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BackgroundCrescendo is a program which identifies functional sites in proteins. The conservation of amino acid residues has been shown to be strongly dependent on the environment in which they occur in the folded protein and amino acid substitution tables that give the likely substitutions of amino acids in particular local environments have been derived.This method uses these substitution tables to distinguish those restraints placed on protein structure from additional restraints due to particular functions mediated by interactions with other molecules. For more information see Chelliah et al. (2004) Running CrescendoThere are two ways in which Crescendo can be run. You can either upload your PDB and multiple sequence alignments to be used or you can simply give the name (and chain identifier if applicable) of your protein of interest and have an alignment created automatically. The first option is recommended as an automatically generated alignment from top scoring BLAST hits may not offer enough sequence variation for Crescendo to accurately identify functionally constrained sites.Scoring SystemsWhen uploading your own files, there are two optional scoring systems used which are sequence-based and rely on the environment-specific substitution tables.
The overall score is calculated for each amino acid. Alignment File Format
>P1;2MM1 structure:2MM1: 1 : : 153 : :myoglobin:Homo sapiens: 2.80:15.8 ----------GLSDGEWQLVLNVWGKVEA--DIPGHGQEVLIRLFKGHPE TLEKFDRFK-HLKSEDEMKASEDLKKHGATVLTALGGILKKKG-----HH EAEIKPLAQSHATKH--KIPVKYLEFISEAIIQVLQSKHPG-DFGADAQG AMNKALELFRKDMASNYKELGFQG* >P1;1PMB structure:1PMB: 1 :A: 153 :A:myoglobin:Sus scrofa:2.50:18.5 ----------GLSDGEWQLVLNVWGKVEA--DVAGHGQEVLIRLFKGHPE TLEKFDKFK-HLKSEDEMKASEDLKKHGNTVLTALGGILKKKG-----HH EAELTPLAQSHATKH--KIPVKYLEFISEAIIQVLQSKHPG-DFGADAQG AMSKALELFRNDMAAKYKELGFQG* >P1;1YMB structure:1YMB: 1 : : 153 : :myoglobin:Equus caballus: 1.90:15.5 ----------GLSDGEWQQVLNVWGKVEA--DIAGHGQEVLIRLFTGHPE TLEKFDKFK-HLKTEAEMKASEDLKKHGTVVLTALGGILKKKG-----HH EAELKPLAQSHATKH--KIPIKYLEFISDAIIHVLHSKHPG-DFGADAQG AMTKALELFRNDIAAKYKELGFQG* >P1;gi|127663|sp|P02181|MYG_INIGE sequence ----------GLSDGEWQLVLNIWGKVEA--DLAGHGQDVLIRLFKGHPE TLEKFDKFK-HLKTEAEMKASEDLKKHGNTVLTALGGILKKKG-----HH EAELKPLAQSHATKH--KIPIKYLEFISEAIIHVLHSRHPG-DFGADAQA AMNKALELFRKDIAAKYKELGFHG* Program OutputOnce Crescendo has run successfully you can view the alignment file (important if you have opted to have Crescendo run automatically). You also have the option of downloading two output files: crescendo.out and crescendo.kin.
Another output option is also available. You can download a PDB file (or your protein of interest) with the Crescendo scores in the B-factor column. Automatic Sequence PartitioningIf you have chosen to run Crescendo automatically, the alignment will have been generated from BLAST top scoring hits. As these may not represent the required sequence diversity for Crescendo to optimally predict the functional sites, we have chosen to split these sequences up based upon their evolutionary relationships. A phylogenetic tree will be generated (using the Neighbor-Joining method) which will then be partitioned at different intervals based on branch length to split the tree up into groups of varying divergence. Crescendo will be run on each of these groups and the results for each will be shown separately on the kinemage applet detailed below. As well as running on each of the subgroups, Crescendo will also run on the whole set of sequences for comparison. A PDF of the phylogenetic tree, with associated partitions can be viewed by clicking on the link.The alignment files containing the sequences in each subgroup are also available to be viewed or downloaded. Predicted Functional ResiduesThe scores of individual amino acids are mapped onto the protein structure, smoothed, converted into a grid of conservation density and this grid contoured. The kinemage of your protein structure is shown and its proposed functional site can be seen by clicking on the "contours" box (if you have uploaded your own files) or by clicking the "ALL_SEQS" box if running Crescendo automatically. The different contour cutoffs can be selected.To see a list of residues which crescendo predicts to be functional at these various cutoffs you can select a value from the drop down box. These residues are then displayed on the screen. The higher scoring regions in the contouring represent residues within the alignment with greater functional restraints on their evolution. Results will be present on the server for at least 30 minutes. References
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