2i2O Methods and Materials

Constructing the evolution of the protein

The protein sequence was blasted in the NCBI website. The blasted sequence was scanned using a non-redundand database to obtain the most significant sequences. 57 sequences with significant E-values were recovered. 55 of these sequences were used to perform a multiple alignment sequence by using the program ClustalX. When this was completed, a distance matrix calculation was performed on the sequences. The program Phylip-3.63 was used. The calculations from the matrix distance calculation were transformed by using a program in Phylip-3.63, Neighbor.exe. Afterwards, the bootstrap values for the sequences were calculated. This was done by running the alignment produced from the Neighbor program through Seqboot. 100 bootstrap samples were produced. The program Protdist.exe from Phylip-3.63 was used with the settings M for multiple, D for multiple data sets, and 100 for the replicates. The file created was run through Neighbor.exe. The same settings needed for Protist.exe were used. The treefile that was created was run through the program Consenses.exe. The resulting file was opened using the program Treefile. The phylogenetic tree created had the bootstrap values displayed on the branch nodes.

Identifying the function of the protein.

The FASTA sequence obtained by BLAST was submitted to the following databases: ProKnow, ProFunc, Locate, String, PDB and DALI. The FASTA sequence or protein name for MIF4G (2i2O) and species Homo sapiens was used in all searches. Searches were performed using all standard limits and conditions unless otherwise specified. Literature covering the protein was further explored using the NCBI database searching by key words HEAT, domain, 2i2O, eIF4G and MIF4G. All results were noted in mediawiki. Following collection of data, queries on all of the previously mentioned databases were performed using The FASTA sequence and protein name for the MIF4G-like protein of Danio Renio (Zebrafish). A sequence of a similar protein in mice identified in the early BLAST search as having a degree of homology was also submitted to LOCATE and Proknow. Significant results from both proteins were compared for similarity. Once again all search results were recorded.

Determining the protein structure

A protein search on PDB, NCBI Entrez, Pfam and InterPro databases was performed as the initial step in identifying the structure of the given protein. The given PDB code 2i2o was used in the searches. Next, proteins with similar structures were identified using the DALI server. A CE comparison was performed among the identified structures.

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