mrcfile.py, which is an open-source, stand-alone Python library for the reading, writing and validation of MRC2014 files. It is available in the ccpem-python environment (see §[link]2.3), but can also be obtained separately from PyPI (https://pypi.python.org/pypi/mrcfile) or GitHub (https://github.com/ccpem/mrcfile).

The main design goals of the mrcfile Python library are to make data from an MRC file available as a NumPy array (van der Walt et al., 2011[Walt, S. van der, Colbert, S. C. & Varoquaux, G. (2011). Comput. Sci. Eng. 13, 22-30.]) via a clear and simple interface, and to allow easy validation of MRC files against the MRC2014 standard. Python's standard file-handling semantics are used as far as possible; for example, MRC files are opened by calling the mrcfile.open() function and closed after use by calling close(). The file header and data arrays are simply accessed via header and data fields on the open MRC file object. Files can be validated for compliance with the MRC2014 standard using the mrcfile.validate() function. Other features include seamless support of gzip-compressed files (as used for maps downloaded from the EMDB) and a memory-mapped file option for fast random access to small chunks of very large files. To make it as simple as possible to install and use, mrcfile is written in pure Python (fully compatible with Python versions 2 and 3) and its only dependency is NumPy. A brief example of its usage is shown in Fig. 3[link]. A full usage guide and a description of the underlying design are available in the online documentation (http://mrcfile.readthedocs.org/).

[Figure 3]
Figure 3
Basic usage of the mrcfile Python library. In this example, a compressed map downloaded from the EMDB is opened and a 2 × 3 slice of data is taken from it. A new MRC file is then created, the data are copied into it and checked, and the file is closed. Finally, the file is validated to confirm that it complies with the MRC2014 standard.

2.3. Python toolkit for EM

A number of other Python modules are available within the CCP-EM software framework. These are used internally in the suite, but may also act as a useful toolkit for programmers wishing to write their own scripts. Currently, Python 2.7.11 is packaged with the suite along with specific libraries developed by CCP-EM and collaborators and additional open-source dependencies. The latter include common scientific modules such as NumPy (van der Walt et al., 2011[Walt, S. van der, Colbert, S. C. & Varoquaux, G. (2011). Comput. Sci. Eng. 13, 22-30.]), SciPy (Jones et al., 2001[Jones, E. et al. (2001). SciPy. http://www.scipy.org.]), Biopython (Cock et al., 2009[Cock, P. J. A., Antao, T., Chang, J. T., Chapman, B. A., Cox, C. J., Dalke, A., Friedberg, I., Hamelryck, T., Kauff, F., Wilczynski, B. & de Hoon, M. J. L. (2009). Bioinformatics, 25, 1422-1423.]) and the Python imaging library Pillow (https://python-pillow.org). This Python `ecosystem' can be accessed by invoking ccpem-python from the command line.

Led by the University of York, clipper-python has been developed to provide Python bindings to the established C++ Clipper library (Cowtan, 2003[Cowtan, K. (2003). IUCr Comput. Comm. Newsl. 2, 4-9.]), which underpins a number of CCP4 programs such as Buccaneer (Cowtan, 2006[Cowtan, K. (2006). Acta Cryst. D62, 1002-1011.]) and which is also used in Coot (Emsley et al., 2010[Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486-501.]). The Clipper library was originally developed to aid the organization of crystallo­graphic data and enhance the performance of crystallographic computation, and as such has many features that are applicable to EM data processing, in particular for high-resolution model building. Of particular relevance is the NXmap class, which is a noncrystallographic map class that stores a map of an arbitrary data type. In contrast to the Xmap crystallo­graphic map class, it is finite in extent and has no symmetry, and is therefore appropriate for EM volumes derived from SPR or tomography (Cowtan, 2003[Cowtan, K. (2003). IUCr Comput. Comm. Newsl. 2, 4-9.]). clipper-python exposes many of the C++ arrays as pythonic NumPy arrays; this in turn conveniently links the specific objects found in MX and EM (such as map volumes) to the NumPy library for the rapid development and deployment of new algorithms.

ConKit (Simkovic et al., 2017[Simkovic, F., Thomas, J. M. H. & Rigden, D. J. (2017). Bioinformatics, https://doi.org/10.1093/bioinformatics/btx148.]), developed at the University of Liverpool, is a Python interface for the analysis, manipulation and visualization of evolutionary contact predictions from several alternative algorithms: HHblits (Remmert et al., 2012[Remmert, M., Biegert, A., Hauser, A. & Söding, J. (2012). Nature Methods, 9, 173-175.]), JackHMMER (Johnson et al., 2010[Johnson, S., Eddy, S. R. & Portugaly, E. (2010). BMC Bioinformatics, 11, 431-438.]), HHfilter (Remmert et al., 2012[Remmert, M., Biegert, A., Hauser, A. & Söding, J. (2012). Nature Methods, 9, 173-175.]), CCMpred (Seemayer et al., 2014[Seemayer, S., Gruber, M. & Söding, J. (2014). Bioinformatics, 30, 3128-3130.]), PSICOV (Jones et al., 2012[Jones, D. T., Buchan, D. W. A., Cozzetto, D. & Pontil, M. (2011). Bioinformatics, 28, 184-190.]) and bbcontacts (Andreani & Söding, 2015[Andreani, J. & Söding, J. (2015). Bioinformatics, 31, 1729-1737.]). This library facilitates the inclusion of additional structure restraints inferred from deep-sequencing data, based on contact predictions made by external programs and provided in one of a number of data formats. Initial efforts using this approach for guiding models into cryo-EM density have proved successful (Schep et al., 2016[Schep, D. G., Zhaoa, J., John, L. & Rubinstein, J. L. (2016). Proc. Natl Acad. Sci. USA, 113, 3245-3250.]).

