Composition stage

The composition of each partial structure (located in previous macrocycles) is compared with the composition of the unit cell. Available models that fit in the missing composition are selected for search. In case the modelled sequence for a search model is known, the program can account for the fact that a model may only be a partial model for a component (e.g. one domain only).

Search stage

Calculations are organized into a search tree that is explored according to depth-first traversal. For the underlying calculations, available functions from phaser [PHASER] are used.

For each partial structure and applicable model, a rotation function is calculated. This is then followed up by a translation function calculation. Each translation function peaks are checked for packing clashes, and if accepted, checked whether are significant, probable solutions or not. Calculations are performed in the order of peak score (RF/TF), model quality (sequence identity) or partial structure quality (LLG score). Exploration continues until a significant solution is found (quick mode) or all possibilities has been exhausted (full mode). There is also no thresholding step for rotation (rationale: weak signal in rotation function) and translation function peaks (rationale: packing check fast). As a consequence, more work is performed, but simultaneously this allows weighting translation function results with packing overlap, and also avoids pipe stalls, which would adversely effect scaling in a parallel environment.

In quick mode, if a significant solution is found, conventional search terminates. However, all models that are alternatives of the model that gave this clear solution are superposed onto the solution and refined. This allows the quick evaluation of model quality for a potentially large number of alternative models. Currently, SSM [SSM] is used for superposition.

Parallelisation is done at a function level. Calculations are dispatched until the number of assigned CPUs are filled up. Execution can either take place in a shared memory environment using threads and processes or using a submission queue (currently SGE, LSF and PBS are supported). The degree of parallelisation scales with the complexity of the search, and given unlimited resources, reduces the runtime to a constant. However, in simple cases (e.g. a good model that gives a clear rotation and translation peak), no parallel resources can be used.

Refinement

Peaks above a certain threshold of the best peak and those that are identified as significant solutions are subjected to refinement. After refinement is completed, a second thresholding step is performed. Top peaks will be subjected to post-processing and also carried forward for the next macrocycle. Significant peaks that are below the threshold (e.g. a correct solution for a small domain) are also input to the post-processing step, but will not be propagated to the next cycle.

Post-processing

Significant solutions are analysed for specific features so that the search could be improved.

• Amalgamation. Assembly of solutions that come from the same starting point. In case the "starting point" is empty, allowed origin shifts are added to the search. In case the origin is undefined and the space group has floating origin, only the rotation component is extracted.
• Rotation peak salvage. Propagates significant rotation peaks to the next stage.
• Regular assembly recognition. Identifies local point group symmetry from the solution.
• Assembly completion. In case assembly definitions are available (e.g. from regular assembly completion), and a partial assembly has been located, missing molecules are filled in.

No actual calculation is performed, only the possibilities are noted. In case the given solution enters the next Search stage (as determined by Composition stage), these possibilities will be scored as a priority.

Space group exploration

Search progress in possible space groups is compared. Space groups having significant solutions or above the threshold (determined by the best score over all space groups) are propagated to the next macrocycle.

Solutions

If the search finishes, solutions are written out in all likely space groups. For each space group, the top solution is selected, and all partial solutions above a threshold relative to the top solution is considered. Solutions that are fully "contained" in a higher scoring solution (i.e. all models correspond to a model in the higher scoring solution) are discarded. This way, the top solution is output even if the composition is overestimated, but at the same time removing incomplete solutions (e.g. 14 molecules out of the total 15) that cannot be distinguised based on the final score.

Solutions are written out in an internal compressed format, which is capable of storing a high number of solutions without onerous storage requirements, and can be manipulated with provided tools.

Graphical interface

As with the generic Phaser GUI, two interfaces are available for running MRage, one designed for single-component searches with minimal customization of input models, and an advanced interface that allows all available features to be used. These are launched from a single button in the main GUI, which will initially pop up an information dialog describing the available options.

The simple GUI is centered around a single input control allowing any combination of files to be dragged in:

• Reflections (mandatory): these can be in any file or data format; only amplitudes or intensities are required.
• Sequence: should contain a single polymer sequence, in any format. This is highly recommended, and required if you want to perform an automatic homology search.
• Template models: unprocessed PDB files, which will be treated by Sculptor as described above.
• Homology search: HHPred or BLAST results.

In the simplest cases, only the data and sequence are required; you should then select a homology search method in the Options section. "NCBI BLAST" requires internet access; "Local BLAST" requires that BLAST is installed and available on the command line. At this point you can run the program without further input.

Other settings:

• Max. number of hits to use controls how many PDB entries from the homology search are processed by the pipeline. Increasing this provides greater coverage, but at the cost of significantly longer runtimes.
• Resolution cutoff determines the resolution used for running Phaser; this does not need to be changed, but increasing the cutoff will result in a faster but less accurate run.
• Parallelization method controls how the jobs are distributed across the available computing resources. multiprocessing and threading are essentially equivalent (although in practice they may behave slightly differently), and run locally. The other options require that you have access to one of the supported queuing systems and can submit jobs from the computer where you are running the GUI.
• Number of CPUs controls how many jobs are run at once. It should not exceed the number of processors available, either on the local system or the queuing system. (Note: if you plan to use your computer for other tasks while MRage is running, you may wish to leave one CPU core free if possible.)
• Search mode: sets the thoroughness of the search, as describe in the Search stage section.
• Symmetry exploration determines the space groups to try (Symmetry exploration)
• Overall count is the number of copies of the component to search for in the asymmetric unit.

Advanced GUI

This version of the GUI moves the description of the crystal contents and search information to a separate tab, with input options grouped by component. The first tab is essentially similar to the simple GUI, but the only input field is for the reflections file.

The composition tab contains another set of tabs for each unique component. These allow the full range of input files to be specified; some of these have additional parameters (such as the mandatory sequence identity for ensembles and model collections). The "Basic info" tab allows the NCBI BLAST search to be run interactively if desired (the results will be loaded as a homology result). A link to the HHPred server, which does not allow automated queries, is available on the "Homology searches" tab.

You must specify the component stoichiometry, i.e. the number of copies of the component in the overall assembly. This is distinct from the "Overall count" parameter in the main tab: for instance, if you are trying to solve a structure of a hexamer bound to a monomer and expect two copies of the complex in the asymmetric unit of the crystal, the overall count is two, and the stoichiometry of the components is 6 and 1 for the hexamer and monomer respectively.

Command line

phaser.MRage [command-line switches] [PHIL-format parameter files]

Command-line switches:

-h, --help            show this help message and exit
--show-defaults       print PHIL and exit
-i, --stdin           read PHIL from stdin as well
-v, --verbosity       set verbosity level (DEBUG,INFO,WARNING,VERBOSE)

PHIL arguments

Everything not starting with a dash('-') is interpreted as a PHIL argument. This can be a PHIL-format file containing parameters or a command-line assignment.