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Kevin Cowtan, [email protected]
CCP4 March 2007
“Pirate applications”...
Pirate: phase improvement software Brigantine:bias removal software Unnamed application
Kevin Cowtan
Kevin Cowtan, [email protected]
CCP4 March 2007
Pirate
Status: Statistical phase improvement software. Included in CCP4 version 6.0.
Updated in 6.0.2
Includes: Basic phase improvement:
Effective handling of order/disorder.
NCS averaging: Automated NCS determination from heavy atoms and density. Per-pair weighted NCS averaging.
Kevin Cowtan, [email protected]
CCP4 March 2007
Pirate
Status (2006): Phase improvement works well:
Slower than ‘dm’ Sensitive to the quality of the input HL coefficients
NCS: When it works, it works well. Buggy: works in < 10% of cases!
Why? Because at this meeting last year, I was told that
Buccaneer was more important.
Kevin Cowtan, [email protected]
CCP4 March 2007
Pirate
Status (2007): Automation further improved:
Log-likelihood weight now calculated automatically by
optimisation of the log-likelihood gain. Next version will also incorporate a better algorithm for
estimating the cell composition.
User interface improved: User no longer has to provide a reference structure
(unless they want to). GUI simplified.
Kevin Cowtan, [email protected]
CCP4 March 2007
Pirate
Status (2007): NCS code heavily updated. Now works fully
automatically in 30-50% of cases. Outstanding issue with NCS along special directions. When it doesn’t work, it does no harm. Minor tuning still required. (Timeconsuming!)
Kevin Cowtan, [email protected]
CCP4 March 2007
Pirate
Future:
Further NCS improvements.
Use of an input model of electron density map to
provided an additional source of information Integration with buccaneer.
Kevin Cowtan, [email protected]
CCP4 March 2007
Brigantine
Brigantine is a bias removal program:“Bias Removal In General Allows New Trace In New Density”
Mainly aimed at tackling MR bias problem, but uses
ideas and code from Pirate, but has applications to
validation and maybe density modification as well.
Kevin Cowtan, [email protected]
CCP4 March 2007
Brigantine
Status:
Currently performs 3 calculations: Simple composite omit map.
Fast: seconds to a few minutes.
Sigmaa-weighted composite omit map. Fairly fast: minute to tens of minutes
Pirate composite omit map. Fairly slow: tens of minutes to a few hours.
All of these are model-free, i.e. Can be used before
model building.
Kevin Cowtan, [email protected]
CCP4 March 2007
Brigantine
Pirate composite omit map. Related to a similar idea in Resolve
(CCP4 study weekend 2007)
But does not involve any density modification. i.e. It can be used in combination with arbitrary density
modification methods. Maybe even recycled with several
different density modification methods.
Code works. One tunable parameter needs to be estimated. It is not clear if the results are useful.
Kevin Cowtan, [email protected]
CCP4 March 2007
Brigantine
Applications: After MR
(to reduce model biad)
Between different density modification procedures. (speculative)
As part of iterated density modification/model
building procedure. (as validation)
In model completion (validation, ligands, etc.)
Kevin Cowtan, [email protected]
CCP4 March 2007
Unnamed application
An unnamed program to provide an improved method
for perturbing a model to reduce bias after
refinement.
An alternative to simulated annealing.
Uses: Unbiasing a free set. Precursor to Brigantine.
Kevin Cowtan, [email protected]
CCP4 March 2007
Tools
Fast anisotropic scaling
(needs tuning)
Omit maps
Sigma-a maps
Refinement?
Kevin Cowtan, [email protected]
CCP4 March 2007
Buccaneer, Coot, and low resolution
Developments in buccaneer and coot
for model building, and application
to low resolution problems.
Kevin Cowtan
Kevin Cowtan, [email protected]
CCP4 March 2007
Buccaneer: Method
Statistical model building software based on the use
of a reference structure to construct likelihood
targets for protein features.
Kevin Cowtan, [email protected]
CCP4 March 2007
Buccaneer
Status (2006): Successful tool for protein chain tracing, even at
low resolutions.
Tasks (2006): Implement sequence assignment/docking. Implement recycling for model completion Iterate with refmac
Kevin Cowtan, [email protected]
CCP4 March 2007
Buccaneer
Status (2007): Sequence assignment/docking implemented. Recycling for model completion implemented. Iteration with refmac implemented.