3. Graphical user interface and job management

Initial interfaces have been provided for a series of model-building tools applicable to high-to-medium-resolution (<10 Å) volumes. These tasks can be accessed via the control GUI, as stand­alone task GUIs or via the CLI. The control GUI (Fig. 4[link]) contains simple project-management utilities allowing users to create new projects, record a chronological list of jobs and monitor the status of ongoing processes. Details of projects and jobs are stored in an SQLite database that is integrated via PyQt4/Qt4 bindings. The GUI is designed such that jobs are launched as detached, separate processes, so that the main GUI thread can be launched and terminated without interfering with long-running subtasks that are launched from it.

[Figure 4]
Figure 4
CCP-EM project and task window. Top: CCP-EM project window showing the taskbar which is used to launch applications on the left and the project job history on the right. Bottom: example of the CCP-EM DockEM task. The toolbar at the top gives rapid access to molecular-graphics programs, job files, documentation and job launch. The input parameter setup tab is shown below, with required inputs highlighted in red. Additional launcher and results tabs appear as the job is launched and completed, respectively.

Each new project created by a user is stored in a separate directory, and child tasks of that project are stored in individual subdirectories. The top project directory stores the SQLite database file used to record the following information for each task: incremental job number, date and time of job initiation and completion, task type, task name, job location and current status. Clicking on the task entry launches its task window. The left-hand toolbar launches a new instance of the selected task. If Test mode is selected, new tasks will be preloaded with parameters and data from that task's unit test to allow new users to trial an application and examine the expected output.

Each task window has a similar basic layout (Fig. 4[link]), with a toolbar and four main tabs: Setup, Pipeline, Launcher and Results. The toolbar provides a Run button for launching the task, a New button for cloning the current task (i.e. preserving any defined inputs) and a Load button for opening previous runs. The Coot, CCP4mg and Chimera buttons provide quick links to commonly used molecular-graphics programs. The Terminal and Output buttons launch a terminal or file-browser instance in the task's subdirectory to allow rapid file navigation, whilst the Info button displays a brief description of the task and provides clickable hyperlinks to the task's online documentation. Finally, there is a Kill button for terminating the task and a green hexagon status indicator. The status indicator is grey when ready, spins when running and is coloured green on successful completion of the task or red upon failure.

The Setup tab allows input parameters to be entered by the user. Appropriate defaults are used wherever possible and required user input is highlighted in red. Each input has a tooltip, which is visible upon mouse hover and gives a brief description to aid new users. Programs originally developed for the CCP4 suite have keyworded input for specifying extended functionality where appropriate.

The Pipeline tab shows the individual jobs that make up the task. For example, in the MOLREP task three processes are launched in series. Initially SFCHECK (Vaguine et al., 1999[Vaguine, A. A., Richelle, J. & Wodak, S. J. (1999). Acta Cryst. D55, 191-205.]) analyzes the input .mrc file, followed by the main MOLREP (Vagin & Teplyakov, 2010[Vagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22-25.]) process and finally a third SFCHECK process comparing the fitted structure with the input map. The status of each subprocess is colour-coded: grey for ready, blue for running and green for finished. Clicking on each job displays information in the right-hand widget, including the job's log file. Double-clicking on the log file opens the text file in the user's standard editor. If the standard error file of the job process is greater than 0 bytes in size then it is also displayed to alert the user to potential problems.

The Launcher tab highlights important files associated with the task, for example in the REFMAC (Murshudov et al., 2011[Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355-367.]) refinement task the input PDB and map files are shown along with the refined output PDB file. A brief description of each file is given and double-clicking will launch an appropriate application to view the file. For standard files (for example text or PDF files) the user's normal desktop application will be launched, while for structural biology files (e.g. coordinate files or map volumes) the user can select their preferred molecular-graphics (MG) program from a list of those available. Clicking Open Selected will open all selected files at once, allowing the rapid visualization of the results of a task.

