The Protein Crystallisation Construct Designer (ProteinCCD) is a tool to help deciding how to choose promising constructs for protein expression and crystallisation. A specific feature of CCD is that since it starts from the DNA sequence, it keeps track of the protein-DNA relationship. Thus, although all the analysis and construct choice is being done in the protein level, CCD can be used to suggest oligonucleotides for PCR amplification of the chosen constructs, since it also knows the DNA sequence.

•CCD gathers various predictions for your protein and displays the information aligned for easy inspection.

•CCD allows you then decide where possible constructs should start and stop by simply clcking with the mouse.

•CCD does not design the construct automatically.

•CCD will suggest oligonucleotides for PCR amplification, based on either TM or simply a user choice for length.

•CCD will not check the oligonucleotides for secondary structure or for corss-hybrization in other sequences and is totally unware of restriction sites since it was designed to help in LIC cloning.

In other words CCD is a tool to help you make smart choices, and will not do anything smart for you.

Usage:

1.Input an oper reading frame of the DNA sequence of interest in the input text field and press submit
The sequence is allowed to contain line breaks, numbers etc
A CCD status window should inform you of the progress of the predictions.

2.Once all the results have been harvested, set any number of start and stop points for your construct. You can do this by clicking 'Mark start' or 'Mark stop' first, followed by clicking on the protein sequence at desired start or stop locations. Selected starts will be highlighted in green, selected stops in red

3.Clicking 'Get' will give you all the selected start and stop oligos. The length of the oligos will be based on melting temperature (when 'T' is selected) or a given number of basepairs (when 'N' is selected). For Tm, the following formula is used: Tm = 64.9 + 41.0 x (number of G's and C's in the primer - 16.4)/N

CCD is a meta server; all predictions are retrieved from other web services
See the web services for details on the methods used

Currently, the following options are available:

•Secondary structure predictions:

◦All from Network Protein Sequence @nalysis (NPS@).

•Disorder predictions:

◦RONN :Regional Order Neural Network

◦IUPRED: Prediction of Intrinsically Unstructured Proteins

•Other features:

◦Lupas : From NPS@

◦Phobius : A combined transmembrane topology and signal peptide predictor

•Domains:

◦DLP-SVM: Domain Linker Prediction using SVM

◦SMART: Simple Modular Architecture Research Tool