In an ad-hoc script, without GUI
Kevin Cowtan, [email protected]
CCP4 March 2007
Buccaneer
Sequence assignment/docking:
Use the same method for classifying side chains by type as
for finding C-alphas in the first place, but with a
difference target function:
ALA CYS HIS MET THR ... x20
Finding:
Classifying:
Kevin Cowtan, [email protected]
CCP4 March 2007
Buccaneer
Recycling for model completion implemented. “Lateral growing”: grow sideways from existing
chain fragments by looking for new C-alphas at an
appropriate distance “sideways” from the existing
chain. Fast recycling within Buccaneer, or slower recycling
with other programs, e.g. refmac.
Kevin Cowtan, [email protected]
CCP4 March 2007
Buccaneer
Steps: (bold are new since 2006) Find C-alphas Grow into chains Join the chains Pre-sequence loop building (up to 2 C-alphas) Assign sequence Correct insertions/deletions Filter based on poor density Remove clashing chains Build side chains
Kevin Cowtan, [email protected]
CCP4 March 2007
Buccaneer
The positive: Successfully applied to 2 real unknown
structures with good phases at ~3.5A. More
than half the model built and sequenced. Successfully applied to 4.0A truncated data. First citation this month!
The negative: Model completeness still not as good as
Resolve. What about bad phases at higher resolutions?
Kevin Cowtan, [email protected]
CCP4 March 2007
Coot
Coot developments in model building:
Improved helix placement Improved handling of low resolution maps Handle ‘UNK’ for use with Buccaneer Developments towards strand placement Future integration of Buccaneer functionality
Kevin Cowtan, [email protected]
CCP4 March 2007
Coot
Improved helix placement: Helix built both ways and refined without Cb. Correct orientation determined from Cb density.
Low resolution: Add terminal residue does RSR Auto-fit sidechain does RSR Secondary structure restraints
Kevin Cowtan, [email protected]
CCP4 March 2007
Coot
Strand placement New algorithms are being examined for both helix
and strand placement. First attempt:
Use skeleton and find where ridges cross a sphere
containing the feature. Faster than current method for helices.
Kevin Cowtan, [email protected]
CCP4 March 2007
Coot
Integration of Buccaneer features: 'UNK' residue type handling. Buccaneer sequencing has been exported to a
standalone ‘cootaneer’ library. For speed, this has to work without a reference structure. Use pre-calculated resolution dependent libraries. Just needs integrating.
Buccaneer growing may be added in future. An alternative engine for current ‘Add terminal residue’? Or an auto-grow algorithm.
Kevin Cowtan, [email protected]
CCP4 March 2007
Wiki and CCP4
Wiki as a tool to organise and
expand CCP4 documentation
and mailing list answers.
Kevin Cowtan
Kevin Cowtan, [email protected]
CCP4 March 2007
What is a Wiki? A website which anyone (or anyone authorised)
can edit. Wikipedia is an example:
Kevin Cowtan, [email protected]
CCP4 March 2007
Why Wiki? Much easier to update than a website...
Edit through the web browser, even remotely. Automatic markup (most of the time). Semi-automatic linking and cross referencing.
Collaborative... Many people doing the bits they know best when it is
convenient to them.
Kevin Cowtan, [email protected]
CCP4 March 2007
Wiki Features Build in version control.
Track and revert changes.
Access control for admin, writing, (reading). Initially wikis were open, these days must register. Per-page access control as well as site wide.
Many allow pages with discussion areas. A page can be read only but allow comments.
Kevin Cowtan, [email protected]
CCP4 March 2007
How could CCP4 use one? Take good bits out of the program documentation,
manual, bulletin board discussions, and gather and
organise them. Turn bulletin board discussions into FAQ pages. Host frequently changing info:
Contact details Useful web resources Problem pages
Kevin Cowtan, [email protected]
CCP4 March 2007
Case studies:
~2002: Tried to use one to document eHTPX
collaboration. Failure: Unfamiliar. People forced to contribute.
~2003: Implemented at York for local documentation
pages. Success: Widely read. A smaller group contribute. Enough enthusiastic contributors to make it work. There must be a ‘critical mass’ of contributors.
Volunteers: EJD, JPT, KDC
Kevin Cowtan, [email protected]
CCP4 March 2007
Deploying a Wiki
Issues: The server is web-facing Wikis are regularly attacked
Software updates must be applied very frequently Better to have a dedicated machine outside the firewall
Choice of wiki software: MoinMoin, Twiki, Mediawiki, etc... Language, database or flat file, access control, version
control, comments, images, math, plugins, search, email
notification of changes, content export, etc...
Kevin Cowtan, [email protected]
CCP4 March 2007
Deploying a Wiki
Issues: Alternative: Wiki hosting sites:
wikia, wikidot, wikispaces. Some are free, or $5-$50/month. Some display adverts Some demand content be licensed, e.g. CC, FDL
Issues: Convenience: can I be auto-logged in when I arrive? Can the content be exported if we need to move?
