AlignMe

Run AlignMe in pair-wise mode

1. Provide sequences
This form allows you to align protein sequences. Each input sequence has to be in fasta format, which consists of a header starting with ">" followed by a name or description of the sequence, and the sequence in the following lines.

Example:

>1KPL.pdb CHAIN_ID: A
TPLAILFMAAVVGTLTGLVGVAFEKAVSWVQNMRIGALVQVADHAFLL
WPLAFILSALLAMVGYFLVRKFAPEAGGSGIPEIEGALEELRPVRWWR
VLPVKFIGGMGTLGAGMVLGREGPTV

In standard submissions, the user is expected to provide a single fasta sequence in each of the two boxes below. The output will be a single pairwise alignment.
However, a batch mode is available to enable the user to easily calculate many pairwise alignments in a single submission. Thus, if more than one sequence is provided in a given box, then all those sequences will be aligned (pairwise) with the sequences in the other box. The results will be reported as a set of pairwise alignments. Every input sequence should be in fasta format as above. Please note that this is not the same as a multiple-sequence alignment.

Enter a sequence in fasta format:		Enter another sequence in fasta format:

...or upload a fasta file:		...or upload another fasta file:

2. Select optimized or user-defined parameters
Gap penalties and the choice of input parameters have been optimized for transmembrane proteins based on the HOMEP 2010 data set. We recommend that you use these parameters only for aligning transmembrane proteins and only if you know the secondary structure of the proteins.

Parameters for α-helical membrane proteins

	AlignMe PST Most accurate alignments for very distantly related proteins, <15% identity; ~9 mins for a 179 and 215-residue sequence pair
	AlignMe PS Most accurate alignments for low-homology proteins, ~15-45% identity; ~5 mins for a 179 and 215-residue sequence pair
	AlignMe P Most accurate alignments for very closely related proteins, >45% identity; ~5 mins for a 179 and 215-residue sequence pair
	Fast, but less accurate alignments (~3 seconds) based on a substitution matrix and a hydrophobicity scale
	slower, but more accurate ~10 min (substitution matrix, hydrophobicity scale & secondary structure prediction)

Or use your own alignment parameters

Define your own parameters

Show detailed alignment parameter options

2.1. Gap penalties
AlignMe uses a Needleman-Wunsch algorithm with an affine gap penalty scheme. Gap penalties define how likely amino acids are to be aligned with one another or against a gap.

In general, affine gap penalties have a gap opening penalty (o) and a gap extension penalty (e). A gap of length n is then given a score of: o + (n-1)e. All gap penalties have to be positive integers or fractions (no commas).

AlignMe allows you to define either 2, 4 or 6 gap penalties:

2: A gap open penalty and a gap extension penalty are used along the entire length of the sequence.

4: Special gap penalties can be assigned to gaps at the end of the alignment, i.e. N- or C-terminal gaps. This can be useful if gaps are more (or less) likely to appear at the termini than within the sequence. Thus the third and fourth parameters are the gap open and gap extension penalities of the terminal gaps.

6: By using 6 gap penalties and a threshold value it is possible to subdivide the sequence into 2 different subsets to which different gap penalties are assigned. Subset 1: below threshold gap opening and extension penalty (first & second params) Subset 2: above threshold gap opening and extension penalty (third & fourth params) The fifth & sixth parameters are the gap open and gap extension penalities of the terminal gaps.

Select number of gap penalties: 2 4 6

Gap opening penalty

Gap extension penalty

Gap opening penalty		Gap extension penalty
Terminal gap opening penalty		Terminal gap extension penalty

Below-threshold gap opening penalty		Below-threshold gap extension penalty
Above-threshold gap opening penalty		Above-threshold gap extension penalty
Terminal gap opening penalty		Terminal gap extension penalty

2.2. Profiles, scales and matrices
AlignMe can calculate alignments based on substitution matrices, hydrophobicity scales or profiles, e.g. secondary structure predictions. In this section you must choose at least one parameter on which your alignment will be based. However, a combination of several inputs is possible.

The option "weights" allows the relative influence of different input parameters to be adjusted. Please see the manual for detailed information.