Molecular-graphics integration is provided for three of the most common MG programs: Coot (Emsley et al., 2010[Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486-501.]), Chimera (Pettersen et al., 2004[Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. & Ferrin, T. E. (2004). J. Comput. Chem. 25, 1605-1612.]) and CCP4mg (McNicholas et al., 2011[McNicholas, S., Potterton, E., Wilson, K. S. & Noble, M. E. M. (2011). Acta Cryst. D67, 386-394.]). Coot is packaged with CCP-EM, while the others are used if available. (CCP-EM searches the system paths to find the expected MG executable, or users can explicitly set this path via the CCP-EM settings.) In the simplest cases, tasks will launch the MG program and the selected files will be automatically loaded. However, for some tasks specialized run scripts have been produced. For example, the MRC to MTZ task loads all calculated map coefficients in Coot so that different degrees of sharpening and blurring can be compared (see §[link]4.8), the TEMPy:DiffMap task loads scaled maps into Chimera and the DockEM task allows selections of best hits to be displayed in Chimera.

Finally, the Results tab uses a PyQt Webkit widget to display an HTML file of the task's results. This HTML file is produced by the jsrview package from CCP4 (Winn et al., 2011[Winn, M. D. et al. (2011). Acta Cryst. D67, 235-242.]) via its Python bindings pyrvapi (log files from CCP4-derived programs are pre-processed using the CCP4 smartie library; Briggs, 2007[Briggs, P. (2007). CCP4 Newsl. Protein Crystallogr. 46, contribution 5.]). The jsrview package was initially developed by E. Krissinel to support dynamic HTML output in CCP4's jsPISA webserver (Krissinel, 2015[Krissinel, E. (2015). Nucleic Acids Res. 43, W314-W319.]). This package allows a developer to create dynamic and interactive HTML pages using a library of high-level C functions featuring various graphical widgets (such as plots, molecular graphics, tables, buttons, comboboxes etc.) and nested layouts (tabs, folders and grids). The functions generate a task file with a pseudo-program for a real-time JavaScript interpreter, based on jQuery, which is loaded in the browser widget using a bootstrap HTML page. The package may be used in programs written in C, C++ and Fortran, as well as Python through the set of corresponding bindings. The resulting output may be served either from the local file system or via a remote server. This would allow CCP-EM to efficiently transfer to web-based applications in the future if required.

4. CCP-EM tasks

The initial set of applications in the CCP-EM suite is focused primarily on model building into volumes derived from single-particle reconstruction and high-resolution subtomogram averaging. Fig. 5[link] shows in detail the task for refinement of atomic models, while Fig. 6[link] gives an overview of possible routes through the suite.

[Figure 5]
Figure 5
CCP-EM REFMAC task for the refinement and validation of atomic models in high-resolution cryo-EM maps. The single task includes the generation of structure factors from an input reconstruction, as well as the application of multiple blurring and sharpening factors. The left panel shows the CCP-EM pipeline for refinement and validation against experimental half-maps. The centre panel shows the results tab and the right panel shows the input and refined model in Coot.
[Figure 6]
Figure 6
Model-building pipeline in CCP-EM. For maps (or segments thereof) with resolutions of less than ∼5 Å and an appropriate model it is suggested to try DockEM followed by Flex-EM. For higher resolution data MOLREP and REFMAC can be used for refinement if a suitable model is available. If no model is available then Buccaneer can be used to build a model de novo. Note that for medium-resolution data sets a combination of approaches is recommended.

If a user has an appropriate atomic model available, such as a homologous domain from a high-resolution crystal structure, then either MOLREP (Vagin & Teplyakov, 2010[Vagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22-25.]) or DockEM (Roseman, 2000[Roseman, A. M. (2000). Acta Cryst. D56, 1332-1340.]) can be used to dock the structure into the cryo-EM volume. If no atomic model is available then Buccaneer (Cowtan, 2006[Cowtan, K. (2006). Acta Cryst. D62, 1002-1011.]) can be used for de novo model building. The next step is to refine the structure, i.e. to optimize the fit of the atomic model to both the experimental volume and the established stereochemical restraints. Either Flex-EM (Topf et al., 2008[Topf, M., Lasker, K., Webb, B., Wolfson, H., Chiu, W. & Sali, A. (2008). Structure, 16, 295-307.]) or REFMAC (Murshudov et al., 2011[Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355-367.]) can be employed here. Initially developed for low-resolution crystallography, additional structural restraints can be helpful when the information content in the map density is low. ProSMART (Nicholls et al., 2012[Nicholls, R. A., Long, F. & Murshudov, G. N. (2012). Acta Cryst. D68, 404-417.]) can used to generate such additional restraints for REFMAC. Flex-EM requires rigid-body definitions and these can be produced using the helper program RIBFIND (Pandurangan & Topf, 2012a[Pandurangan, A. P. & Topf, M. (2012a). J. Struct. Biol. 177, 520-531.],b[Pandurangan, A. P. & Topf, M. (2012b). Bioinformatics, 28, 2391-2393.]). CCP-EM also includes the TEMPy library (Farabella et al., 2015[Farabella, I., Vasishtan, D., Joseph, A. P., Pandurangan, A. P., Sahota, H. & Topf, M. (2015). J. Appl. Cryst. 48, 1314-1323.]), and task interfaces for TEMPy:DiffMap (difference map) and TEMPy:SMOC (Segment-based Manders' Overlap Coefficient) for structural validation are provided.

