MOLPROBITY: structure validation and all-atom contactanalysis for nucleic acids and their complexesIan W. Davis, Laura Weston Murray, Jane S. Richardson and David C. Richardson*Department of Biochemistry, Duke University, Durham, NC 27710-3711, USAReceived February 20, 2004; Revised and Accepted March 25, 2004ABSTRACTMOLPROBITY is a general-purpose web service offeringquality validation for three-dimensional (3D) struc-tures of proteins, nucleic acids and complexes. It pro-vides detailed all-atom contact analysis of any stericproblems within the molecules and can calculate anddisplay the H-bond and van der Waals contacts in theinterfaces between components. An integral step inthe process is the addition and full optimization of allhydrogen atoms, both polar and nonpolar. The result sare reported in mult iple forms: as overall numericscores, as lists, as downloadable PDB and graphicsfiles, and most notably as informative, manipulable 3Dkinemage graphics shown on-line in the KING viewer.This service is available free to all users at http://kinemage.biochem.duke.edu.INTRODUCTIONExperimental structure determination has rather differentstrengths and weaknesses for nucleic acids than for the proteincase, where most validation methods were developed. Posi-tions and interactions of the bases can be quite accuratelydetermined, but for both X-ray and NMR methods, much ofthe sugar–phosphate backbone is quite difficult and ambigu-ous, with too many degrees of freedom relative to the observ-able data. Figure 1 contrasts the reproducibly well-fit all-atomcontacts of RNA bases with the frequent steric clashes ofH-atoms seen in RNA backbone in the 2.5–3 A˚resolutionrange typically attained for large, biologically importantnucleic acids. Structural biologists fully appreciate the diffi-culty with backbone, but so far have lacked good tools fordiagnosis or remediation. While existing torsion angle ana-lyses (1,2) are substantially correct, errors in one or moreangles, resulting in impossible conformations, are common(3,4). Traditional clash analysis tools (5,6) do not use thehydrogens, which are especially revealing in this case. Theall-atom contact analysis (7) featured on the MOLPROBITY siteprovides a simple but powerful diagnostic tool for nucleic acidbackbone, and its local and directional nature can even suggesthow to make improvements. That same analysis gives theend-users of nucleic acid structures an easy way to assesslocal accuracy in a region of interest. An all-atom contactvisualization of the interface between two molecules alsogives a direct, intuitive way to see the H-bond and van derWaals interactions.METHODSMOLPROBITY is implemented using the scripting language PHPin conjunction with the Apache web server. External programswritten in C, Java and other languages are invoked byMOLPROBITY to analyze the structures and generate kinemagevisualizations. The MOLPROBITY PHP code collects and parsesthe output of these programs and presents the results in ameaningful way. PHP code is also responsible for creatingthe user interface (in the form of web pages), controllingprogram flow (e.g. which tools are available when) and manag-ing user data over the lifetime of a session.Input is a PDB-format macromolecular coordinate file fromthe Protein Data Bank (8) or the Nucleic Acid Database (9), orcan be uploaded from the results of a structure determination.We identify structures used in the examples here by both PDBand NDB codes (e.g. 1JJ2/rr0033). All hydrogen atoms, bothpolar and nonpolar, are added by the REDUCE program (10).REDUCE’s expert system uses the information from both hydro-gen bonding and all-atom steric compatibility to fully optimizelocal H-bond networks and correct 180‘flips’ for Asn, Glnand His orientations in the proteins. Base tautomers are notvaried, and only the first layer of waters is considered, tominimize sensitivity to errors in positioning and to keep theH-bond networks small enough for deterministic analysis. Thisstep produces a commented, modified PDB file and also agraphic display of the consequences of each proposed side-chain flip; any changes deemed unacceptable can be overrid-den by the user.With all hydrogens present, all-atom contacts are then cal-culated by PROBE (7), which uses traditional van der Waalsradii (11) for most atoms and 1.0 A˚for polar H, in a rolling-probe algorithm that leaves a dot when the 0.25 A˚-radius probe*To whom correspondence should be addressed: Tel: +1 919 684 6010; Fax: +1 919 684 8885; Email: [email protected] online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open accessversion of this article provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the originalplace of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivativework this must be clearly indicated.ª 2004, the authorsNucleic Acids Research, Vol. 32, Web Server issue ª Oxford University Press 2004; all rights reservedNucleic Acids Research, 2004, Vol. 32, Web Server issue W615–W619DOI: 10.1093/nar/gkh398intersects another not-covalently-bonded atom (7). The resultsinclude a clustered list of disallowed atom pair overlaps>0.4 A˚, an overall clash score (number of bad overlaps per1000 atoms) and two kinemage graphics displays (12,13) ofcontacts for the whole structure. Van der Waals contacts areshown as back-to-back patches of green or blue dots on thesurfaces of non-covalent atom pairs within 0.5 A˚of touching(as in Figure 1a); hydrogen bonds are shown as lenses of palegreen dots outlining the interpenetrating surfaces of a donorand acceptor. Steric clash overlaps are emphasized with spikesrather than dots, progressing from yellow to hot pink as theclash becomes truly serious beyond 0.4 A˚overlap (Figure 1b).The graphics in MOLPROBITY are displayed interactively on-line in the KING Java kinemage viewer. KING can smoothlyrotate very large structures, with extensive user control ofdisplay and measurement options, and it shows all of thecontact dot surfaces, H-atoms, animations, ribbons, two-dimensional (2D) plots and so on that are produced by theMOLPROBITY analyses. Electron density maps (e.g. from theElectron Density Server at http://fsrv1.bmc.uu.se/eds/) can