Vol. 20 no.
5 2004, pages 758–769
BIOINFORMATICS
STAM: simple Transmembrane Alignment Method
Yinon Shafrir∗ and H. Robert Guy
National Cancer Institute, LECB, MSC 5677, 12 South Drive, Bethesda,
MD 20892-5677, USA
Received on August 5, 2003; revised on October 2, 2003; accepted an October 14, 2003
Advance Access publication January 29, 2004
ABSTRACT
Motivation: The database of transmembrane protein (TMP)
structures is still very small. At the same time, more and more
TMP sequences are being determined. Molecular modeling is
an interim answer that may bridge the gap between the two
databases.
The first step in homology modeling is to achieve a good
alignment between the target sequences and the template
structure. However, since most algorithms to obtain the
alignments were constructed with data derived from globular
proteins, they perform poorly when applied to TMPs.
In our application, we automate the alignment procedure and
design it specifically for TMP. We first identify segments likely
to form transmembrane α-helices. We then apply different
sets of criteria for transmembrane and non-transmembrane
segments. For example, the penalty for insertion/deletions in
the transmembrane segments is much higher than that of a
penalty in the loop region. Different substitution matrices are
used since the frequencies of occurrence of the various amino
acids differ for transmembrane segments and water-soluble
domains.
Results: This program leads to better models since it does not
treat the protein as a single entity with the same properties,
but accounts for the different physical properties of the various
segments. STAM is the first multisequence alignment program
that is directly targeted at transmembrane proteins.
Availability: Source code and installation package are avail-
able on request from the authors. Web access is currently
implemented.
Contact: yinon@nih.gov
INTRODUCTION
Homology modeling has become a central tool in investigating
proteins structure. In many cases when protein structure is
not available through standard experimental techniques (such
as X-ray crystallography), homology modeling may provide
significant information regarding the protein’s structure and
function. The need for computational modeling becomes more
acute when one considers transmembrane proteins (TMPs).
While they may account for up to one-third of the proteins in
∗ To whom correspondence should be addressed.
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DOI: 10.1093/bioinformatics/btg482
the cell, TMPs’ comprise a much smaller fraction of known
protein structures in the PDB database (Arai et al., 2003; Boyd
et al., 1998).
The first step towards a successful homology model is
aligning the target proteins sequence with a sequence of
a protein with a known structure. An ‘optimal’ alignment
maximizes a ‘matching’ function between the sequences.
There are many approaches to solve this fundamental problem
in bioinformatics, for a basic review see (Mount, 2001).
Most methods treat the protein’s sequence as a single entity
and manipulate it as such. A set of alignment parameters
is applied to the entire sequence without any weight given
to different regions. While this approach is successful when
applied to water-soluble proteins, the situation is different
for membrane proteins. TMPs reside in two different physio-
chemical environments simultaneously. As a consequence,
most membrane-spanning segment has a regular secondary
structure [simple transmembrane alignment method (STAM)
deals only with transmembrane α-helices], while the extra-
cellular and intracellular regions are more likely to possess
flexible irregular loops. Furthermore, the transmembrane seg-
ments are composed primarily of hydrophobic residues, in
stark contrast to that of the connecting loops, which are gen-
erally much more hydrophilic. These two major differences
have a significant impact on the choice of parameters for multi-
sequence alignment process performed on TMP sequences.
The two main parameters in multi-sequence alignment are
gap penalties associated with opening gaps in the sequence
(and hence in the putative structure), and a substitution mat-
rix that is related to the probabilities of residue type mutating
to another residue type.
The secondary structure restriction implies that introduction
of insertions/deletions in the transmembrane segments should
be rare (such as when a single proline kinks the helix), while
gaps could be applied more liberally in the loop regions that
lacks any regular secondary structure.
Substitution matrices reflect the tendency of different
residue types to mutate. Residues exposed on protein sur-
faces mutate more than those buried within the protein
(Komiya et al., 1988; Overington et al., 1992). Residues of
transmembrane segments that are exposed to lipids tend to
be very hydrophobic, whereas surface residues of soluble
Published by Oxford University Press