Other general utility tasks that are provided include MRC to MTZ for the conversion of map files to structure factors, and MRCEdit for viewing and editing MRC header information. CCP-EM also maintains the MRC image-processing system (Crowther et al., 1996[Crowther, R. A., Henderson, R. & Smith, J. M. (1996). J. Struct. Biol. 116, 9-16.]), and GUIs are provided for the mrc2tif and mrcallspacea routines.

4.1. MOLREP

MOLREP (Vagin & Teplyakov, 2010[Vagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22-25.]) was originally developed as an automated program for molecular replacement in CCP4 and has since been adapted for rigid-body docking into cryo-EM maps. It was extensively used by Amunts et al. (2014[Amunts, A., Brown, A., Bai, X.-C., Llácer, J. L., Hussain, T., Emsley, P., Long, F., Murshudov, G., Scheres, S. H. W. & Ramakrishnan, V. (2014). Science, 343, 1485-1489. (2014). ]) for the placement of homologous structures into the yeast mitochondrial large ribosomal subunit and other large complexes (for further methodological details, see Brown et al., 2015[Brown, A., Long, F., Nicholls, R. A., Toots, J., Emsley, P. & Murshudov, G. (2015). Acta Cryst. D71, 136-153.]). MOLREP works in reciprocal space, is relatively fast and is best suited to high-resolution maps. Although by default the single best-fit structure is returned, multiple solutions can be examined by specifying the number of copies expected in the cryo-EM volume. MOLREP uses a rotation function with a phased translation function. A spherically averaged phased translation function can also be used in which the centre of mass of the input model is found before optimizing the orientation (Vagin & Isupov, 2001[Vagin, A. A. & Isupov, M. N. (2001). Acta Cryst. D57, 1451-1456.]), which can be advantageous when using distantly related search models or with lower resolution maps.

4.2. DockEM

DockEM (Roseman, 2000[Roseman, A. M. (2000). Acta Cryst. D56, 1332-1340.]) is an exhaustive rigid-body docking algorithm. With a defined angular sweep, all possible orientations of the search model are examined within a defined area of the target map and the cross-correlation (CC) score is calculated. The top ten best poses are returned (as ranked by CC) and can be selected to be viewed in Chimera. Once the DockEM scoring has been completed, the analysis can be repeated to return more poses and/or the peak radius, which defines the exclusion threshold for neighbouring solutions, can be altered.

Owing to the exhaustive nature of DockEM it is slower than MOLREP, but it is useful for low-resolution data sets where MOLREP cannot produce an unambiguous solution. The scoring function, along with visualization in Chimera, allows users to consider the relative quality of multiple possible fits.

4.3. Buccaneer

Buccaneer (Cowtan, 2006[Cowtan, K. (2006). Acta Cryst. D62, 1002-1011.]) is used for automated de novo model building and originates from the CCP4 suite. Required inputs are an EM map (MRC format) and an expected sequence (FASTA format or similar). Partial PDB models can also be specified for extension by the program. The Buccaneer pipeline runs multiple iterations of statistical chain tracing (identifying connected Cα atoms and docking sequence) followed by coordinate refinement using REFMAC. Currently, input maps should have a resolution of ∼5 Å or better. Users can adjust the overall sharpening level, and this can significantly improve the number of residues that are able to be correctly positioned. Models built by Buccaneer can be loaded into Coot for validation and further manual model building.

4.4. Flex-EM

Flex-EM (Topf et al., 2008[Topf, M., Lasker, K., Webb, B., Wolfson, H., Chiu, W. & Sali, A. (2008). Structure, 16, 295-307.]; Joseph et al., 2016[Joseph, A. P., Malhotra, S., Burnley, T., Wood, C., Clare, D. K., Winn, M. & Topf, M. (2016). Methods, 100, 42-49.]) provides flexible fitting of rigid-body domains against EM volumes using real-space restraints. It requires an atomic model that has been rigid-body fitted and the corresponding map. Rigid-body domains must also be supplied, either defined manually or by using the RIBFIND task. Flex-EM is suitable for medium-resolution data sets, ∼15 Å or better, and two modes of refinement are available: conjugate-gradient minimization (CG) and simulated-annealing molecular dynamics (MD). By default, Ramachandran φψ restraints are included, but these can be switched off as required. The CCF score (real-space cross-correlation) is recorded per iteration and is shown in the Results tab. Flex-EM is an extension of Modeller (Webb & Sali, 2016[Webb, B. & Sali, A. (2016). Curr. Protoc. Bioinform. 54, 5.6.1-5.6.37.]) and requires Modeller to be installed separately (https://www.salilab.org/modeller/).

