QSCOP-BLAST--fast retrieval of quantified structural information for protein sequences of unknown structure.

Abstract

QSCOP is a quantitative structural classification of proteins which distinguishes itself from other classifications by two essential properties: (i) QSCOP is concurrent with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank and (ii) QSCOP covers the widely used SCOP classification with layers of quantitative structural information. The QSCOP-BLAST web server presented here combines the BLAST sequence search engine with QSCOP to retrieve, for a given query sequence, all structural information currently available. The resulting search engine is reliable in terms of the quality of results obtained, and it is efficient in that results are displayed instantaneously. The hierarchical organization of QSCOP is used to control the redundancy and diversity of the retrieved hits with the benefit that the often cumbersome and difficult interpretation of search results is an intuitive and straightforward exercise. We demonstrate the use of QSCOP-BLAST by example. The server is accessible at http://qscop-blast.services.came.sbg.ac.at/

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Nucleic Acids Research, 2007, Vol. 35, Web Server issueW411–W415
doi:10.1093/nar/gkm264
QSCOP-BLAST—fast retrieval of quantified
structural information for protein sequences
of unknown structure
Stefan J. Suhrer, Markus Gruber and Manfred J. Sippl*
Center of Applied Molecular Engineering, Department of Bioinformatics, University of Salzburg, Hellbrunnerstrasse
34, 5020 Salzburg, Austria
Received January 31, 2007; Revised March 30, 2007; Accepted April 8, 2007
ABSTRACT
QSCOP is a quantitative structural classification
of proteins which distinguishes itself from other
classificationsbytwo
(i)QSCOPisconcurrent
Collaboratory for Structural Bioinformatics (RCSB)
Protein Data Bank and (ii) QSCOP covers the
widely used SCOP classification with layers of
quantitative structural information. The QSCOP-
BLAST web server presented here combines the
BLAST sequence search engine with QSCOP to
retrieve, for a given query sequence, all structural
information currently available. The resulting search
engine is reliable in terms of the quality of results
obtained, and it is efficient in that results are
displayed instantaneously. The hierarchical organi-
zation of QSCOP is used to control the redundancy
anddiversity oftheretrieved
benefit that the often cumbersome and difficult
interpretation of search results is an intuitive and
straightforwardexercise.
use of QSCOP-BLAST by example. The server
is accessible at http://qscop-blast.services.came.
sbg.ac.at/
essential
with
properties:
Researchthe
hitswith the
Wedemonstrate the
INTRODUCTION
The retrieval of structural information from current
databases for the annotation of protein sequences with
unknown structure is a fundamental challenge of struc-
tural and molecular biology. The task faces numerous
problems. The available structural classifications are
incomplete having a large backlog of unclassified struc-
tures and they lack clear quantitative rules that can be
used to quantify and judge family membership. Many
complex proteins are available only as complete chains
rather than individual domains so that the scanning of hit
lists and the analysis of single hits is cumbersome and
time consuming. In addition, the reliability of the various
sequence and structure classification schemes is difficult
to judge in general, and accuracy of annotations and
classifications may vary widely depending on the protein
family of interest.
Theproteinstructure
addresses some of these problems. It endows classic
SCOP [Structural Classification Of Proteins (2,3)] with
quantified structural information and it is concurrent with
Protein Data Bank (PDB) (4), containing all available
structures in the public domain. To build QSCOP,
the protein chains not contained in SCOP are cut
into domains and the resulting domains are classified
against the domains contained in the SCOP database.
QSCOP is updated every week with the newly released
PDB entries.
The intention of the QSCOP-BLAST server is to
provide access to all available protein structures through
a search engine which retrieves structural information
for a given query sequence. Since QSCOP is organized
in hierarchical layers defined by quantitative structural
relationships, the redundancy and structural diversity
of the result obtained is conveniently controlled by the
user.
