Figure 10. RCM analysis of FLS2, EFR, and PGIP performs as well as other computational methods. Y-axis: scores for each amino acid according to (A) positive selection analysis (nonsynon- ymous/synonymous substitution rates); (B) optimal docking area (scale is negative so that more likely docking sites are higher along y-axis); and (C) Consurf; all are graphed with respect to RCM score on the x- axis. Residues that were mutationally tested in the present study or in [35] are indicated as having no significant impact on function (white) or a significant impact on function (black) when mutated. Functional residues (in black) scored significantly higher than non-functional residues (in white) in all analyses completed (T-test; p,.05). doi:10.1371/journal.pone.0021614.g010 impact function for reasons other than overall protein structure and integrity. Future improvements to RCM are anticipated. Output of results onto a generic 3D LRR model as a pdb file may be useful, for viewing and manipulation in PyMOL, Swiss-PdbViewer or similar programs. The LRR repeat models developed in an RCM run currently require hand-curation, but greater automation of LRR matrix building is anticipated, for instance through use of HMMER (http://hmmer.janelia.org) to build consensus logos of the proteins being compared. RCM at this stage is limited to use with LRR domains, but because RCM utilizes typical repeat References Repeat Conservation Mapping domain structures rather than precise spatial data, the method is likely to be adaptable to other repeat proteins that have a known repetitive structure. This includes armadillo, tetratricopeptide, and ankyrin repeats. Adaptation of conservation mapping to ankyrin repeats could be particularly insightful, as these domains are the focus of artificial evolution towards novel ligands [90]. The repeat conservation mapping approach is a predictive method that can be utilized to identify key similarities and differences among groups of homologous LRR-containing pro- teins. The RCM program predicts functional sites in LRR domains, which may facilitate basic structure-function studies or in vitro protein evolution toward modified functions for these widespread and biologically significant domains. The current implementation is functional for LRRs, but it should be modifiable for use with other repeat-containing protein domains. Supporting Information Figure S1 Additional RCM maps for LRR domains for which there are solved LRR+ligand structures. (PDF) Figure S2 Maps of individual residue conservation scores, at an intermediate step in RCM prior to calculation of regional conservation scores. (PDF) Figure S3 Identification of an EFR ortholog. (PDF) Figure S4 Examples of ROS and callose assays of EFR double-alanine mutant alleles expressed under control of native EFR promoter in transgenic Arabidopsis T1 seedlings. (PDF) Figure S5 Presence of EFR-HA double-alanine mutant proteins, whether functional or not, in transgenic Arabidopsis seedlings. (PDF) Figure S6 RCM maps of FLS2 generated using different FLS2 ortholog sets. (PDF) Figure S7 Analysis of FLS2 and PGIP by other compu- tational methods. (PDF) Description of the RCM method. Text S1 (DOC) Acknowledgments The authors thank Dan Huttenlocher for initial contributions to the RCM programming approach, and Mark Craven for helpful conversations. Author Contributions Conceived and designed the experiments: LH AB. Performed the experiments: LH TK LF JF-R. Analyzed the data: LH AB TK LF JF-R. Contributed reagents/materials/analysis tools: LF JF-R. Wrote the paper: LH AB. Implemented RCM Program: VR XC RG. Provided editorial comments on the draft manuscript: LF VR XC TK LF JF-R RG AB. 1. Kobe B, Kajava AV (2001) The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol 11: 725–732. 2. Schmitz-Linneweber C, Small I (2008) Pentatricopeptide repeat proteins: a socket set for organelle gene expression. Trends Plant Sci 13: 663–670. PLoS ONE | www.plosone.org 12 July 2011 | Volume 6 | Issue 7 | e21614