4.5. RIBFIND

RIBFIND was developed by Pandurangan & Topf (2012a[Pandurangan, A. P. & Topf, M. (2012a). J. Struct. Biol. 177, 520-531.],b[Pandurangan, A. P. & Topf, M. (2012b). Bioinformatics, 28, 2391-2393.]) to cluster rigid-body domains for Flex-EM. DSSP (Kabsch & Sander, 1983[Kabsch, W. & Sander, C. (1983). Biopolymers, 22, 2577-2637.]) is used to identify secondary-structure elements (SSEs) via neighbourhood-based clustering. The number of SSEs generated by the clustering is tuneable via two spatial proximity parameters: the residue-contact distance, which represents the contact between any two residues (side-chain centroid–centroid distance) enforcing the minimum distance between the clustered SSEs, and the cluster cutoff, which clusters any two SSEs based on the percentage of residues in contact with them. The contact distance is set as an a priori input and the cluster cutoff can be tuned visually, post-processing, using a JSmol (Hanson et al., 2013[Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. & Sussman, J. L. (2013). Isr. J. Chem. 53, 207-216.]) plugin widget.

4.6. REFMAC

REFMAC has been used extensively for high-resolution structure refinement of macromolecules against X-ray crystallographic data (Murshudov et al., 2011[Murshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355-367.]). It has recently been re-optimized for use with high-resolution EM maps from SPRs (Brown et al., 2015[Brown, A., Long, F., Nicholls, R. A., Toots, J., Emsley, P. & Murshudov, G. (2015). Acta Cryst. D71, 136-153.]). REFMAC uses a maximum-likelihood target function to simultaneously optimize the agreement of the input model with the experimental density and with expected stereochemical restraints. Here, the experimental gradients are calculated in reciprocal space. This has several advantages compared with real-space refinement beyond the convenience of use for pre-existing MX-derived applications, which include the following: all parameters can be refined against all data, resolution-dependent weighting can be applied, and overall quality metrics, in particular Fourier shell correlation (FSC), become available (Brown et al., 2015[Brown, A., Long, F., Nicholls, R. A., Toots, J., Emsley, P. & Murshudov, G. (2015). Acta Cryst. D71, 136-153.]). Real-space refinement also has advantages such as local parameter optimization and rapid local adjustments (for example rotamer searches), and allows user visualization and intervention. Therefore, it is recommended that REFMAC is used in concert with the real-space refinement and validation routines available in Coot, and the CCP-EM GUI provides quick launching from the REFMAC task window for this.

For use with EM data, the REFMAC process itself requires some data manipulation. The CCP-EM task (Fig. 5[link]) uses the pipeline functionality to handle this automatically for the convenience of the user. The input map (.mrc) is converted internally to structure factors (.mtz) and map sharpening can be applied at this point. If selected, the `find in map' option will add a MOLREP process to the pipeline to perform rigid-body docking. The `local' refinement mode extracts the map volume around the input model coordinates and refines against this volume only (as opposed to the whole volume). This is useful for very large systems or where atomic models are only available for specific domains; however, careful manipulation is required to place the refined model back into the correct orientation (with respect to the whole volume) and this is handled automatically after refinement in the CCP-EM pipeline. If the macromolecule includes nucleic acids, LIBG (Brown et al., 2015[Brown, A., Long, F., Nicholls, R. A., Toots, J., Emsley, P. & Murshudov, G. (2015). Acta Cryst. D71, 136-153.]) can be added to the pipeline to automatically generate additional restraints to maintain base pairing and stacking. Finally, if appropriate half-maps are available after three-dimensional map reconstruction, cross-validation processes, as described by Brown et al. (2015[Brown, A., Long, F., Nicholls, R. A., Toots, J., Emsley, P. & Murshudov, G. (2015). Acta Cryst. D71, 136-153.]), are added to the pipeline and run automatically.

REFMAC has a number of other options which are helpful for EM refinement and are exposed in the CCP-EM task window. These include setting the relative weight of the experimental and stereochemical restraints (along with an option to automatically determine weights), pre-refinement map sharpening and the use of additional restraints, including jelly-body restraints or external restraints from ProSMART.

4.7. ProSMART

ProSMART (Procrustes Structural Matching Alignment and Restraint Tool) was originally developed by Nicholls et al. (2012[Nicholls, R. A., Long, F. & Murshudov, G. N. (2012). Acta Cryst. D68, 404-417.]) to aid the modelling of low-resolution X-ray structures via the generation of additional restraints, and has since been successfully applied to model refinement from cryo-EM data (Brown et al., 2015[Brown, A., Long, F., Nicholls, R. A., Toots, J., Emsley, P. & Murshudov, G. (2015). Acta Cryst. D71, 136-153.]). The external restraints generated by ProSMART can be added to REFMAC to supplement its standard dictionary of restraints. The additional interatomic distances can be generated in a number of ways: from high-resolution homologous structures, secondary-structure restraints or multiple chains within the target model, or the modelled structure can be self-restrained to the current conformation. The CCP-EM task allows the generation of these restraints, which can then be visualized in Coot and edited as required. They can be then be added to the REFMAC process via the input in the `external restraints' section of the setup task.

