1. Common Coot (Fulica atra) (Fulica atra) Common Coot (Fulica atra) (Fulica atra)

2. Introduction to Coot

3. Model-Building with Coot An Introduction, low resolution tools(, NCS) Bernhard Lohkamp Karolinska Institutet Mar 2011 Shanghai (Paul Emsley) (University of Oxford)

4. Coot Molecular Graphics application Molecular Graphics application Protein crystallographic model-building tools (Crystallographic Object-Oriented Toolkit) Protein crystallographic model-building tools (Crystallographic Object-Oriented Toolkit) Aims: Aims: Model building, completion, validation Model building, completion, validation Fill the gap where automated methods fail Fill the gap where automated methods fail “Slick and powerful” interface to CCP4 “Slick and powerful” interface to CCP4 Interface to other programs: SHELXL, Refmac, Libcheck, Probe&Reduce (Molprobity), EBI, EDS, Povray, Raster3D, PHENIX,... Interface to other programs: SHELXL, Refmac, Libcheck, Probe&Reduce (Molprobity), EBI, EDS, Povray, Raster3D, PHENIX,... Several model-building and validation tools Several model-building and validation tools

5. Eugene Krissinel Kevin Cowtan Alexei Vagin Stuart McNicholas + Bernhard Lohkamp Paul Emsley

6. Refinement Validation External e.g. REFMAC Internal Internal External e.g. MolProbity Feature Integration Validation, Model Building and Refinement should be used together

8. What is “Refinement”? The adjustment of model parameters (co- ordinates) so that the calculated structure factors match the observations as nearly as possible The adjustment of model parameters (co- ordinates) so that the calculated structure factors match the observations as nearly as possible In “one-shot” real-space refinement, such as in Coot, this translates to: In “one-shot” real-space refinement, such as in Coot, this translates to: move the atoms into as high density as possible while minimizing geometrical distortions move the atoms into as high density as possible while minimizing geometrical distortions

9. Distorted Geometry Pre-Refinement

10. Refinement Gradients

11. Refinement: Cycle 3

12. Refinement Cycle 200: Minimized

13. Major feature of Coot Major feature of Coot Gradient minimizer (BFGS derivative) Gradient minimizer (BFGS derivative) Based on mmCIF standard dictionary Based on mmCIF standard dictionary Minimizing bonds, angles, planes, non-bonded contacts, torsions, [chiral volumes, Ramachandran] Minimizing bonds, angles, planes, non-bonded contacts, torsions, [chiral volumes, Ramachandran] Provides “interactive refinement” Provides “interactive refinement” Different minimizer to Refmac… Different minimizer to Refmac… …means “nice & tight” geometry …means “nice & tight” geometry Chi squareds Chi squareds Real Space Refinement Diamond, R. (1971). Acta Cryst. A 27, 436-452. Faster & Animated

14. Refinement “Traffic Lights” “Traffic Lights” represent the chi-squared values for each of the refined geometry types

15. Refinement Techniques Auto-zone Auto-zone Single-Atom Drag Single-Atom Drag Over-dragging Over-dragging Ramachandran Refinement Ramachandran Refinement Sphere refinement Sphere refinement Coming Soon..? Coming Soon..? Dials, PowerMate, spaceballs Dials, PowerMate, spaceballs Wii Refinement Wii Refinement

16. Wii Refinement?

18. 9/29/2016 On the fly symmetry

19. Electron Density: “Continuous Crystal”

20. Rotamer Database Molprobity Option

21. Some more Coot Tools… Alternate Conformations Alternate Conformations Ligand fitting/search Ligand fitting/search Rigid-body Fitting Rigid-body Fitting Steepest Descent Steepest Descent Simplex (slower but better) Simplex (slower but better) “Move Molecule Here” “Move Molecule Here” Water Search Water Search Fill-partial-residues (after MR) Fill-partial-residues (after MR) Dots, ball&stick representation Dots, ball&stick representation

23. Superpositions S(econdray) S(tructure) M(atching) S(econdray) S(tructure) M(atching) Least Squares Superposition: Least Squares Superposition:

24. Low Resolution Tools

25. Extra Restraints....

26. Peptide plane Peptide plane Ramachandran restraints Ramachandran restraints Secondary Structure restraints Secondary Structure restraints Remove degree of freedom Remove degree of freedom Torsion angle restraints Torsion angle restraints Backrub rotamers Backrub rotamers

28. Ramachandran Restraints Scenario: Scenario: I have a loop, with poor density, I know the atoms are there somewhere and I want to provide a “reasonable” model I have a loop, with poor density, I know the atoms are there somewhere and I want to provide a “reasonable” model Controversial Feature? Controversial Feature? Ramachandran Plots have been used for “validation” - but here we are deliberately optimizing them Ramachandran Plots have been used for “validation” - but here we are deliberately optimizing them Ramachandran Plots can be added to the geometry target function Ramachandran Plots can be added to the geometry target function

33. Ramachandran Restraints Controversial? Controversial? “... the Ramachandran Plot is one of the simplest and most sensitive means for assessing the quality of a protein model...” “... the Ramachandran Plot is one of the simplest and most sensitive means for assessing the quality of a protein model...” Gerard Kleywegt & Alwyn Jones (1996) Gerard Kleywegt & Alwyn Jones (1996) But to quote Jane Richardson: But to quote Jane Richardson: Do you want a better structure – or a better idea of the quality of your structure? Do you want a better structure – or a better idea of the quality of your structure?

