EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Validation - proteins EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Electron density maps oWhen looking at PDB structures Electrons Density (ED) maps are more/as important as the 3D atomic model! oED is a 3D map of where the scattering electron cloud is according to the measured X-ray data. o2Fo-Fc map indicates where electrons are (according to SF and model). Normally colour blue or grey. oFo-Fc difference map: •green for positive difference: where the current model fails to place sufficient electrons •Red for negative difference: where the current model places too many electrons •www.ebi.ac.uk/pdbe/entry/pdb/4z9l/bound/ANP EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Electron density for a ligand with poor fit 2h7p.pdb: ED visualized in coot Notice difference density around ligand 2h7p has been obsoleted and replaced 4tzt with really superb ED and ligand fit 2h7p.pdb: ED around ligand, as visualized in buster-report EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Data resolution affects electron density detail: Well placed/refined sucrose ligand at different data resolutions: 4 1ylt 1.2Å resolution “atomic resolution” 2Fo-Fc ED shows atoms as Individual blobs. Need higher resolution for hydrogen atoms 2pwe 2.0Å resolution Typical medium resolution for ligand studies. Can see ring pucker 2qqv 3.0Å resolution Low resolution. Ligand placement unambiguous but fine detail cannot be seen EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Model improvement •Basically interpret electron density maps in real space to improve model •Initially automated methods (warp/arpwarp) used •But mostly manual corrections to the model are done using the Coot program •Look at Fo-Fc difference map for both negative and positive features •Build into 2Fo-Fc partF_coot_fix_A186.png EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Why do crystallographers make mistakes? •Limitations to the data •Incomplete •Weak •Limited resolution •Space and time averaged • • •The human factor •Subjectivity and bias involved in map interpretation and refinement (even at atomic resolution!) •Inexperienced people do the work, use of black boxes, … •Not everybody is a good chemist •Even experienced people make mistakes Kleywegt, Acta Cryst. D65, 134 (2009) trp_nodiff.png trp.png PDF report for depositor & referees - Statistics and plots for the entry, per chain, per residue, and list of unusual features wwPDB X-ray validation pipeline Validation pipeline 1.0 MolProbity EDS Xtriage Mogul Deposited data (coordinates & reflections) Percentiles Fig6.tif PDF maker Validation XML file External reference files (e.g., Engh & Huber) Gore et al., Acta Cryst. D68, 478 (2012) EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png wwPDB validation reports •Pipeline produces PDF report and XML output •Slider graphic useful • • • • •Current PDF is “rather verbose” • •pdbe.org/valrep/1cbs EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Ramachandran plot •Look at main chain dihedral angles phi and psi •Ramachandran et al. (1963) worked out only certain combinations of phi/psi cause clashes •https://en.wikipedia.org/wiki/Ramachandran_plot https://upload.wikimedia.org/wikipedia/commons/thumb/9/97/Protein_backbone_PhiPsiOmega_drawing.svg/ 320px-Protein_backbone_PhiPsiOmega_drawing.svg.png http://xray.bmc.uu.se/gerard/embo2001/modval/phirot_anim.gif Rotation around φ with ψ=0o http://xray.bmc.uu.se/gerard/embo2001/modval/psirot_anim.gif Rotation around ψ with φ=0o (Images kindly provided by David Sanders, University of Saskatchewan.) EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Summary ‘Sliders’ Validation information for users Atoms bumping into each other Surprising torsion angles Atoms not in electron density EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Sidechain outliers •Just like the main chain phi and psi dihedral angles amino acid sides chains have chi angles with have preferred and disallowed regions •The 5 chi angles of an arginine side chain •http://www.ccp14.ac.uk/ccp/web-mirrors/garlic/garlic-1.5/commands/dihedrals.html http://www.ccp14.ac.uk/ccp/web-mirrors/garlic/garlic-1.5/commands/chi_angles.gif EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Summary ‘Sliders’ Validation information for users Atoms bumping into each other Surprising torsion angle Atoms not in electron density EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png MolProbity – clash score •Idea is to look for bad non-bonded contacts after hydrogen atoms have been added to the model •Very powerful method •Suggests NQH flips •Included in wwPDB validation reports •Or Use from: •Molprobity web site •Or within coot • • •http://molprobity.biochem.duke.edu/ EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Validation information for users Summary ‘Sliders’ Atoms bumping into each other Surprising torsion angles Atoms not in electron density EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Real-space fit •Quantitative, real-space measure of how well a residue fits its local density (Jones et al., 1991) •Express as R-value (RSR) or correlation coefficient (RSCC) •RSR = S | robs - rcalc | / S | robs + rcalc | •Sums extend over all grid points inside a mask around the residue • Jones, T.A., Zou, J.Y., Cowan, S.W. and Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47, 110-119. • http://alpha2.bmc.uu.se/dombo/pics/alwyn_jones.jpg EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png RSR - real-space R-value •pdbe.org/1cbs •RSR = S |robs - rcalc| / S |robs + rcalc| • rsr-1 Example: retinoic acid (REA) in 1CBS (1.8Å) EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png RSR - real-space R-value rsr-2 rsr-3 rsr-4 rsr-5 Define mask (1.1Å) ~500 Grid points Observed density Calculated density RSR = S |robs - rcalc| / S |robs + rcalc| pdbe.org/1cbs EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png RSRZ is reported in Summary ‘Sliders’ Atoms bumping into each other Surprising torsion angles Atoms not in electron density EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png PDBe simplification of validation sliders Entry percentile scores Entry percentile scores EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png “Best molecule” – integration of validation information in PDBe query system •www.ebi.ac.uk/pdbe/entry/search kinase-molecules.tiff EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png NMR validation •NMR VTF recommendations published •Global quality scores reported for“well-defined residues” only •As averages over the ensemble •Medoid model only •Montelione et al., Structure 21, 1563 (2013) NMR entry 2MP2 EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png EM validation reports •Prototype EM map-validation reports •Most of the PDBe “Visual analysis” functionality implemented •Compare to: pdbe.org/emd-8116/analysis emdatabank-logo.png PDB 5IRE – EMDB 8116 (Zika virus) Visual Analysis functionality developed in 2009-2012 BBR with OME/Jason Swedlow EMBL_EBI_RGB_InversedUpdate.png PDBe_logo_white.png Ligands in proteins •So you have successfully navigated all the hazards so far have great data, well integrated, successful MR, refinement model building, Ramachandran analysis …… •You have density in the active site and the whole point of the structure is to find how the interesting drug candidate ligand binds ……. •Here be dragons! https://upload.wikimedia.org/wikipedia/commons/5/54/Liber_Floridus_page_scan_A%2C_ca._1460.jpg