4.8. Map to MTZ

This task runs the `sfcalc' mode of REFMAC and has two specific roles: the conversion of real-space maps in MRC format to reciprocal-space structure factors in MTZ format, and map sharpening/blurring (Nicholls et al., 2012[Nicholls, R. A., Long, F. & Murshudov, G. N. (2012). Acta Cryst. D68, 404-417.]). Presently, the optimum sharpening coefficient (where `optimum' means maximizing the interpretable density features) cannot be analytically determined either locally or globally, although attempts are ongoing. Visual inspection of maps with different sharpening factors, however, reveal significant differences, which can impact both manual and automated model building. Therefore, the Map to MTZ CCP-EM task applies an array of sharpening factors for assessment. A Wilson plot is displayed, allowing inspection of potential truncation pathologies arising from over-sharpening (Nicholls et al., 2012[Nicholls, R. A., Long, F. & Murshudov, G. N. (2012). Acta Cryst. D68, 404-417.]), and the task is linked to Coot for visual inspection. This process is recommended for new data sets and it should be noted that local areas may have different optimal sharpening values.

4.9. TEMPy

TEMPy implements a wide variety of scoring functions for model-to-map and map-to-map fits (Vasishtan & Topf, 2011[Vasishtan, D. & Topf, M. (2011). J. Struct. Biol. 174, 333-343.]; Farabella et al., 2015[Farabella, I., Vasishtan, D., Joseph, A. P., Pandurangan, A. P., Sahota, H. & Topf, M. (2015). J. Appl. Cryst. 48, 1314-1323.]; Joseph et al., 2017[Joseph, A. P., Lagerstedt, I., Patwardhan, A., Topf, M. & Winn, M. D. (2017). J. Struct. Biol. 195, 294-305.]), as well as other functions for map and model manipulations. It was designed as a Python library with a series of command-line scripts for useful routines. The CCP-EM suite currently has two TEMPy interfaces: TEMPy:DiffMap and TEMPy:SMOC. TEMPy:DiffMap produces difference density maps via the scaled subtraction of two experimental maps or an experimental map and a coordinate model (where the model is converted into a calculated volume). TEMPy:SMOC (Segment-based Manders' Overlap Coefficient) is a local validation metric that produces a correlation score per residue calculated on segments of overlapping residue windows. This method of calculating localized fit to density can alert users to areas of a model which require attention, and is particularly suited to medium-resolution data sets (∼4–10 Å).

4.10. MRC image-processing system

The MRC–LMB have provided a comprehensive software library for EM since the early days (Crowther et al., 1996[Crowther, R. A., Henderson, R. & Smith, J. M. (1996). J. Struct. Biol. 116, 9-16.]), together with a large set of programs and utilities (for example XIMDISP; Smith, 1999[Smith, J. M. (1999). J. Struct. Biol. 125, 223-228.]). CCP-EM has taken over long-term maintenance of this software and it is distributed as part of the suite. The majority of the routines are written in Fortran and are available via a CLI; however, two programs (mrc2tif and mrcallspacea) have CCP-EM GUIs and more will be produced if requested by the community.

5. Availability and future plans

Currently, CCP-EM binary installations are available to download from http://www.ccpem.ac.uk and are available for 64-bit Linux and Apple platforms, with plans to extend to 64-bit Windows in the near future. The Linux distributions are built on a nightly basis using a Jenkins CI (continuous integration) platform hosted by SESC Build Service (STFC). This autonomous system compiles the code, runs the unit tests and reports the status of the distribution, highlighting any potential errors that have inadvertently been introduced. This allows the rapid development and deployment of new functionality.

Here, we have described the current functionality of the CCP-EM software suite, which is focused on the fitting and building of atomic models, while also providing generic tools for manipulating and visualizing image and volume data. We are currently working on extending the range of applications for single-particle reconstruction, and have plans to cover subtomogram averaging as well. We are working closely with the EMDB (Patwardhan, 2017[Patwardhan, A. (2017). Acta Cryst. D73, 503-508.]) on providing tools for map and model validation as part of structure determination, i.e. to be applied prior to deposition (Henderson et al., 2012[Henderson, R. et al. (2012). Structure, 20, 205-214.]). Documentation on the CCP-EM software is available from the website, and further information, feedback and user questions can be obtained from the mailing list at http://www.jiscmail.ac.uk/CCPEM. Software developers who wish to discuss including their programs in the CCP-EM distribution should contact the authors of this article.

Acknowledgements

We would like to thank the UK Medical Research Council for the award of Partnership Grants MR/J000825/1 and MR/N009614/1 to support the work of CCP-EM. We thank Alan Roseman, Maya Topf, Kevin Cowtan, Garib Murshudov, Oleg Kovalevskiy, Ardan Patwardhan, Eugene Krissinel, Jude Short, Agnel Joseph, Arun Prasad Pandurangan and many others for their contributions to the suite. We also thank the Research Complex at Harwell for hosting the core CCP-EM team, and the EPSRC-funded CCPForge service for hosting the developmental version of the CCP-EM software. Finally, we are very grateful to the members of the cryo-EM community for their support of the project.