34. Adding Torsion Angle Restraints Torsion angle refinement is slow (relatively) Torsion angle refinement is slow (relatively) Simple addition of these restraints to the geometry target function Simple addition of these restraints to the geometry target function often makes the region “stuck and unsatisfied” often makes the region “stuck and unsatisfied” i.e. trapped in Local Minimum i.e. trapped in Local Minimum Add Pseudo-bonds (c.f. Brigcogne & Buster)

35. Restrain the Hydrogen-bonding atom distances

38. “Backrub” Rotamers

39. Previous

44. Helix-Building

45. Alpha Helix Placement Scenario: Looking at a new map, not built with automatic tools : Scenario: Looking at a new map, not built with automatic tools : “I can see that there’s a helix here - build it for me!” “I can see that there’s a helix here - build it for me!” From a given point: From a given point: Move to local averaged maximum Move to local averaged maximum Do a 2D MR-style orientation search on a cylinder of electron density Do a 2D MR-style orientation search on a cylinder of electron density Build a helix (both directions) Build a helix (both directions) 1D Rotation search to find best fit 1D Rotation search to find best fit Score based on density at CB positions Score based on density at CB positions Trim ‘n Grow Trim ‘n Grow

48. 2 x 1-D Helix orientation searches

50. All search models (for the “up” orientation)

53. Fitting Strands

54. Placing Strands Unlike Helices, Strands have to be treated as non-idealized Unlike Helices, Strands have to be treated as non-idealized Repeating a single phi/psi value doesn't make a structure that fits “real-world” density Curvature of strands should be taken into account Curvature of strands should be taken into account Use selections from a “database” of good structures

55. Strand fitting algorithm Cylinder search Cylinder search Get N fragments of length l from database Get N fragments of length l from database 1-D Translation search along the tube 1-D Rotation search around the tube Direction flip search Rigid body refine best solutions Rigid body refine best solutions Real-space refine best solution Real-space refine best solution

59. Not all is rosy...

61. Fitting Strands caveat In the case of strand-fitting, the initial translation search centring the cylinder is not performed (the search cylinder is too thin) In the case of strand-fitting, the initial translation search centring the cylinder is not performed (the search cylinder is too thin) The user is responsible for centring the search point “in the middle of the tube” The user is responsible for centring the search point “in the middle of the tube” Not at a C-alpha position Not at a C-alpha position

62. Automated Fast Secondary Structure Search

63. Coot Futures… Aim: Aim: Slick, easy to use Slick, easy to use Powerful Powerful Smooth interface to external applications Smooth interface to external applications Under Development Under Development Interesting things move quickly Interesting things move quickly There may be bugs There may be bugs

64. Further information Coot WIKI Coot WIKI http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Coot http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Coot Coot BB (mainling list) Coot BB (mainling list) http://www.biop.ox.ac.uk/coot/mailing-list.html http://www.biop.ox.ac.uk/coot/mailing-list.html Coot documentation Coot documentation http://www.biop.ox.ac.uk/coot/docs.html http://www.biop.ox.ac.uk/coot/docs.html

65. Acknowledgements Paul Emsley Paul Emsley Kevin Cowtan Kevin Cowtan Eleanor Dodson Eleanor Dodson Keith Wilson Keith Wilson Libraries, dictionaries Libraries, dictionaries Alexei Vagin, Eugene Krissinel, Stuart McNicholas Alexei Vagin, Eugene Krissinel, Stuart McNicholas Dunbrack, Richardsons Dunbrack, Richardsons Coot Builders and Testers Coot Builders and Testers William Scott, Ezra Peisach William Scott, Ezra Peisach York YSBL, Dundee, Glasgow (early adopters) York YSBL, Dundee, Glasgow (early adopters) Coot Mailing List subscribers Coot Mailing List subscribers http://www.biop.ox.ac.uk/coot/or Google: Coot or for WinCoot http://www.ysbl.ac.uk/~lohkamp/coot

66. Handling NCS...

67. What is Non-Crystallographic Symmetry? 2 or more copies of a molecule in the unit cell not related by crystallographic symmetry 2 or more copies of a molecule in the unit cell not related by crystallographic symmetry Crystallographic copies of molecules are (of course) treated as if they were exactly the same across the unit cell – and indeed across the whole crystal Crystallographic copies of molecules are (of course) treated as if they were exactly the same across the unit cell – and indeed across the whole crystal Non-crystallographically related molecules provide different representations of the same molecule Non-crystallographically related molecules provide different representations of the same molecule This can be useful for model-building This can be useful for model-building But difficult to use in practice But difficult to use in practice

68. Handling NCS What are the Problems? What are the Problems? Strict NCS: Strict NCS: NCS should appear like crystallographic symmetry does [exact copies] NCS should appear like crystallographic symmetry does [exact copies] Non-Strict NCS: Non-Strict NCS: Molecules are different Molecules are different How to cope with differences, but minimize unnecessary rebuilding? How to cope with differences, but minimize unnecessary rebuilding?

69. Handling NCS Typical Scenario: I have done an LSQ overlap of my NCS- related molecules and from the graph, have seen significant deviations in the positions of some side-chains. I have done an LSQ overlap of my NCS- related molecules and from the graph, have seen significant deviations in the positions of some side-chains. Why are they different? Why are they different?

70. …or Kleywegt Plots [*] [*] Named by George Sheldrick

71. ...or NCS Differences graph

75. NCS Model-modification Tools Automatic detection of NCS Automatic detection of NCS And their operators And their operators Copy Master NCS molecule to others Copy Master NCS molecule to others Applies NCS transformation Applies NCS transformation Copy NCS Master residue-range Copy NCS Master residue-range Change NCS Master chain Change NCS Master chain NCS Skipping NCS Skipping