Figure 2. Illustration of how GeNMR is able to generate the structure of ubiquitin using only experimental NOE constraints [scenario (d) in the article]. The initial structure (purple) is a largely random coil poly- peptide ( 18 A˚ RMSD), an intermediate structure (cyan) contains most of the secondary structure elements ( 3.9 A˚ RMSD), while the lowest energy structure (yellow), reﬁned via simulated annealing, is within 1.2 A˚ of the known X-ray structure. The last panel shows the ﬁnal ensemble (or bundle) of structures generated by GeNMR. torsion-based genetic algorithm using a chemical shift- based, a NOE-based and a knowledge-based potential. Table 1 lists the results of tests conducted on ﬁve proteins with experimental NOE and experimental chemical shift data as chosen from the BioMagResBank with 85–99% sequence identity to known structures (the exact match was removed in this test). These data show that GeNMR produces structures that are very similar to the known structures (<1.5 A˚ RMSD). Typical structure gen- eration times for this scenario are about 15–20 min, depending of the server load and the size of the structure. Scenario (f): NOE+Shift with no homologue In this situation, homology modeling has failed and, as a result, several alternative routes to structure modeling are attempted including chemical shift threading and sub-fragment assembly. If these approaches also fail to quickly identify a model, GeNMR employs the progres- sive NOE reﬁnement technique described in ‘Scenario d)’. Nucleic Acids Research, 2009, Vol. 37, Web Server issue W675 Speciﬁcally, an initial structure is generated using shift- derived (as well as NOE-derived) torsion angle con- straints. Subsequently a genetic algorithm reﬁnes these initial structures using a progressive three-step approach that attempts to satisfy the short range NOEs ﬁrst, then the medium range NOEs and ﬁnally the long range NOEs. The resulting ‘rough’ structure is then reﬁned using simu- lated annealing with XPLOR-NIH. Table 1 lists the results of tests conducted on ﬁve proteins with experimen- tal NOE and chemical shift data where the homology modeling step has been removed from the GeNMR pipe- line. These data show that GeNMR produces structures that are very similar to the known structures (<2.0 A˚ RMSD). Typical structure generation times for this sce- nario are about 20–25 min, depending of the server load and the size of the structure. LIMITATIONS GeNMR was primarily designed to make NMR-based structure determination simpler, faster and easier. However, in striving towards a simpliﬁed, user-friendly solution certain compromises had to be made. One com- promise concerns GeNMR’s limited support for ambigu- ous NOE constraints. While some stereo-speciﬁc ambiguity in NOEs is allowed in the data input ﬁles, GeNMR does not have the capacity of programs such as ARIA (7) to handle unassigned NOEs. Rather, GeNMR assumes that the NOE input and/or chemical shift assignments are mostly complete and mostly correct. With the possible exception of certain high throughput structural genomics data sets, we believe this is the most prevalent situation for NMR data sets. A second compromise concerns GeNMR’s capacity to handle other NMR-derived constraints. Currently GeNMR does not handle or process J-coupling or residual dipolar coupling (RDC) restraints. While J-couplings are commonly measured and widely accommodated by many other NMR structure generation programs, we have found that this information is generally less accurate than the information that can be derived directly or indir- ectly from chemical shifts (10,12). Since chemical shifts are a necessary pre-requisite to measuring J-couplings, we believe that by excluding J-couplings from GeNMR’s input ﬁelds we are actually making structure determina- tion a little simpler and a little less laborious for the experimentalist. On the other hand, residual dipolar cou- plings (RDCs) do provide somewhat more information than J-couplings (26). In particular, RDCs provide criti- cal, long range information about the relative orientation of secondary structure elements within proteins. Currently, GeNMR does not yet support the inclusion of RDC data. Eﬀorts are underway to add this feature to the server and it is expected that RDC calculations will be supported by the summer of 2009. While not expli- citly supported with a separate data entry ﬁeld, H-bond constraints can be submitted to the GeNMR server as a part of the NOE distance restraint ﬁle. In addition, GeNMR is also able to extract H-bonding data from