proteins are exposed to water and tend to be hydrophilic.
These propensity and conservation differences suggest that
substitution matrices that were constructed using water-
soluble proteins alignments as reference data, are inappropri-
ate for the analysis of TMPs. Various groups have developed
substitution matrices specifically tailored to TMP, and indeed
they differ significantly from the standard matrices (Jones
et al., 1994; Ng et al., 2000).
Alignment of sequences within the same family is usually a
straightforward process. The sequences’ identity in such cases
is high enough to achieve a meaningful alignment without
substantial effects due to the choice of substitution matrix or
insertion/deletion penalties. However, when the sequences set
include members that belong to the same superfamily but not
necessarily to the same family, the homology identity can fall
below the 30% threshold, and the importance of the alignment
parameters in obtaining a correct alignment becomes more
evident. For example, two ion-channel proteins can belong
to two distinct families with little or no obvious sequence
identity; yet share the same folding topology. In this situ-
ation, standard alignment programs encounter problems, and
in many cases fail to align functionally important domains.
Jones and colleagues already wrote in 1994: ‘Considering
the differences in the mutability patterns observed between
a lipid and non-lipid environment, clearly when trying to
align distantly related transmembrane segments it is vital to
bear these differences in mind. Alignment programs that use
the transmembrane matrix for transmembrane regions (either
experimentally determined or predicted) and a general muta-
tion data matrix for the polar flanking regions are likely to
perform much better than programs that use a single matrix’.
(Jones et al., 1994).
In addition, the question of variable penalty gaps as a
function of the secondary structures is an old one. In 1989,
Henneke published an algorithm that takes into account the
known secondary structure of a sequence and adjust the gap
penalty accordingly. In 1992, Smith and Smith published the
pattern-induced multi-sequence alignment (PIMA) algorithm
and showed that ‘secondary structure information can signific-
antly improve the accuracy of aligning structure boundaries
in a set of homologous sequences even when the structure
of only one member of the family is known’. However, as
was mentioned before, only a handful of TMPs have known
structure (Smith and Smith, 1992).
At the same time, many methods of identifying the pos-
ition of the transmembrane segments in the sequence have
been developed. The simplest ones rely on scales that indic-
ate the relative hydrophobicity of the different amino acids.
The basic assumption is that long stretches of hydrophobic
residues in a TMP correspond with the actual transmembrane
segments. Many scales have been suggested based on different
approaches, with similar success in predicting the location of
the transmembrane parts. Later on, the prediction algorithms
shifted towards approaches based on neural networks and
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STAM: simple Transmembrane Alignment Method
adaptive systems. This technique relies on pre-constructed
profiles of transmembrane segments and comparing the target
sequences against these profiles. The rate of successful identi-
fication is slightly higher in all these methods and depends on
the quality of the profiles alignment (Chen et al., 2002; Kall
and Sonnhammer, 2002).
While in theory the concept of parameters dependency
on topology is not new, and applied manually (and sub-
jectively) for many years, STAM is the first application to
combine secondary structure prediction (not known structure)
with alignment parameters variability, in order to address the
special problem of multi-sequence alignment of transmem-
brane proteins. In this sense STAM is different from global
alignment programs, in that it breaks the sequences into seg-
ments. It is also different from local alignment algorithms by
maintaining the structural integrity of the transmembrane seg-
ments without much emphasis on local similarities. It differs
from both classes of programs by the applications of different
alignment parameters at different sections of the sequence.
ALGORITHM
The basic structure of the algorithm is described in the flow
chart (Fig. 1). The first step is to predict the general topo-
logy of each sequence by distinguishing between two types of
segments: transmembrane and non-transmembrane. This sort-
ing is achieved by applying improved hydrophobicity analysis
(von Heijne, 1992) to the sequences.
Different scales can be used (e.g., Guy, 1985 or Kyte and
Doolittle, 1982), as well as sliding windows sizes. Standard
hydropathy analysis with a single window has been shown to
be unreliable (Fariselli et al., 2003). STAM uses two sliding
windows (the trapezoidal method) and a sensitive peak detec-
tion method that allows it to identify segments even with
low hydrophobic signature. From our experience, using this
improved method, the success rate of correctly identifying the
true type of the segment seems to be relatively insensitive to
the specific scale used as long as it is a realistic one.
