W236 Nucleic Acids Research, 2006, Vol. 34, Web Server issue uses Monte Carlo optimization with simulated annealing. Starting from a random sequence, single amino acid sub- stitutions or rotamer switches are accepted based on the Metropolis criterion. The simulation starts at a very high temperature where almost all substitutions are accepted and ﬁnishes at 0 . Approximately 1 million rotamer substitutions are attempted per 100 residues being varied. Independent simulations in which every residue in the protein is allowed to vary generally converge to sequences that are 70–80% identical to each other. SERVICES Protein design The RosettaDesign server returns low energy sequences for target protein structures. The protein backbone remains ﬁxed during the simulation. Side chain conformation prediction Given a protein structure and sequence, the RosettaDesign server can be used to predict the lowest energy conformations of the side chains. INPUTS, OUTPUTS AND JOB OPTIONS Registration To receive results via email users must register. Alternatively, users can access the web server as a ‘guest’. In this case they must return to the web site to retrieve results. Input files PDB ﬁle: users must submit a ﬁle with the atomic coordinates of the protein that will be the template for design. The coordin- ates must be in PDB format. There can be gaps in the structure, but each residue must have a complete set of backbone heavy atoms—N’, C’, O and Ca. The residues can be missing side chain atoms. Resﬁle: the resﬁle speciﬁes which sequence positions will be varied, and which amino acids will be considered at each position. Users can also request that the native amino acid be kept at a particular sequence position, but allow the side chain to adopt a new conformation. The resﬁle can be created on the web site using point-and-click operations (Figure 1) or a user can upload his or her own resﬁle. The server will check the integrity of the uploaded resﬁle to ensure the correct format. The resﬁle created on the web site with point-and-click opera- tions can also be saved for future use. A full description of the format for a resﬁle is provided in the documentation section of the web site. Figure 1. Interface for choosing which sequence positions to vary.