Funding information

Funding for this research was provided by: Medical Research Council (award Nos. MR/J000825/1, MR/N009614/1).

References

First citationAdams, P. D. et al. (2010). Acta Cryst. D66, 213–221.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationAmunts, A., Brown, A., Bai, X.-C., Llácer, J. L., Hussain, T., Emsley, P., Long, F., Murshudov, G., Scheres, S. H. W. & Ramakrishnan, V. (2014). Science, 343, 1485–1489. (2014).   CrossRef CAS PubMed Google Scholar
First citationAndreani, J. & Söding, J. (2015). Bioinformatics, 31, 1729–1737.  Web of Science CrossRef CAS PubMed Google Scholar
First citationBiyani, N., Righetto, R. D., McLeod, R., Caujolle-Bert, D., Castano-Diez, D., Goldie, K. N. & Stahlberg, H. (2017). J. Struct. Biol. 198, 124–133.  CrossRef CAS PubMed Google Scholar
First citationBriggs, P. (2007). CCP4 Newsl. Protein Crystallogr. 46, contribution 5.  Google Scholar
First citationBrown, A., Long, F., Nicholls, R. A., Toots, J., Emsley, P. & Murshudov, G. (2015). Acta Cryst. D71, 136–153.  Web of Science CrossRef IUCr Journals Google Scholar
First citationCheng, A., Henderson, R., Mastronarde, D., Ludtke, S. J., Schoenmakers, R. H., Short, J., Marabini, R., Dallakyan, S., Agard, D. & Winn, M. D. (2015). J. Struct. Biol. 192, 146–150.  CrossRef PubMed Google Scholar
First citationCock, P. J. A., Antao, T., Chang, J. T., Chapman, B. A., Cox, C. J., Dalke, A., Friedberg, I., Hamelryck, T., Kauff, F., Wilczynski, B. & de Hoon, M. J. L. (2009). Bioinformatics, 25, 1422–1423.  CrossRef PubMed CAS Google Scholar
First citationCowtan, K. (2003). IUCr Comput. Comm. Newsl. 2, 4–9.  Google Scholar
First citationCowtan, K. (2006). Acta Cryst. D62, 1002–1011.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationCrowther, R. A., Henderson, R. & Smith, J. M. (1996). J. Struct. Biol. 116, 9–16.  CrossRef CAS PubMed Web of Science Google Scholar
First citationEmsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486–501.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationFarabella, I., Vasishtan, D., Joseph, A. P., Pandurangan, A. P., Sahota, H. & Topf, M. (2015). J. Appl. Cryst. 48, 1314–1323.  CrossRef CAS IUCr Journals Google Scholar
First citationHanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. & Sussman, J. L. (2013). Isr. J. Chem. 53, 207–216.  Web of Science CrossRef CAS Google Scholar
First citationHenderson, R. et al. (2012). Structure, 20, 205–214.  Web of Science CrossRef CAS PubMed Google Scholar
First citationJohnson, S., Eddy, S. R. & Portugaly, E. (2010). BMC Bioinformatics, 11, 431–438.  CrossRef PubMed Google Scholar
First citationJones, D. T., Buchan, D. W. A., Cozzetto, D. & Pontil, M. (2011). Bioinformatics, 28, 184–190.  CrossRef PubMed Google Scholar
First citationJones, E. et al. (2001). SciPy. http://www.scipy.orgGoogle Scholar
First citationJoseph, A. P., Lagerstedt, I., Patwardhan, A., Topf, M. & Winn, M. D. (2017). J. Struct. Biol. 195, 294–305.  CrossRef Google Scholar
First citationJoseph, A. P., Malhotra, S., Burnley, T., Wood, C., Clare, D. K., Winn, M. & Topf, M. (2016). Methods, 100, 42–49.  CrossRef CAS PubMed Google Scholar
First citationKabsch, W. & Sander, C. (1983). Biopolymers, 22, 2577–2637.  CrossRef CAS PubMed Web of Science Google Scholar
First citationKrissinel, E. (2015). Nucleic Acids Res. 43, W314–W319.  Web of Science CrossRef PubMed Google Scholar
First citationKühlbrandt, W. (2014). Science, 343, 1443–1444.  Web of Science PubMed Google Scholar
First citationLander, G. C., Stagg, S. M., Voss, N. R., Cheng, A., Fellmann, D., Pulokas, J., Yoshioka, C., Irving, C., Mulder, A., Lau, P.-W., Lyumkis, D., Potter, C. S. & Carragher, B. (2009). J. Struct. Biol. 166, 95–102.  Web of Science CrossRef PubMed CAS Google Scholar
First citationLawson, C. L., Patwardhan, A., Baker, M. L., Hryc, C., Garcia, E. S., Hudson, B. P., Lagerstedt, I., Ludtke, S. J., Pintilie, G., Sala, R., Westbrook, J. D., Berman, H. M., Kleywegt, G. J. & Chiu, W. (2016). Nucleic Acids Res. 44, D396–D403.  Web of Science CrossRef PubMed Google Scholar
