Nucleic Acids Research, 2008, Vol. 36, Web Server issue W497 infrequent. With the emergence of structural genomics and structural proteomics, there have been a number of eﬀorts aimed at accelerating the NMR structure determi- nation process or extending the upper size limits for which NMR can be used. Almost all of these have focused on either eliminating the chemical shift assignment step (7,8) or automating the assignment of NOEs (9,10). Rather than looking at ways of improving NOE measurements, a small minority of NMR researchers have been looking at ways of skipping the NOE step altogether and going directly from chemical shift assign- ments to 3D structures (11–14). Unlike NOEs, chemical shifts are easy to measure and not particularly sensitive to protein size restraints. Furthermore, chemical shifts provide exquisitely detailed information about the cova- lent structure of atoms and molecules. Indeed, when properly analyzed, chemical shifts can be used to infer secondary structure, ﬂexibility, dihedral angles, side-chain orientations, hydrogen-bonding interactions and electro- static or ionic interactions (11). Recently, several papers have appeared which suggest that reasonably accurate protein structures can be determined directly from chem- ical shifts (11–14). Of particular interest is the recent work by Cavalli et al. (13) and Shen et al. (14), both of which showed that accurate 3D structures for a number of small (<120 residue) proteins could be calculated using a combination of NMR chemical shifts, fragment assembly and heuristic potential functions. However, the computa- tional eﬀort required for this process is quite extreme, with individual structures requiring 1000s of CPU hours of calculation. Here, we wish to describe a simple web server, called CS23D (chemical shift to 3D structure), that allows the rapid ( 15 min on 1 CPU) and accurate determination of protein structures using only assigned chemical shifts as input. CS23D builds on nearly 15 years of research in our lab related to using chemical shifts to identify secondary structures (15), to predict torsion angles (16), to identify protein folds (11), to predict protein ﬂexibility (17), to reﬁne protein structures (11) and to correct chemical shift referencing (18). In particular, CS23D combines a techni- que we call maximal subfragment assembly with other techniques such as chemical shift threading, de novo struc- ture generation (via Rosetta), chemical shift-based torsion angle prediction and chemical shift reﬁnement to generate and reﬁne the protein coordinates. Tests conducted on >100 proteins from the BioMagResBank (for which chemical shifts and 3D structures are available) indicate that CS23D converges (i.e. ﬁnds a solution) for about 95% of protein queries. The resulting structures have a back- bone RMSD <2 A˚ of the known structure and generally exhibit better geometry and chemical shift agreement than conventionally determined NMR structures. Additional details about CS23D are given subsequently. PROGRAM DESCRIPTION CS23D is composed of two parts, a front-end web interface (written in Perl and HTML) and a back-end consisting of eight diﬀerent alignments, structure generation and Figure 1. A ﬂow chart outlining the general structure of the CS23D web server and the programs that it calls to generate protein structures from chemical shift data. The speciﬁc function of each of the named programs (THRIFTY, PEPMAKE, ROSETTA, etc.) is explained in the text. structure optimization programs (written in Java, Perl, Python and C/C++) along with three local databases (Figure 1). The front-end accepts both SHIFTY (18) and BMRB (19) formatted chemical shift ﬁles. The shift ﬁles may be either pasted or typed into the text box or uploaded through a ﬁle browse button. The output for a typical CS23D structure calculation consist of a set of 10 lowest energy PDB coordinates in a simple, downloadable text format. A hyperlink to view the single lowest energy structure through the WebMol viewer (20) is also provided. In addition, details about the overall energy score (prior to and following energy minimization), chemical shift corre- lations (between the observed and calculated shifts) and torsion angle violations is provided at the top of the output page. If the structure calculation failed to converge to a reasonable value, a warning is printed at the top of the page. Details about the CS23D energy function, reason- able values for chemical shift ‘energies’ and reasonable values for torsion angle violations is provided in the Documentation link on the CS23D home page. ALGORITHMS, DATABASES AND TESTING A ﬂow chart describing the processing logic used in CS23D is shown in Figure 1. As seen in this diagram,