While most NMR-based structure determination packages use NOE data almost exclusively, it has recently been shown that it is possible to determine protein struc- tures using only chemical shift data (i.e. without NOEs). Indeed, several stand-alone programs and web servers have been developed for this purpose, including Cheshire (8), CS-Rosetta (9) and CS23D (10). However, chemical shift-based structure determination is not as robust or as rapid as NOE-based structure determination—especially for novel protein folds (10) or large structures (8,9). Indeed, some of these programs take hundreds or even thousands of CPU hours to complete their calculations. Furthermore, none of these shift-based structure determination techniques takes advantage of the additional information contained in NOE constraint data. Ideally, what is needed for NMR-based structure deter- mination is a tool that is: (i) easy to use (a ‘single click’ solution); (ii) platform independent (a web server); (iii) fast (generating structures in minutes, not hours); (iv) capable of handling both NOE and chemical shift data; and (v) smart enough to perform the NMR equiva- lent of ‘molecular replacement’ by taking advantage of well-known structural bioinformatics techniques such as threading, homology modeling and fragment-based assembly. Here, we wish to describe a simple web server, called GeNMR, that addresses these needs. In particular, GeNMR is a next generation, highly generalizable system for rapidly generating protein structures by NMR using a combination of genetic algorithms and simulated anneal- ing. It allows the rapid ( 15 min) and accurate determi- nation of protein structures using any combination of chemical shifts and/or NOEs as input. GeNMR builds on nearly 15 years of research in our lab related to using chemical shifts and other NMR data to identify protein secondary structures (11), to predict torsion angles (12), to identify protein folds (13), to predict protein ﬂexibility (14) and to calculate protein structures (10). GeNMR greatly simpliﬁes the task of protein structure determina- tion as users do not have to install or become familiar with complex stand-alone programs or obscure format conver- sion utilities. Tests conducted on a sample of 90 proteins from the BioMagResBank indicate that GeNMR pro- duces high-quality models for all protein queries, regard- less of the type of NMR input data. Additional details about GeNMR are given below. PROGRAM DESCRIPTION GeNMR is composed of two parts, a front-end web-inter- face (written in Perl and HTML) and a back-end consist- ing of eight diﬀerent alignment, structure generation and structure optimization programs (written in Java, Perl, Python and C/C++) along with three local databases (Figure 1). Users must provide at least two types of data: (i) sequence data (in raw or FASTA format); (ii) chemical shift data in either SHIFTY (15) or BMRB (16) formats and (iii) NOE data in either CYANA (4) or XPLOR (5) format. The sequence data is needed to handle Nucleic Acids Research, 2009, Vol. 37, Web Server issue W671 Figure 1. A ﬂow chart outlining the general structure of the GeNMR web server and the programs that it calls to generate protein structures from chemical shift and/or NOE data. The speciﬁc function of each of the named programs is explained in the text. the (common) situation where there are missing NOE or chemical shift assignments. Without the complete protein sequence, GeNMR would typically generate concatenated (i.e. incorrect) structures in the regions where NOE or chemical shift data was missing. The sequence, chemical shift and/or NOE ﬁles may be either pasted or typed into the text box or uploaded through a ﬁle browse button. The output for a typical GeNMR structure calculation consists a user-deﬁned set of lowest energy PDB coordinates in a simple, download- able text format. A hyperlink to view the single lowest energy structure through the WebMol viewer (17) is also provided. In addition, details about the overall energy score (prior to and following energy minimization), NOE violations, torsion angle violations and chemical shift correlations (between the observed and calculated shifts) is provided at the top of the output page. If the structure calculation fails to converge to a reasonable value, a warning is printed at the top of the page. Generally, if a structure fails to converge or exhibits a high number of violations, users should investigate the correctness of their NOE or chemical shift assignments. There is no limit on the number of queries a user may send and so resubmitting to the server with new or corrected data is generally encouraged. Details about the GeNMR energy function, reasonable values for chemical shift cor- relations and reasonable values for torsion angle and NOE violations is provided in the Documentation link on the GeNMR home page.