W496–W502 Nucleic Acids Research, 2008, Vol. 36, Web Server issue doi:10.1093/nar/gkn305 Published online 30 May 2008 CS23D: a web server for rapid protein structure generation using NMR chemical shifts and sequence data David S. Wishart1,2,3,*, David Arndt1, Mark Berjanskii1, Peter Tang 1 , Jianjun Zhou1 and Guohui Lin1 1Department of Computing Science, 2Department of Biological Sciences, University of Alberta and 3National Research Council, National Institute for Nanotechnology (NINT), Edmonton, AB, Canada T6G 2E8 Received February 1, 2008; Revised April 10, 2008; Accepted April 30, 2008 ABSTRACT CS23D (chemical shift to 3D structure) is a web server for rapidly generating accurate 3D protein structures using only assigned nuclear magnetic resonance (NMR) chemical shifts and sequence data as input. Unlike conventional NMR methods, CS23D requires no NOE and/or J-coupling data to perform its calculations. CS23D accepts chemical shift files in either SHIFTY or BMRB formats, and produces a set of PDB coordinates for the protein in about 10–15 min. CS23D uses a pipeline of several preexisting programs or servers to calculate the actual protein structure. Depending on the sequence similarity (or lack thereof) CS23D uses either (i) maximal subfragment assembly (a form of homology modeling), (ii) chemical shift threading or (iii) shift-aided de novo structure prediction (via Rosetta) followed by chemical shift refinement to generate and/or refine protein coordinates. Tests conducted on more than 100 proteins from the BioMagResBank indicate that CS23D converges (i.e. finds a solution) for >95% of protein queries. These chemical shift generated structures were found to be within 0.2–2.8 A˚ RMSD of the NMR structure generated using conventional NOE-base NMR methods or conventional X-ray methods. The performance of CS23D is dependent on the com- pleteness of the chemical shift assignments and the similarity of the query protein to known 3D folds. CS23D is accessible at http://www.cs23d.ca. INTRODUCTION Over the past 20 years, nuclear magnetic resonance (NMR) spectroscopy has emerged as one of the most powerful physical methods to study the structure and dynamics of proteins. It combines the strengths of such methods as ﬂuorescence and circular dichroism for characterizing proteins in solution with the power of X-ray crystallography to perform these characterizations atom by atom. Aside from X-ray crystallography, NMR is the only other method available that allows the 3D structure of proteins to be determined to atomic resolution (1). Furthermore, NMR is the only known method that allows protein structures and dynamics to be determined in solution (i.e. near physiological conditions). To date, more than 7000 peptide and protein structures have been determined by NMR and deposited into the PDB (2). Each one of these structures was determined using a three- step process pioneered by Kurt Wuthrich and colleagues (3) in the early 1980s. This process involves: (i) determin- ing the chemical shift assignments of the target protein; (ii) measuring the inter- and intra-residue 1H NOEs (nuclear Overhauser enhancements) to generate short- range distance constraints and (iii) using the NOE-derived constraints to perform molecular dynamics or distance geometry to calculate the 3D structure of the protein. Over the years, slight improvements to this process have occurred with the inclusion of more experimental con- straints such as heteronuclear J-couplings (4) and residual dipolar couplings (5) or the introduction of improved conformational sampling protocols such as simulated annealing (6). Nevertheless, the central concept of using NOE-based methods to determine the 3D structure of proteins has remained essentially unchanged for nearly a quarter century. While NOE-based methods are generally robust and well-proven, they are not without their faults. In partic- ular, the measurement of 1H NOEs is both time- consuming and error-prone. Furthermore, NOEs becomes progressively less useful and more diﬃcult to measure as the size of the proteins increases. This constraint means that the determination of 3D structures by NMR for proteins larger than 200 residues is very diﬃcult and *To whom correspondence should be addressed. Tel: +780 492 0383; Fax: +780 492 5205; Email: david.wishart@ualberta.ca 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.