The hydrophobicity-based method is enhanced further by
helicity propensity analysis that tries to minimize the number
of false positives (over-prediction of transmembrane seg-
ments) or false negatives (under-prediction). For example,
many hydropathy-based programs often fail to identify the
S4 segment in voltage gated 6TM ion channels. The added
helicity analysis helps STAM to identify it in most cases. This
propensity scale is described in the work by Persson and Argos
(1994).
STAM’s goal is not to predict accurately the transmem-
brane topology of all the sequences in the sequences set,
but rather to achieve a biophysical meaningful alignment.
As long as a sizable number of the sequences have the
same topology and are cataloged as such, the resulting align-
ment will not exhibit dependence on the individual sequences
that comprise the profile. Thus, the method of establishing
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 Y.Shafrir and H.R.Guy
Fig. 1. The flowchart of STAM’s algorithm. See text for more details.
the transmembrane topology could be any of the common
algorithms available today or will be available in the future.
In addition to this automated prediction and assignment step,
we gave the user the option to impose his/her own prediction
before the alignment step using a combination of methods and
via a ‘majority consensus vote’ obtain better confidence in the
prediction (Nilsson et al., 2002) or preferably experimentally
obtained data.
STAM should not be used as a general alignment pro-
gram. Water-soluble proteins that have core hydrophobic
regions will be misidentified and cause major alignment
errors. For water-soluble proteins, the standard alignment
methods are adequate, and should be used. When TMPs
are aligned (with STAM or any other alignment program),
it is practical to separate the transmembrane region from the
large soluble domains. This is done for two main reasons:
(1) soluble domains are often highly variable (among different
families) and therefore their alignment is meaningless from a
modeling point of view. (2) Large soluble domains typically
have properties of water-soluble proteins, thus making them
poor candidates for alignment by a program that was designed
specifically for transmembrane regions.
Once the hydrophobic segments have been resolved, an
additional process of ‘segments’ conditioning’ is preformed.
This process takes into account the propensity of the residues
at the edges of the segments to be part of a transmembrane
helix (Persson and Argos, 1994).
STAM then sorts the predicted topologies into distribution
bins. In the best case, if all sequences share the same topo-
logy, the distribution will include only one bin. The sequences
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Fig. 2. The identification and extraction of the putative helical core
from the alignment. The ‘chopped’ segments will be realigned with
the ‘chopped’ segments from the neighboring helices as loops.
that belong to the distribution bin that has most members will
constitute a basis for the overall alignment. The rest of the
sequences are stored for future alignment.
The segments are then aligned in sequential order. All the
corresponding transmembrane segments are aligned together
under very restrictive conditions. The gap-opening penalty is
set to a very high value and the substitution matrix is set to
reflect the different substitution probabilities in a transmem-
brane region. From this alignment, a transmembrane core is
extracted. This core is the block of columns that have no gaps
at any positions. The ‘fringe residues’ are then chopped and
assigned to the surrounding loop regions. This ensures that
errors in identifying the edges of the transmembrane seg-
ments may have a second opportunity to be aligned correctly
(example at Fig. 2). STAM also detects segments that diverge
significantly from the block form (such as to make the core
region smaller than preset number of residues) and removes
them from the alignment on the assumption that they are false
positives.
Rather then reinvent the wheel, we used ClustalW algorithm
(Thompson et al., 1994) to align the segments, though the user
can easily modify the source code to use a different alignment
method. We chose ClustalW, since the source code is freely
available and allows the user to modify ClustalW parameters
(such as extension gap penalty) and its integration into STAM
was fairly easy. However, studies have shown that the differ-
ences between various alignment programs are comparatively
small (Elofsson, 2002; Thompson et al., 1999).
Once all the transmembrane segments are aligned, the modi-
fied loop segments are analyzed. The process is identical to the
alignment process of the transmembrane segments. This time
however, the alignment parameters are more lax. The penalty
for opening a gap is substantially lower and the substitution
matrices are the standard PAM or BLOSUM.
With this, the major part of the alignment process is
complete. The aligned segments are recombined to aligned
sequences to compose a profile. The remainder of the
sequences that did not participate in the previous alignment
procedure (due to different identified topology) is now aligned
sequentially to the profile. By imposing a secondary structure

