Automated phase improvement with Parrot
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Parrot is a program which takes an initial set of phase probability distributions from experiment phasing (or occasionally molecular replacement), and produces an improved set of phase probability distributions which may be used to calculate a clearer and more interpretable map. It does this by applying real space constraints based on known features of a protein electron density map.
Parrot is an evolved traditional density modification method, and as such is a very fast calculation. It is also highly automated, estimating solvent content from the sequence, and NCS operators from a heavy atom, molecular replacement or partial model if required.
- Reference: Zhang K. Y. J., Cowtan K., Main P. (1997) Methods in Enzymology, 277, 53-64. Combining constraints for electron-density modification.
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[edit] Running Parrot
Pirate performs phase improvement to improve the phase estimates (or phase probability distributions) obtained from experimental phasing or molecular replacement, and thereby improve the electron density map. To run Parrot, you need an MTZ file containing a set of observed structure factors and phase information.
Open the 'Parrot' task from the Density improvement module. You can now enter the following information:
In order to run the program, you must provide 2 files:
- A sequence file. This must contain the sequence of the protein. The file may simply contain a list of 1-letter residue codes, or it may contain multiple chains, each specified by a chain ID (preceded with '>'), followed by the residue codes on subsequent lines (FASTA format).
If there is NCS present, then the NCS related chains need not be given (the program will work either way, however supplying NCS chain sequences will help the program in determining the correct solvent content). - An MTZ file. This must contain the stucture factor magnitudes, and phase probability distributions from experimental phasing or molecular replacement. Normally these will be given as Hendrickson Lattman coefficients, although phase and figure of merit may also be used. (When rebuilding in a molecular replacement map, the phase and figure-of-merit may be obtained from the rigid-body-refinement.)
An output MTZ filename is generated automatically. You may change this if you wish.
The remainder of the fields should be filled out automatically. Select 'Run now'. The calculation may take a few seconds to a few minutes.
[edit] Program output
Parrot produces some dignostics in the output. The most useful of these are probably the Sigma-A statistics, which give an indication of phase reliability. Values are in the range 0...1 with greater values representing better phases. However, while these may be useful for comparing different runs of Parrot (e.g. to determine hand), they do not generally give a good indication of how well the program has worked. The best ways to determine whether the resulting map is interpretable is to:
- Look at it in a graphics program, such as Coot.
[edit] Advanced options
[edit] Phase improvement and NCS averaging with Parrot
Parrot can also perform automatic NCS averaging. Some additional information is required in order to determine the NCS operators. This can be in one of two forms:
- A PDB file containing heavy atom coordinates. In general at least 3 atoms per monomer are required, although in the case of proper NCS fewer may be sufficient. In practice more atoms are often required.
- A PDB file containing a model. The model may be incomplete and fragmentary, as long as overlapping sequence fragments of ~20 residues are available from each molecule. An unsequenced chain trace is not sufficient.
To perform NCS averaging, check one of the 'Get NCS from...' boxes at the top of the task window, and specify the input PDB file in the files section of the task window. Then run the job as normal.
[edit] Program output concerning NCS
Any NCS operators determined from the input PDB file will be displayed in the program log file, before the start of the first cycle of density modification, as shown in the frst two fragments on the right (corresponding to NCS from heavy atoms and NCS from a model respectively. You should expect to see a number of operators related to the number of NCS copies as follows:
| Number of NCS copies | Number of operators (proper NCS) | Number of operators (improper NCS) |
|---|---|---|
| 2 | 1 | 2 |
| 3 | 2 | 6 |
| 4 | 3 | 12 |
| N | N-1 | N(N-1) |
When using heavy atoms to determine the NCS operators, it is not uncommon for a few operators to be missed, but this does not usually affect the results.
Two other values from the log file can be used as a check on the NCS calculation: the fraction of the asymmetric unit (ASU) which obeys each NCS operator, and the correlation between the NCS related desnities within that region (shown on the right). An ASU fraction of more than 20% is usually correct, less than 10% is usually wrong, although exceptions are possible.
NCS correlations are also an indicator of reasonable operators. Greater than 0.2 is usually correct, less than 0.1 may be wrong.
[edit] Multi-domain averaging
Multi-domain averaging should be handled automatically if there are enough heavy atoms in each domain to determine the operators. When using a partial model, break the chains between domains and give each domain a different chain name.
[edit] Related pages
[edit] Program documentation
The latest version of the documentation is available here.
This page describes Pirate version 0.8.1 (CCP4 version 6.1.0 extension).
[edit] Program availability
Parrot is available as an extension to CCP4 6.1.0 from here. Installation is by automatic script.
--Kevin Cowtan 09:54, 27 August 2008 (CDT)
