dimple

Macromolecular crystallography pipeline

for refinement and ligand screening

based on CCP4 programs.

How it works

You provide the reflection data (merged mtz) and Apo model (pdb) and tell the pipeline where it should dump the results:

$ dimple my.mtz apo.pdb output-dir

Actually you can give any number of models or pdb codes, but only one of them will be used – the one with most similar unit cell:

$ dimple my.mtz apo.pdb my-other.pdb 4uqi output-dir

DIMPLE will do what it can to quickly return a refined model and difference density map pointing to unmodelled electron density blobs – potential ligand sites.

Simplifying a bit: the pipeline runs macromolecular refinement after a few usual preparatory steps (I to F, choosing Rfree set, reindexing if needed). Sometimes it needs to run Molecular Replacement before refinement. And at the end it checks for unmodelled blobs – suspected ligands.

It’s quick. Run time depends of course on the data, such as resolution, size of unit cell, etc., the model and computer, but about 3 minutes is typical. With MR it is usually 3-10 minutes, but from time to time much, much longer.

In Diamond (UK’s national synchrotron) Dimple is automatically run after auto-processing if the user provided a PDB file before the experiment.

Options

DIMPLE has a lot of options (dimple -h lists all of them), but since the goal of the pipeline is to make things simple, we present here only three of them:

--slow (or -s for short) – recommended if you are not in hurry. DIMPLE will take twice as long, spending more time on extra cycles of refinement. If this is still too fast, give this option twice – to get 100 cycles of jelly-body refinement.

-M is also quite popular. It decides when MR should kick in. -M 0.4 (the default value) runs MR if the R-factor after rigid-body refinement, in data up to 3.5Å, is above 0.4. Edge cases: -M0 – always run MR, -M1 – never.

--libin CIF passes your own ligand descriptions to Refmac.

Selected Details

MTZ columns – by default, Dimple expects either intensities labeled as IMEAN or amplitudes labeled as F or FP. To use different columns specify options --icolumn / --fcolumn. Intensities are used only when amplitudes are not in the file, or when the user explicitely sets --icolumn or --ItoF-prog.

Reindexing – if the data and model are in compatible but different spacegroups, we use pointless to change the spacegroup in the MTZ file. We also check all possible settings – pointless calculates structure factors from the model and compares the CC on E^2 to find the best matching settings, reindexing data if necessary. In some cases this steps saves us a couple minutes by avoiding MR.

Free reflections – Rfree statistics depend to some degree on how lucky is the pseudo-random set of free flags. Therefore one may prefer to use consistent free flags when comparing data collected from different crystals. That is why Dimple, by default, assigns the same flags when the same pdb file is used (ignoring flags in the input mtz file).

The consistent flags are generated by the CCP4 freerflag program under the assumption that the data is in the same point group as the model and that the resolution is above 1Å (we had to pick an arbitrary limit). For higher resolution the free set will be consistent only up to 1Å.

As an alternative, you may use an external set of reference flags (--free-r-flags external.mtz) or existing flags from the input mtz (--free-r-flags - or -R -). You may also specify the column name and the value corresponding to excluded data (e.g. --freecolumn FreeR_flag=5).

Different asu volume – if asu in the data is much larger than in the model, we search for multiple copies of the model in MR. In the opposite case, when asu in the data is smaller, we make a single ensemble from all the chains (phaser.ensembler) before MR.

NCS – The PDB file format stores non-crystallographic symmetry transformations in the MTRIXn records. The coordinates of NCS copies may or may not be in the file, as reflected (with some exceptions) by the MTRIXn/iGiven field. Dimple is generally aware of these things and should work reasonably in all cases, although as of Q2 2016 it does not, waiting for a bugfix in refmac.

Rigid-body refinement – both refmac and phaser can do it. We could simplify our pipeline by always running phaser before refmac, but it would be slower (on average). Not by a large margin, though. Phaser checks if the input model is already placed correctly and skips the search if it is.

Since we only use data up to 3.5Å for rigid-body, in some cases 5% of free reflections was not enough to give reliable statistics. On the other hand there is no danger of overfitting in rigid-body. Thus, we stopped using Rfree set in this step at all.

Actual refinement – we run refmac restrained refinement twice. The first run (labelled as jelly) has jelly-body restraints, no hydrogens and ignores very high resolution reflections. In the default mode we run only 4 cycles with these settings, and 8 cycles of the final refinement. Note that the usual recommendation for jelly-body refinement is 100+ cycles. We picked the numbers 4 and 8 after testing various combinations on hundreds of datasets. This split happened to give slightly better results than other combinations within the same time limit.

Scoring blobs – it is rather simplistic now, we need to work on it

Generating pictures – we have an option (-f) to generate static images (PNG or JPEG) of the blobs. They are used by SynchWeb in DLS. Pictures are generated with Coot+Raster3d - this combines the familiar look and feel of Coot with nicer graphics and headless rendering.

Currently we are working on interactive web-based viewer that emulates Coot and hopefully works fast enough to replace static images.

Contaminants – in the end, if the data does not match the model, we check if the unit cell matches any of the unit cells corresponding to this list of crystallization contaminants.

FAQ

Installation

DIMPLE is part of the CCP4 suite. All the work is done by other programs from the suite, which are run underneath. Most importantly refmac and phaser. So you need to have CCP4 installed, and dimple is already there.

But if you’d like to try the very latest version that hasn’t filtered through to the CCP4 suite yet, get it from github.com/ccp4/dimple.

If you do not want to use git, download master.zip, unpack it, rename dimple-master to dimple and run ./dimple/dimple.

Comments?

Any comments and thoughts how to improve this tool are genuinely welcome. If it doesn’t work as expected or doesn’t work at all – let us know asap. Use the issue tracker or email CCP4 helpdesk or email wojdyr@gmail.com or chat at https://gitter.im/ccp4/dimple.