Pipelining Ligands in PHENIX: elbow and REEL Nigel W. Moriarty Lawrence Berkeley National Laboratory Physical Biosciences Division
Ligands in Crystallography Drug design Biological function studies Generate ligand restraints Fit ligand to density Refine macromolecule and ligand
e-lbow Goals Fast, simple and flexible procedure to include ligands in refinement Reduce the tedium of building 3D ligand models Automate generation of restraints for ligands Comparison of ligand structures with PDB models Chemical input format Reflection Data Protein Information Chemical restraints file (CIF) Cartesian coordinates file (PDB) phenix.refine
Ligand pipeline flowchart Chemical input Reflection data Protein model elbow Geometry LigandFit Ligand model phenix.refine Restraints Final model
angles elements names atom dihedrals connectivity bond residue name centres chiral orders bond charges formal planes coordinates cartesian Input Formats SMILES PDB xyz Mol2D, Mol3D (v2000, v3000) GAMESS input and output files Sequences PRODRG TXT format Monomer library CIF PDB Ligand
The Algorithm
The Algorithm Parse SMILES or other input Post process Molecular object Atomic centres Bonds Determine ring and chiral structures Construct a Z-Matrix model of heavy atoms Z-Matrix is an internal coordinate representation Chemically intuitive Provide direct control of bonds, angles and dihedrals
Z-Matrix C1 H1 C1 1.1 H2 C1 1.1 H1 109.6 H3 C1 1.1 H1 109.6 H2 120.0 H4 C1 1.1 H1 109.6 H2-120.0 H1 Degrees of freedom = 3n-6 H4 C1 H2 H3
The Algorithm Optimise Z-Matrix geometry using simple force field (requires cartesian coordinates) Add hydrogens Optimise just hydrogens using simple force field Optimise using AM1 semiempirical quantum chemical method Check structure and repeat optimisation if necessary Generate geometry restraints
Comparison with experiment MSD Chem ligand library contains approximately 6443 ligands SMILES strings a smaller number of experimental cartesian coordinates approximately 3285 comparable ligands Input SMILES string to elbow Compared AM1 geometry to experimental results from the PDB
Refinement Tests Use the restraints for ligand obtained from elbow in phenix.refine Refined at resolution 1.2Å or better and 2.5Å (data truncated) Compare to structure deposited in PDB Legend PDB High Low Calculate RMSD using cartesian coordinates, bond, angle & dihedrals e-lbow run time 194 seconds
Challenges Disordered or weak density Poor deposited ligand geometry
Map Density Correlation Compare the correlation of the model density to the 2mFo-DFc map phenix.refine and elbow provide provide ligand geometries that match the experimental data equally as well as the original libraries used in refinement Results are also good for low resolution, but the comparison is difficult (PDB refinements were at high resolution)
Histograms Mean, Sigma xyz: 0.11, 0.07 bond: 0.037, 0.017 dihedral: 6.0, 4.8 Differences between the original and new geometries are within the restraint standard deviations
Flexibility Simple command line interface Scriptable using Python Covalently bound ligands Automatically treats all ligands in a PDB file Close integration with refinement
Start COOT with elbow in COOT coot --script $PHENIX/elbow/elbow_in_coot.scm coot --script $PHENIX/elbow/elbow_in_coot.py Build a ligand from SMILES or use the coordinates from an internal molecule Manipulation of ligand geometry to provide a starting geometry AM1 geometry optimisation
SMILES Simple run phenix.elbow --smiles c1ccccc1c phenix.elbow --smiles filename.smi phenix.elbow --msd atp PDB file phenix.elbow --file filename.pdb phenix.elbow filename.pdb Atom naming phenix.elbow --smiles c1ccccc1c --template names.pdb phenix.elbow --msd atp --template atp.pdb
Output PDB file contains optimised geometry CIF file contains restraints filename.bonding.py is a Python script to change the bonding via --bonding --tripos will output a TRIPOS file --output will change the name of the output files
Adjusting geometry phenix.elbow --smiles FC=CF --opt - -view pymol --view pymol run pymol filename.pdb Edit geometry and save as fixed.pdb elbow will read the new geometry as starting geometry for AM1 optimisation
Providing geometry Starting geometry for AM1 optimisation phenix.elbow --initial-geometry file.pdb Final geometry to get a CIF file phenix.elbow --final-geometry file.pdb Provide corresponding SMILES for a better CIF file
PDB options A PDB file can have many ligands To list all residues in a file phenix.elbow file.pdb --all-residues To run elbow on all unknown ligands phenix.elbow file.pdb --do-all To run elbow on a known ligand phenix.elbow file.pdb --residue ATP
PDB options (cont.) To control the auto bond determination -- auto-bond-cutoff=2.5 To control hydrogen addition --add-hydrogens=true To use a large PDB file as template phenix.elbow --smiles O --template large.pdb --residue HOH
elbow is Python scripts from elbow.command_line import builder molecule = builder.run(smiles= O, opt=true, quiet=true) print molecule.display()
Misc. features --read-only to exit after reading input --pickle to read topology file --pipe to use or open a pipe shell --pymol to use progress in PyMOL if PHENIX installed --quiet & --silent --name sets ligand name --id defaults to LIG
elbow & phenix.refine Run phenix.elbow ensuring that the atom naming is correct using a protein-ligand complex PDB file Run phenix.refine using the CIF file Possible to generate CIF link file using the CONECT or LINK record in the proteinligand complex PDB file Add hydrogens to a PDB containing protein model and ligands
Other elbow programs phenix.metal_coordinate elbow.join_cif_files, join_pdb_files elbow.join_cif_files combined.cif file1.cif file2.cif elbow.doc
elbow Summary e-lbow provides easy to use methods to generate novel ligands and known ligands AM1 provides a computationally efficient method for geometry optimisation of molecules containing any main group elements Provides geometries comparable to existing methods Readily pipelined into automatic refinement using phenix.refine and phenix.ligandfit
What is REEL? Restraints Editor elbow Ligands Loads any restraints file Loads any elbow input Restraints Editor Especially Ligands No linking or modifications Restraints Editor Exclusively Ligands Loads a small PDB file Restraints Editor Effectively Ligands REEL will make a real difference A. White, Ph.D., Boehringer-Ingelheim GmbH
Visualisation PHENIX User Meeting March 28th 2008
REEL features Generate a geometry for a set of restraints Modify restraints and generate new geometry Fast editing in menu items Highlight atom and restraints are highlighted Multiple ligands simultaneously Highlight restraint and atoms are highlighted Save restraints (CIF) Save geometry (PDB) Run elbow
elbow GUI
REEL flowchart Chemical input elbow Geometry Restraints REEL Geometry Restraints
Demonstration
Summary elbow converts many inputs to geometry and restraints REEL allows restraints editing and several elbow features
Acknowledgments Computational Crystallography Initiative Paul Adams Pavel Afonine (phenix.refine) Ralf Grosse-Kunstleve (cctbx, phenix.refine, HySS,.) Nick Sauter (iotbx, labelit) Peter Zwart (mmtbx.xtriage, phenix.refine) Other PHENIX developers Cambridge University, Los Alamos National Laboratory, Texas A&M, Duke University Others CCP4 developers (MTZ library) Alexei Vagin & Garib Murshudov (Monomer Library) Funding: LBNL (DE-AC03-76SF00098) NIH/NIGMS (P01GM063210) NIH/NIGMS (P01GM064692) PHENIX Industrial Consortium