RosettaBackrub--a web server for flexible backbone protein structure modeling and design

Abstract

The RosettaBackrub server (http://kortemmelab.ucsf.edu/backrub) implements the Backrub method, derived from observations of alternative conformations in high-resolution protein crystal structures, for flexible backbone protein modeling. Backrub modeling is applied to three related applications using the Rosetta program for structure prediction and design: (I) modeling of structures of point mutations, (II) generating protein conformational ensembles and designing sequences consistent with these conformations and (III) predicting tolerated sequences at protein-protein interfaces. The three protocols have been validated on experimental data. Starting from a user-provided single input protein structure in PDB format, the server generates near-native conformational ensembles. The predicted conformations and sequences can be used for different applications, such as to guide mutagenesis experiments, for ensemble-docking approaches or to generate sequence libraries for protein design.

Figure 1.

Application (I) Modeling of Point Mutations. Shown are the input form for a prediction of multiple point mutations (top) and the corresponding output (bottom). The form contains fields to enter a job name to identify jobs later and fields to upload or provide a link to a PDB file. The general simulation settings are identical for all three applications, i.e. the Rosetta version (if applicable) and the number of structures generated. The application-specific settings are the chain identifier (Chain ID), the residue position of the mutation site (Res ID), the target amino acid (AA) and the Backrub radius in Ångströms. The output shows the table of all mutations made in a PDZ-domain from Mus musculus (PDB ID code: 2PDZ) and the Jmol plug-in with the resulting modeled structures. The initially uploaded structure is shown in red.

Figure 2.

Application (II) Backrub Ensemble Design. The input form (top) for Backrub Design contains the general settings (as in Figure 1) and in addition application-specific fields for the simulation temperature (controlling the amplitude of the structural variation in the modeled conformational ensemble), and the segment length for the Backrub simulation, as well as the number of sequences generated per structure. The output (bottom) features the backbone traces of 10 generated ensemble members in green and a representation of the dynamic regions of an Ubiquitin ensemble (from blue to white to red with increasing variability) mapped onto the ubiquitin starting structure (PDB ID code: 1UBQ). Furthermore a sequence motif of the designed core residues is shown.

Figure 3.

Application (III) Interface Sequence Plasticity Prediction. The input form (top) holds the general settings (Figure 1) and parameters for the definition of the interface partners and the designed residues. The output (bottom) shows an overlay of the structures of the best scoring sequence for each of 10 initial backrub structures of the human growth hormone receptor complex (PDB ID code: 1A22) and a box-plot of the predicted frequencies of designed amino acid residues at a given designed sequence position (position 67 in chain A).