Enter at least one, or more as required:

Matrix	Upload a custom matrix	Weight

Add more substitution matrices

Substituion matrices consist of substituion rates describing how likely one residue type in a sequence will become another residue type over an evolutionary period. A matrix should look like this:

   A  C  D  E  F  G  H  I  K  L  M  N  P  Q  R  S  T  V  W  Y
A  4  0 -2 -1 -2  0 -2 -1 -1 -1 -1 -2 -1 -1 -1  1  0  0 -3 -2
C  0  9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2
D -2 -3  6  2 -3 -1 -1 -3 -1 -4 -3  1 -1  0 -2  0 -1 -3 -4 -3
E -1 -4  2  5 -3 -2  0 -3  1 -3 -2  0 -1  2  0  0 -1 -2 -3 -2
F -2 -2 -3 -3  6 -3 -1  0 -3  0  0 -3 -4 -3 -3 -2 -2 -1  1  3
G  0 -3 -1 -2 -3  6 -2 -4 -2 -4 -3  0 -2 -2 -2  0 -2 -3 -2 -3
H -2 -3 -1  0 -1 -2  8 -3 -1 -3 -2  1 -2  0  0 -1 -2 -3 -2  2
I -1 -1 -3 -3  0 -4 -3  4 -3  2  1 -3 -3 -3 -3 -2 -1  3 -3 -1
K -1 -3 -1  1 -3 -2 -1 -3  5 -2 -1  0 -1  1  2  0 -1 -2 -3 -2
L -1 -1 -4 -3  0 -4 -3  2 -2  4  2 -3 -3 -2 -2 -2 -1  1 -2 -1
M -1 -1 -3 -2  0 -3 -2  1 -1  2  5 -2 -2  0 -1 -1 -1  1 -1 -1
N -2 -3  1  0 -3  0  1 -3  0 -3 -2  6 -2  0  0  1  0 -3 -4 -2
P -1 -3 -1 -1 -4 -2 -2 -3 -1 -3 -2 -2  7 -1 -2 -1 -1 -2 -4 -3
Q -1 -3  0  2 -3 -2  0 -3  1 -2  0  0 -1  5  1  0 -1 -2 -2 -1
R -1 -3 -2  0 -3 -2  0 -3  2 -2 -1  0 -2  1  5 -1 -1 -3 -3 -2
S  1 -1  0  0 -2  0 -1 -2  0 -2 -1  1 -1  0 -1  4  1 -2 -3 -2
T  0 -1 -1 -1 -2 -2 -2 -1 -1 -1 -1  0 -1 -1 -1  1  5  0 -2 -2
V  0 -1 -3 -2 -1 -3 -3  3 -2  1  1 -3 -2 -2 -3 -2  0  4 -3 -1
W -3 -2 -4 -3  1 -2 -2 -3 -3 -2 -1 -4 -4 -2 -3 -3 -2 -3 11  2
Y -2 -2 -3 -2  3 -3  2 -1 -2 -1 -1 -2 -3 -1 -2 -2 -2 -1  2  7

This example is for the BLOSUM62 matrix.

Scale	Upload a custom scale	Window type	Size	Weight	Use?	Threshold

Add more hydrophobicity scales

Hydrophobicity scales assign a relative hydrophobicity to each amino acid type. These values are obtained by experimental, theoretical or combined approaches. However, importantly, the sign must be reversed relative to the free energies. Thus, an input scale should look like this:

                C       0.13
                N       -2.05
                Q       -2.36
                S       -0.84
                .     ....
                .     ....

This example is of an excerpt of the Hessa et al scale (HWvH).

It is possible to smooth the hydrophobicity profile with 4 different types of window averaging: triangular, rectangular, sinusoidal and zigzag. In that case, the size of the window must also be provided. Detailed information about window types is provided in the online manual.

Profile type	First profile	Second profile	Column	Weight	Use?	Threshold

Add more profiles (e.g. transmembrane or secondary structure predictions)

Profiles can be secondary structure predictions, transmembrane predictions, or hydrophobicity values for each amino acid position etc. The first profile must have the same length as the first sequence that you submitted. (same is true for the second profile).
The profile file should look like this:

        1   S    U   0.87  0.06  0.08
        2   G    U   0.84  0.06  0.1
        3   F    U   0.73  0.06  0.21
        4   E    U   0.58  0.05  0.37
        .   .    .   ....  ....  ....
        .   .    .   ....  ....  ....

The user must specify the column from which the values should be extracted (first column = 1, second column = 2...).
Comment lines need to be removed

3. Anchors

Anchors allow for aligning certain positions or motifs that might not be aligned running an unbiased alignment.
Each anchor consists of the residue identifier in the first sequence, the residue ID in the second sequence and a weight, specifying the strengths of the anchor.
The first amino acid has the residue ID '1'.
A high weight will enforce the anchor.
The default in the form is set to 1000, which will enforce this anchor.
Either enter them using the form above or as a file with the following format:

	12	24	10.0
	123     78       5.0
	....................

For batch submissions (i.e. multiple pairwise alignments) anchors can also be specified, by uploading a file and indiciating the sequence number next to the anchor position. For example:

        1:7     5:7     1000
        ....................

will fix position 7 in sequences 1 and 5 in the corresponding alignment and assumes that at least 5 sequences have been provided in the second box.

Comment lines need to be removed!

Residue 1	Residue 2	Weight

Add more anchors

...or upload an anchor file?