In the annotation and characterization of protein
sequences of unknown structure frequent questions are
(i) is there a known structure for a related protein, (ii) how
many related structures are available that may serve as a
model for the unknown structure of the query sequence
and (iii) what is the domain structure of the query
sequence and for which domains is structural information
available. These and related questions are critical in many
areas of protein structure research. Reliable answers are
particularly important for large-scale initiatives like
structural genomics projects, where the decision of
whether or not a particular protein target should be
channeled into the structure determination pipeline
critically depends on the effective and reliable retrieval
of all structural information available for that target.
classificationQSCOP (1)
*To whom correspondence should be addressed. Tel: 0043-662-8044-5796; Fax: 0043-662-8044-176; Email: sippl@came.sbg.ac.at
? 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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The QSCOP-BLAST server is specifically designed to
address such questions and to make the interpretation of
the retrieved results intuitive and straightforward. In the
following sections, we review the components of QSCOP-
BLAST and demonstrate its use by a worked out example.
METHODS
QSCOP
SCOP is one of the major protein structure classification
schemesused in genome
In applications, it is generally assumed that the hierarch-
ical organization of domains in SCOP families, super-
families, folds and classes reflects quantitative structural
relationships. This is not the case. Many SCOP families
are structurally diverse containing folds that are quite
dissimilar, and the extent of diversity varies strongly
among the various SCOP families (1). However, for the
implementation of efficient search engines and the
straightforward interpretation of hit lists clearly defined
quantitative relationships among protein domains are
indispensable. QSCOP endows classic SCOP families with
quantitative structural relationships (1) which are essential
in protein structure research.
QSCOPconsists ofhierarchical
decreasing structural similarity of protein domains.
These layers are defined by the number of structurally
equivalent residues (5) shared among two domains.
The first layer covers all structures that have at least
99% equivalent residues in common. This basic layer
combines identical and very similar structures of a SCOP
family into a single group. The successive layers are
defined by progressively smaller numbers of equivalent
residues. The current version of QSCOP computes these
layers in steps of 10% down to 30%. The structural
diversity of a particular SCOP family is quantified by its
granularity which is defined as the number of distinct
groups on a given layer (1). The hierarchical organization
of the classification layers obtained in this way provides
a convenient data structure for the classification of new
domains and for searches against the QSCOP database.
and protein research.
layersreflecting
ConcurrentQSCOP
The latest SCOP version 1.71, released at the end of 2006,
contains 75930 domains derived from 59719 protein
chains found in 27600 PDB files. On the other hand,
in January 2007 PDB contains over 41200 files, where
?2000 of these files contain only non-protein chains.
Hence, although recently updated, SCOP has a backlog
of 13000 files corresponding to more than a quarter of
currently available PDB files.
Concurrent QSCOP contains all protein chains found in
all available PDB files. The PDB files not represented in
SCOP are classified against the 75930 domains contained
in the most recent SCOP release. The update yields 45045
new domains so that the total number of domains in
QSCOP is 120975 (75930 SCOP domainsþ45045 new
domains). The QSCOP classification is updated with every
new PDB release and therefore, it stays concurrent with
PDB. Consequently, the QSCOP-BLAST service always
matches a protein sequence against the complete volume
of available knowledge on protein structures.
QSCOP-BLAST search
As the name implies QSCOP-BLAST uses the BLAST
program (6,7) to search the QSCOP classification.
The sequences of all QSCOP domains are extracted
from the respective PDB files and the standard BLAST
database files required by the BLAST engine are
constructed using the BLAST suite of programs. The
behavior of the BLAST program can be controlled by
several parameters which affect the search results. The
QSCOP-BLAST web service uses the recommended
default parameters (the score matrix is BLOSUM62,
gap open and extension penalties are set to 11 and 1,
respectively, and the e-value cutoff is 10).