mask on the profile, we take advantage of this feature in
many profile-sequence alignment programs, such as ClustalW
(Thompson et al., 1994) or Hmmer (Durbin et al., 1998; Eddy,
1995).
STAM is designed to reduce the impact of topology pre-
diction errors on the final alignment. There are two common
prediction errors. False positives, which is the assignment
of transmembrane status to a loop or the breakage of a long
segment into two shorter ones, and false negatives, which is
the assignment of loop status to a transmembrane segment.
In the vast majority of cases this will lead the sequence to
be cataloged in a different distribution bin than the major-
ity of the sequences (and therefore will not be part of the
core profile) unless all the sequences share the same char-
acteristics. If they do, the consistent nature of the prediction
algorithm will make the same prediction mistake in all of them
and the alignment will not be much effected. For that reason,
the consistency of the topology analysis is key to reduce
the effect misprediction will have on the overall alignment.
Still, unidentified false positives disrupt the final alignment
more than false negatives, since regions identified as loops
are aligned again after the putative transmembrane helices.
Consequently, STAM will choose a consensus topology with
the least number of segments between two bins with the
same number of sequences. The last defense against predic-
tions errors is the removal from the core-alignment segments
that cause the core helical region to be smaller than a pre-
set number of residues. These misalignments are often the
result of a prediction mistake along the sequence. However,
even with these measures, prediction mistakes can effect the
final alignment. Hence, the user is encouraged to inspect the
prediction results and correct them manually if an error is
detected.
STAM was implemented in Microsoft Visual Basic
version 6 and currently is available by request from the author
for Windows 2000/XP-based machines (and will be available
on our future website). We currently are working to port the
algorithm to more environments.
RESULTS
Comparing the accuracy of alignments is a difficult task even
when the sequences involved are of globular proteins. A
comparison is usually made between the proteins’ sequences
alignments and the proteins’ three-dimensional (3D) struc-
tural alignments. This approach is not adequate when TMPs
are concerned. The number of known structures of TMP is
very small, and the subset of known TMP structures that are
homologs is almost empty.
However, we first examined our method versus ClustalW
by aligning sequences from families for which at least one
structure has been determined. We looked at three different
major improvements that STAM introduces to the alignment
of TMPs.
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STAM: simple Transmembrane Alignment Method
Correct positioning of the helices
We compared STAM alignment of eukaryotic (bovine)
rhodopsin (Palczewski et al., 2000), and three bac-
terial rhodopsins: bacteriorhodopsin (Belrhali et al., 1999),
halorhodopsin (Kolbe et al., 2000) and sensory rhodopsin II
(Royant et al., 2001).
Even though, the structures of the prokaryotic and euka-
ryotic families are distant from one another they share many
structural characteristics (Nero and Louis, 1992). Both famil-
ies have a seven transmembrane helix motif, but the bacteri-
orhodopsin helices tend to be shorter and more perpendicular
to the membrane.
As we can see in Figure 3A, STAM identified correctly all
the transmembrane segments and aligned them without gaps
in their helical cores. Figure 3B is the ClustalW alignment
of the same sequences. While ClustalW produced essentially
the same alignment for the three bacterial sequences, it failed
to position the helices between the families correctly. The
first three helices of the bovine rhodopsin are completely mis-
aligned with the helices of the three bacterial rhodopsins. It
is important to note that STAM identified the topology of all
the sequences correctly using the KD hydrophobicity scale,
and failed to do so using Guy’s scale. Yet, even with the
misidentification of the topology, STAM managed to align
the sequences correctly because two sequences were enough
to build a meaningful secondary-structure mask to include in
the profile/sequence alignment procedure.
Insertions/deletions in known helices
Crystal structure have been determined for the pore forming
domain of four distantly related bacterial K + channels: KcsA
(Doyle et al., 1998), MthK (Jiang et al., 2002), KirBac1.1
(Kuo et al., 2003) and KvAP (Jiang et al., 2003). Figure 4
compares the alignments obtained using STAM to ClustalW of
these proteins and the three closest homologs to each that have
<50% identity to each other, as was determined by a BLAST
search (Altschul et al., 1990) of the non-redundant database.
Due to their low level of sequence conservation, the more
peripheral M1 helices (of the 2-TM channels) and S5 helix of
the KvAP, are difficult to align. ClustalW (Fig. 4A) introduces
gaps in the known helices in the crystallized structures, and
in the putative helical regions of some of the other sequences.
Based on the crystal structures, both programs misaligned M1
of the KirBac family relative to the other families. However,
while STAM places the M1 of the KirBac against the M1 of
the other families, ClustalW places it mostly against insertions
in the other sequences. If one was to model KirBac on the
backbone of any of the other families, using STAM will retain
the secondary structure of the sequence while using ClustalW
will result in a disorganized coil spanning the lipid bilayer.
Sensitivity to motifs in distantly related sequences
The final example is the alignment of a small number of dis-
tantly related 6TM ion channels. ClustalW perform relatively
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 Y.Shafrir and H.R.Guy

