CING: an integrated residue-based structure validation program suite

Abstract

We present a suite of programs, named CING for Common Interface for NMR Structure Generation that provides for a residue-based, integrated validation of the structural NMR ensemble in conjunction with the experimental restraints and other input data. External validation programs and new internal validation routines compare the NMR-derived models with empirical data, measured chemical shifts, distance- and dihedral restraints and the results are visualized in a dynamic Web 2.0 report. A red-orange-green score is used for residues and restraints to direct the user to those critiques that warrant further investigation. Overall green scores below ~20 % accompanied by red scores over ~50 % are strongly indicative of poorly modelled structures. The publically accessible, secure iCing webserver ( https://nmr.le.ac.uk ) allows individual users to upload the NMR data and run a CING validation analysis.

Fig. 1

Schematic outline showing the data flow and software components involved in CING (yellow boxes). External programs interfaced to CING, CCPN services/APIs and wwPDB repositories are represented in brown-shades, green and dark-orange, respectively. External programs (olive green) can access the iCing web user interface (purple) through the dedicated iCing robot

Fig. 2

Overview of the CING analysis for PDB entry 2kq3 (Wang et al. 2010). a Project page of entry 2kq3. b Ribbon representation of the first conformer of the 2kq3 ensemble, colour-coded according to residue ROG score. Residues not included by the range selection are coloured grey. c Per-residue circular variance and positional RMSD values as function of residue number. DSSP-derived secondary structure analysis is shown on top. Coloured bars indicate relative solvent accessibility of each residue, as calculated by the WHAT_CHECK’s INOCHK routine, where red indicates “more exposed than usual” and blue means “more buried than usual”. Residues not included by the range selection are coloured grey

Fig. 3

Residue analysis of 2kq3 residue Leu14. a The CING Residue page for Leu14. Structure analysis is displayed on the left and shown in detail in panels (b)–(d); experimental data involving Leu14 shown on the right. b Ramachandran plot of Leu14. Residue specific background colouring derived from the WHAT_CHECK protein reference database for helical (blue); β-sheet (yellow) and coil (green) regions as determined by DSSP. Grey areas define low-density transition regions. Experimental ϕ/ψ restraints are indicated by the transparent orange box. Experimental ϕ/ψ values of the individual members of the ensemble are indicated by green plus signs; its average value by an open circle. Open square box denotes an automatically Talos+ derived ϕ/ψ region on the basis of the experimental chemical shifts. c Janin plot of Leu14; colouring as in (b). D) D1D2 plot (see text) of Leu14; colouring as in (b)

Fig. 4

CING analysis of experimental data. Expandable, searchable, tabular displays are used, that can be sorted on any column. Table entries are directly hyperlinked to the corresponding Residue and Atom pages. a ‘Distance restraints’ page. Check boxes allow for additional information to be displayed. Only critiqued restraints are shown. b Atoms page. Only critiqued atoms are shown

Fig. 5

Comparison of the fraction of well-defined residues with a CING ROG score green (good) versus the fraction of residues that fall in the most favoured region of the Ramachandran plot according to PROCHECK-NMR. The horizontal line at 20 % indicates the CING cut off for the minimum percentage of green residues, beyond which the molecule as a whole is flagged red regardless of the allocation of orange and red residues (see Table 2). The plot shows a significant number of entries on the bottom right for which a high percentage of ‘PC core’ residues are not ‘green’ in CING. Data is shown for the 6,383 NMR PDB entries selected from NRG-CING to have at least ten models and 30 or more amino acid residues. Indicated with an open circle (at 77.9 %) is PDB entry 2kq3 that is discussed as an example in the text