To align two sequences:

• Read into ICM two or more sequences.
• Bioinfo/Align Two Sequences

• Enter the name of your first sequence in the 'Sequence 1' data entry box.
• Enter the name of your second sequence in the 'Sequence 2' data entry box.

NOTE: Any sequences already loaded into ICM can be seen by clicking on the down arrow next to the 'Sequence 1 and 2' data entry boxes. This can save typing and trying to remember what you called your sequence.

• Enter a unique alignment name in the 'alignmentName' data entry box.
• Select a comparison matrix from the list shown below by clicking on the arrow next to the 'comp matrix' data entry box.

• Select the alignment algorithm you wish to use from the list shown below by clicking on the arrow next to the 'alignmentAlgorithm' data aentry box.

ZEGA - a Zero End-gap Global Alignment, that is a pairwise alignment method based on the Needleman and Wunsch algorithm modified to use zero gap end penalties. This type of alignment was first described by Michael Waterman, who called it the "fit" alignment. The paper of Abagyan and Batalov, 1997 describes the statistics of the structural significance of the alignment score and optimization of the alignment parameters for the best recognition of structurally related proteins.

H-Align - alignment method used in the Align and Score functions and find database command (as described in Batalov and Abagyan, 1999)

• Enter the values you wish to use for Gap Open, Gap Extension and the maximum penalized gap penalty.

Gap Open The absolute gap penalty is calculated as a product of gapOpen and the average diagonal element of the residue comparison table You may vary gapOpen between 1.8 and 2.8 to analyze dependence of your alignment on this parameter. Lower pairwise similarity may require somewhat lower gapOpen parameter. A value of 2.4 (gapExtension=0.15) was shown to be optimal for structural similarity recognition with the Gonnet et. al.) matrix, while a value of 2.0 was optimal for the Blosum50) matrix ( Abagyan and Batalov, 1997).

Gap Extension The absolute gap penalty is calculated as a product of gapExtension and the average diagonal element of the residue comparison table.

maxPenalizedGap The maximum penalized gap which is used for Gap Open and Extension

• Click OK and the alignment will be displayed in the alignment editor window at the bottom of the graphical user interface.
• Remember to save the project or write the alignment if you wish to keep the alignment for use at another time.