Fig. 3. (A) STAM alignment of rhodopsins. (B) ClustalW alignment of the same sequences. The helices’ locations in the crystal structures
are denoted by a black underline. The structures codes in the PDB database are as follows: bacteriorhodopsin—1QHJ, halorhodopsin—1E12,
sensory rhodopsin II—1H68 and bovine rhodopsin—1F88.

well when the number of sequences is large and the proteins
are somewhat closely related. Since ClustalW obtains its final
alignment by building a phylogenic guiding tree and clustering
the sequences accordingly, the quality of the final alignment
depends on the amount of input sequences their relative evol-
utionary distances from one another. When the number of
sequences is small and the sequences are very distant, the
quality of the alignment deteriorates. In this case, we aligned
small number of 6TM ion channels that are very distant from
one another.
Eight different 6TM sequences were aligned: four K+ Chan-
nels, two Ca2+ channels and two Na+ channels. KvAP and
Shaker were introduced to the alignment since KvAP is the
only known structure of a 6TM channel and Shaker is one of
the most-studied channels. The results are shown in Figure 5.
In this alignment, ClustalW failed to identify the signature
repetitive motif of arginines at every third position in the
S4 helix of the voltage-sensitive channels, which is respons-
ible for the charge movement through the membrane during
gating. The highest relative amount of arginine or lysine in
an alignment position is 3/8 [the Escherichia coli segment
does not have the (RXX)n signature]. In addition, some of
the other helices are misaligned as well (especially S5). In
this example, STAM avoids these mistakes. It aligns well the
S4 region (7/8 ratio in three positions and 4/8 in one other
position). The gaps are missing in the putative transmembrane
regions, and the gaps concentrate at the highly variable loop
regions or at the edges of the helices.
DISCUSSION
When aligning sequences of TMP, there are added difficulties
to the ones that are already associated with multi-sequence
alignment algorithms. Instead of developing a new alignment
algorithm, we constructed an algorithm that uses contem-
porary alignment procedures, and applies certain criteria to
treat the special cases of TMP. It has been shown already that
most available alignment programs today achieve about the
same rate of success regardless of the underlying algorithms
(Thompson et al., 1999). The basic concept behind this
project was not to reinvent the wheel when it comes to multi-
sequence alignments, but rather use the available tools and