4. Submit

Enter an e-mail address to receive a message containing your results.
This is recommended for "Most accurate alignments" and alignments with
automated PSSM calculations, PSIPRED or OCTOPUS predictions.

AlignMe

Run AlignMe in pair-wise mode

ERROR: Enter 2 fasta sequences....

ERROR: First sequence missing - please upload a fasta file or paste in a sequence

ERROR: Second sequence missing - please upload a fasta file or paste in a sequence

ERROR: Please choose your first fasta file to upload

ERROR: Please choose your second fasta file to upload

ERROR: If you want to upload a fasta file please select the corresponding checkbox (first)

ERROR: If you want to upload a fasta file please select the corresponding checkbox (second)

ERROR Sequence1: The input sequence has to be in fasta format

ERROR Sequence2: The input sequence has to be in fasta format

ERROR batch mode: it is not possible to submit more than 1000 alignments per batch.

Parameters for α-helical membrane proteins

Parameters for β-barrel membrane proteins

Or use your own alignment parameters

ERROR: You have to select a type of alignment parameters for your alignment!

ERROR: Please choose gap penalties!!

ERROR: Please enter a gap opening penalty

ERROR: Format of gap opening penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a gap extension penalty

ERROR: Format of gap extension penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a gap opening penalty

ERROR: Format of gap opening penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a gap extension penalty

ERROR: Format of gap extension penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a gap opening penalty for the termini

ERROR: Format of terminal gap open penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a gap extension penalty for the termini

ERROR: Format of terminal gap extension penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a minimum gap open penalty

ERROR: Format of minimum gap open penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a minimum gap extension penalty

ERROR: Format of minimum gap extension penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a maximum gap open penalty

ERROR: Format of maximum gap open penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a maximum gap extension penalty

ERROR: Format of maximum gap extension penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a termini gap open penalty

ERROR: Format of termini gap open penalty incorrect - positive integers or fractions only (no commas)

ERROR: Please enter a termini gap extension penalty

ERROR: Format of termini gap extension penalty incorrect - positive integers or fractions only (no commas)

ERROR in input line 1: Please enter a weight

ERROR in input line 1: Format of weight is incorrect - positive integers or fractions only (no commas)

ERROR in input line 1: Please choose a matrix to upload

ERROR in input line 1: Please select a matrix

ERROR in input line 1: Please enter a weight

ERROR in input line 1: Format of weight is incorrect - positive integers or fractions only (no commas)

ERROR in input line 1: Please select a scale

ERROR in input line 1: Please choose a scale to upload

ERROR in input line 1: Please select a window type

ERROR in input line 1: Please enter a window size

ERROR in input line 1: Format of window size is incorrect - positive integers except 0 (no commas)

ERROR in input line 1: Please enter a threshold value

ERROR in input line 1: Format of threshold value is incorrect - integers or fractions only (no commas)

ERROR in input line 1: Please select a profile

ERROR in input line 1: Please enter a weight

ERROR in input line 1: Format of weight is incorrect - positive integers or fractions only (no commas)

ERROR in input line 1: Please choose your first profile

ERROR in input line 1: Please choose your second profile

ERROR in input line 1: Please enter a column

ERROR in input line 1: Format of column is incorrect - positive integers except 0 (no commas)

ERROR in input line 1: Please enter a threshold value

ERROR in input line 1: Format of threshold is incorrect - integers or fractions only (no commas)

ERROR in input line 1: Please select a type for the PsiPred prediction!

ERROR in input line 1: Please select a type for the OCTOPUS prediction!

ERROR: Please enter at least one input (profile, scale or matrix) before submitting a job

ERROR: A threshold has to be set!! (If you selected 6 gap penalties, you have to use either a scale or a profile and define a threshold for it.)

ERROR: First residue ID missing

ERROR: First residue ID is zero or negative

ERROR: Second residue ID missing

ERROR: Second residue ID is zero or negative

ERROR: Anchor weight missing

ERROR: If you want to upload an anchor file please select the corresponding checkbox

ERROR: Please choose your anchor file to upload

ERROR: Some of the anchors you defined are incompatible: avoid crossings such as anchor1 = (5, 5, w), anchor2 = (6, 4, w)

4. Submit

You have to provide an E-Mail address if you want to include in your alignment: - an automated secondary structure prediction from PSIPRED - and/or a PSSM from a PSIPRED prediction - and/or a transmembrane prediction from OCTOPUS

Submission failed! Please see the error messages displayed above on this page, change the inputs accordingly, and resubmit.

You have to provide an E-Mail address if you want to include in your alignment:
- an automated secondary structure prediction from PSIPRED
- and/or a PSSM from a PSIPRED prediction
- and/or a transmembrane prediction from OCTOPUS

Submission failed!
Please see the error messages displayed above on this page,
change the inputs accordingly, and resubmit.