Modeller protocol BCKDHA

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Modeller

To get Modeller started, we first had to create a few starting files.

For each of our template sequences 1w85 and 2r8o we had to create a pairwise alignment with our target sequence 1u5b. This pairwise alignment should be in the modeller PIR format. The alignment files were obtained by JALVIEW through the following procedure:

  • Go to > Tools > Preferences > Output. Check if "PIR" output format and "Use Modeller Output" are activated.
  • Go to File > Fetch Sequences and select the PDB database. To retrieve a pairwise alignment, enter the corresponding two pdb entries in the field and seperate them by a semi colon. If you want to specify a specific chain insert the pdb id and the chain, seperated by a colon.
    Example: 1UB5:A;1W85:A
  • The generated alignment can be downloaded as in PIR format.

Modeller takes an py-file which specifies the sequences and alignment file to be used. The model-default.py can be found in /apps/modeller9.9/examples/automodel. Here we changed 'alignment.ali' to 1w85_1u5b.pir, knowns = '1W85' (our first target) and sequence = '1U5B' (out template). The file was saved as model1w85.py (for more information see [1])

Make sure to provide the PDB coordinate files in the folder /atom-files.

Modeller is executed by the following command:

mod9.9 model1w85.py


Modeller can also be called with more than one template. A description how this is done can be found here:[2]

Swissmodel

Swissmodel offers three different modes to model Proteins (automated mode, alignment mode, project mode).

The automated mode uses fully automated modelling and can therefore be only used when the template is very similar to the target.<ref>http://swissmodel.expasy.org/?pid=smd03&uid=&token=</ref>

As an Input for the automated mode, only an amino acid sequence (raw or FASTA format) or the Uniprot AC of the target is required. Optional a template PDB id can be given. Swissmodel automatically selects templates from a Blast run which are suitable due to their E-values if no template is given.



The alignment mode can be used for multiple sequence alignments where at least one PDB structure is known.

'Modelling' Steps:

1. Create an (multiple) alignment of at least the target and the template sequence (Format: FASTA, MSF, CLUSTALW, PFAM and SELEX) We created a multiple alignment using ClustalW<ref>http://www.ebi.ac.uk/Tools/msa/clustalw2/</ref>.

2. Submit the alignment to Swissmodel's alignment mode and make sure it is interpreted correctly


TM Score

The TM scores were calculated using the program 'TMScore'.

References

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