First citationMcNicholas, S., Potterton, E., Wilson, K. S. & Noble, M. E. M. (2011). Acta Cryst. D67, 386–394.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationMurshudov, G. N., Skubák, P., Lebedev, A. A., Pannu, N. S., Steiner, R. A., Nicholls, R. A., Winn, M. D., Long, F. & Vagin, A. A. (2011). Acta Cryst. D67, 355–367.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationNicholls, R. A., Long, F. & Murshudov, G. N. (2012). Acta Cryst. D68, 404–417.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationPandurangan, A. P. & Topf, M. (2012a). J. Struct. Biol. 177, 520–531.  CrossRef CAS PubMed Google Scholar
First citationPandurangan, A. P. & Topf, M. (2012b). Bioinformatics, 28, 2391–2393.  CrossRef CAS PubMed Google Scholar
First citationPatwardhan, A. (2017). Acta Cryst. D73, 503–508.  CrossRef IUCr Journals Google Scholar
First citationPettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. & Ferrin, T. E. (2004). J. Comput. Chem. 25, 1605–1612.  Web of Science CrossRef PubMed CAS Google Scholar
First citationRemmert, M., Biegert, A., Hauser, A. & Söding, J. (2012). Nature Methods, 9, 173–175.  CrossRef CAS Google Scholar
First citationRosa-Trevín, J. M. de la et al. (2016). J. Struct. Biol. 195, 93–99.  Web of Science PubMed Google Scholar
First citationRoseman, A. M. (2000). Acta Cryst. D56, 1332–1340.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationSchep, D. G., Zhaoa, J., John, L. & Rubinstein, J. L. (2016). Proc. Natl Acad. Sci. USA, 113, 3245–3250.  CrossRef CAS PubMed Google Scholar
First citationScheres, S. H. W., Núñez-Ramírez, R., Sorzano, C. O. S., Carazo, J. M. & Marabini, R. (2008). Nature Protoc. 3, 977–990.  Web of Science CrossRef CAS Google Scholar
First citationSeemayer, S., Gruber, M. & Söding, J. (2014). Bioinformatics, 30, 3128–3130.  Web of Science CrossRef CAS PubMed Google Scholar
First citationSimkovic, F., Thomas, J. M. H. & Rigden, D. J. (2017). Bioinformatics, https://doi.org/10.1093/bioinformatics/btx148Google Scholar
First citationSmith, J. M. (1999). J. Struct. Biol. 125, 223–228.  Web of Science CrossRef PubMed CAS Google Scholar
First citationStuart, D. I., Subramaniam, S. & Abrescia, N. G. A. (2016). Nature Methods, 13, 607–608.  CrossRef CAS PubMed Google Scholar
First citationTagari, M., Newman, R., Chagoyen, M., Carazo, J. M. & Henrick, K. (2002). Trends Biochem. Sci. 27, 589.  Web of Science CrossRef PubMed Google Scholar
First citationTopf, M., Lasker, K., Webb, B., Wolfson, H., Chiu, W. & Sali, A. (2008). Structure, 16, 295–307.  Web of Science CrossRef PubMed CAS Google Scholar
First citationVagin, A. A. & Isupov, M. N. (2001). Acta Cryst. D57, 1451–1456.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationVagin, A. & Teplyakov, A. (2010). Acta Cryst. D66, 22–25.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationVaguine, A. A., Richelle, J. & Wodak, S. J. (1999). Acta Cryst. D55, 191–205.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationVasishtan, D. & Topf, M. (2011). J. Struct. Biol. 174, 333–343.  Web of Science CrossRef CAS PubMed Google Scholar
First citationWalt, S. van der, Colbert, S. C. & Varoquaux, G. (2011). Comput. Sci. Eng. 13, 22–30.  Google Scholar
First citationWaterman, D. G., Winter, G., Gildea, R. J., Parkhurst, J. M., Brewster, A. S., Sauter, N. K. & Evans, G. (2016). Acta Cryst. D72, 558–575.  Web of Science CrossRef IUCr Journals Google Scholar
First citationWebb, B. & Sali, A. (2016). Curr. Protoc. Bioinform. 54, 5.6.1–5.6.37.  CrossRef Google Scholar
First citationWinn, M. D. et al. (2011). Acta Cryst. D67, 235–242.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationWood, C., Burnley, T., Patwardhan, A., Scheres, S., Topf, M., Roseman, A. & Winn, M. (2015). Acta Cryst. D71, 123–126.  Web of Science CrossRef IUCr Journals Google Scholar

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