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Fig. 3. Continued.
techniques and apply the restrictions arising from the unique
physio-chemistry of TMP.
We chose not to base our program on Hidden-Markov-
Models (HMM) approach because HMMs necessitate an
initial seed alignment on which the final alignment will be
constructed. This requires the user to provide a good initial
alignment for the TMP’s in question. If the seed alignment is
unambiguous, no complications arise. Things become more
problematic when the sequences in question have little overlap
and highly variable. Then the user has to inject his subjective
criteria and this in turn will propagate throughout the final
alignment.
We prefer to use the more conventional approach of
matrix-based alignments and to use standard criteria that are
general to TMPs and based on empirical results (such as the
hydrophobic scales).
Yet HMMs are extremely fast and powerful tools that need
a good initial alignment. STAM may be just the objective tool
to provide these initial alignments.
As its name implies, STAM, the approach is very
simplistic in nature and not perfect. The method is based
on the following assumptions: (1) long hydrophobic seg-
ments form transmembrane α-helices and (2) short segments
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STAM: simple Transmembrane Alignment Method
between the helices have no regular secondary structure and
long water-exposed segments can be aligned with standard
unconditioned methods. As one can see, these are very basic
assumptions that cover most of the TMP cases (but not
all). Once the hydrophobic regions identification process is
complete, the segments are aligned according to their classi-
fication. Transmembrane segments are aligned with high gap
penalties to prevent distortions in the secondary structure and
with a substitution matrix that represents the substitution prob-
abilities in a transmembrane segment. However, we must note
that the gap penalty value is more important to the alignments
then is the selected matrix. This may reflect the situation today
of which only a handful of substitution matrices, tailored spe-
cifically for TMPs, exist. To compound this problem, most
of these matrices have close entropy values due to their being
developed from closely related proteins. They therefore rep-
resent only a narrow band in the evolution process of TMPs.
STAM can be the tool needed to align more distant sequences
in order to obtain more distant matrices.
Even this naive approach has shown improvement over
standard alignment programs. We chose to compare its results
versus these of ClustalW for two reasons: (1) ClustalW is
one of the most successful and popular alignment programs.
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(2) We used ClustalW algorithm as the alignment engine and,
therefore, it is reasonable to observe the changes STAM brings
to a ClustalW alignment. However, STAM is a very modular
program and replacing the alignment engine with the user’s
favorite alignment program is a straightforward procedure.
For example, we found that at the last phase of the alignment,
when the problem type becomes a sequence-profile align-
ment, SAM (Karplus et al., 1998), which is a HMM-based
program, performs better than ClustalW in some cases.
STAM does not guarantee a ‘correct’ alignment and some-
times it exhibits sensitivity to different input parameters.
STAM is very flexible in this regard. It can either work in full
automatic mode (in which no input from the user is required),
or manual mode (in which the different parameters and topo-
logy predictions can be altered by users). Users are advised
to consider alternative alignments obtained with different
inputs.
Nonetheless, the method that is the basis of STAM strives
to minimize the effects of possible mistakes. Since the
segments are aligned individually, there is no drift of mis-
alignment errors to the neighboring segments. A major mis-
alignment error in transmembrane proteins is the existence of
a gap in the transmembrane regions. ClustalW tends to prefer
to extend gaps. If one gap is opened, the cost of opening
an adjacent gap drops significantly. Though single gaps can
occur in the transmembrane segments, longer gaps are rare.
STAM assigns the same penalty to the extension gap as to the
initial one. This discourages the appearance of long stretches
of gaps in a transmembrane segment. Because of this, most
of the misalignments will appear at the ends of the helices
and not in the core. This is due to errors in the identification
of the helices edges. Since proteins tend to lose their ordered
secondary structure near the membrane boundaries, this is an
acceptable error.

Fig. 4. ClustalW alignment (A) and STAM alignment (B) of the pore-forming domain of different K + channels with solved structure. The
first helices in the known structures are underlined. ClustalW open gaps in many sequences at the first transmembrane helix (S5 in the
KvAP, M1 in all others), making a homology modeling based on its alignment a difficult task. In comparison, STAM maintains the structural
integrity of the helices. The NCBI accession numbers and PDB codes for sequences used in this alignment: 1ORQ (KvAP), gi:15024246
(KvAP-Like1), gi:16411529 (KvAP-Like2), gi:24350062 (KvAP-Like3), 1BL8 (KcsA), gi:27364075 (KcsA-Like1), gi:9657586 (KcsA-
Like2), gi:22406479 (KcsA-Like3), 1LNQ (MthK), gi:1498918 (MthK-Like1), gi:23129256 (MthK-Like2), gi:2648884 (MthK-Like3),
1P7B (KirBac), gi:17130300 (KirBac-Like1), gi:23042578 (KirBac-Like2) and gi:23015860 (KirBac-Like 3).

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Fig. 4. Continued.
Another aspect that STAM brings to the forefront is the
importance and relevancy of the alignment score. Most pro-
grams treat the alignment score as a function that has to be
maximized. Once a maximal value has been attained, the pro-
gram declares the corresponding alignment as ‘optimal’. This
approach is somewhat misleading. Since the program does not
know ‘a-priori’ the secondary structure of the sequence, it can-
not take it into account when maximizing the score function.
This can lead to major misalignments of helices and breaking
up the secondary structure. STAM does not try to maximize
the score over the entire sequences but forces the helices cores
to be aligned first. The alignments may not look as ‘nice’ and
be less in a ‘block’ form but they will retain the biophysical
properties of the sequences.
ClustalW approach of building a phylogenic tree enhances
the accuracy of the alignments by aligning the closest
sequences first before progressing to more distant ones. This
is done by pairwise alignment of all the sequences and meas-
uring the percentage of matched positions. This method is
valid if all the segments in a sequence share a common
evolutionary pathway. However, in TMPs in general, and
ion channel proteins in particular, this assumption in not
necessarily valid. Lets take for example the 6TM ion channel
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STAM: simple Transmembrane Alignment Method
fold. The pore lining helices (S5-P-S6) of K+ channels exhibit
high degree of conservation since these segments control
the channel selectivity and ions permeation. Comparing dif-
ferent potassium channels based on the pore lining helices
point unambiguously to a common ancestor. Nevertheless,
this domain is much less conserved among channels with
different ion selectivity, e.g., the pore-forming domain of
voltage-gated Ca2+ and K+ channels are quite dissimilar. On
the other hand, the composition of the preceding four helices
(S1–S4) depends on the specific gating mechanism of the
channels. Thus, this domain is fairly similar for voltage-gated
K+ , Ca2+ and Na+ channels but can be very divergent among
K+ channels that are gated differently (Fig. 5).
STAM solves this dilemma by generating a new phylogenic
tree for each segment’s alignment. This ensures that more
similar segments will be aligned first (Fig. 6). In the example
above, if a 6TM voltage-gated K+ channel is aligned with
a 6TM voltage-gated Ca2+ channel and a 6TM Ca-activated
K+ channel, the S4 of the voltage-gated K+ channel will be
first aligned with the S4 of the voltage-gated Ca2+ channel.
The order changes at the S5-P-S6 domain; these segments of
the K+ channels are aligned with each other first, and only
than are aligned with the Ca2+ channel and the rest of the
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 Y.Shafrir and H.R.Guy

Fig. 5. ClustalW alignment of distantly related 6TM ion channels (A) and STAM’s alignment of the same sequences (B). The helices’
locations in the known structure (KvAP) are underlined. The ovals denote the wide gaps ClustalW introduces in the middle of S2 and S5.
The black rectangles denote the location of S4 of the voltage sensor. ClustalW fails to identify the repetitive motif of charged residues in
the voltage sensor. NCBI accession numbers for the sequences used: gi:30749953 (KvAP), gi:157063 (Shaker), gi:103086 (Ca-activated K),
gi:1787503 (E.coli K), gi:4633669 (T-Type Ca), gi:6751839 (Ascidian Ca), gi:1771576 (Rat Na) and gi:749324 (Yeast Na).

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 STAM: simple Transmembrane Alignment Method

Fig. 5. Continued.

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 Y.Shafrir and H.R.Guy

Fig. 6. The phylogenic trees that guide the alignments in Figure 5. (A) The phylogenic tree for the ClustalW alignment. This tree is applied
to the entire sequence. (B) The phylogenic tree of the S4 segment in the STAM alignment. The channels with the repetitive arginines are
grouped together. The channels with almost no such motif (The Ca-activated K + and the E.coli K + channels) are also grouped separately.
(C) The phylogenic tree for the P segment in the STAM alignment of Figure 5B. Since the P segment function as the selectivity filter, the
channels are sorted according to their selectivity. Note: in the STAM alignment, KvAP was aligned to the profile of the rest of the sequences
and therefore does not appear in the phylogenic tree.

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