Processing ofQSCOP-BLAST hits
A major problem in the interpretation of hit lists is the
redundancy of protein families. Some SCOP families
contain several hundred domains of varying degree of
similarity and frequently subsets of families have identical
or very similar sequences (8–11). On the other hand, there
are proteins that have identical sequences but quite
dissimilar structures. Examples are domain-swapped
proteins or proteins having multiple conformations in
active and inactive states. Although in such cases the
sequences are identical, the corresponding structures are
generally found in distinct QSCOP groups, which is
a consequence of the fact that QSCOP classifies structures
as opposed to sequences.
The QSCOP-BLAST engine scans a query sequence
against all available protein domains, but the resulting hit
list can be manipulated so that only the hits corresponding
to groups on specified layers are reported. The user
controls the desired granularity or redundancy of the
reported hit list by choosing the appropriate layer in
the QSCOP hierarchy. The advantage is 2-fold. On the
one hand, the redundancy of families having a large
number of members of similar sequence and structure
is reduced to the desired level and on the other hand,
hits that are scattered over several SCOP families,
which frequently happens for sequences corresponding
to multi-domainproteins,
In addition, proteins having similar or identical sequences
but multiple conformations are easily spotted in the
reduced hit lists.
areeasily recognized.
WEB SERVER USAGE
Submission of queriesand display ofresults
The QSCOP-BLAST server accepts query sequences in
any format compatible with BLAST or FASTA (12). The
query sequence is pasted into the sequence entry widget,
and the desired QSCOP layer is chosen from a drop down
menu. Submission triggers the QSCOP-BLAST engine
and the resulting hit list is returned immediately. The hit
list summarizes BLAST and QSCOP information on the
domains found in the search, including the sequence
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location of domains, their SCOP classification string,
alignment length and sequence identity and the BLAST
e-value. BLAST alignments of query sequence and
domains are displayed in the familiar BLAST format.
The domain identifier used in SCOP starts with the letter ‘d’.
In contrast, domain names of domains which are classified
in QSCOP but not in SCOP start with the letter ‘c’.
Query example
The typical application of QSCOP-BLAST is the retrieval
of structural information for a given protein sequence of
unknown structure. In the following example we study
the sequence of the a subunit of methylmalonyl-CoA-
decarboxylase of Pyrococcus furiosus, which has been
elected as a structural genomics target, code name
Pfu-683389-001ofthe Southeast
Structural Genomics. The status of this target is found
to be selected and cloned.
When submitted to QSCOP-BLAST the server returns
a hit list, sorted by BLAST e-values, where the first
95 domains have BLAST e-values smaller than 1:0 ? 10?5,
a conservative threshold to indicate significant hits.
To reduce the redundant information among the domains,
we apply the ‘Related’ filter which removes all domains
which have 475% structurally equivalent residues in
common with some other entry in the hit list. The reduced
list still contains four domains with e-values below
1:0 ? 10?5(Figure 1).
Note that this reduction of redundancy is not a trivial
step since it requires quantitative information on the
structural similarity among the domains contained in the
complete hit list. All four remaining domains are classified
as members of SCOP family c.14.1.4, called biotin-
dependent carboxylase carboxyltransferase domain. The
top hits with the most significant BLAST e-values are two
domains of the A chain of 1vrg (http://dx.doi.org/10.2210/
pdb1vrg/pdb), the b subunit of propionyl-CoA carbox-
ylase of Thermotoga maritima at 2.30A˚
Incidentally 1vrg is the structural genomics target
TM0716 of the Joint Center of Structural Genomics
(JCSG) with the PDB release date 22 February 2005.
The chain is not classified in SCOP.
The top hit, c1vrgA2, corresponding to the C-terminal
domain of the A chain matches residues 269–522 of
the query sequence. The second domain, c1vrgA1,
(N-terminal domain) matches the N-terminal residues
1–257 of the query. Hence, it is immediately clear, that the
query consists of two domains. The respective e-values
of 1:59 ? 10?98and 1:37 ? 10?92correspond to sequence
identities of 71 and 66%, respectively. Hence, the hits are
highly significant. A look up in the QSCOP classification
shows that the two domains c1vrgA1 and c1vrgA2 have
similar structures although their sequence similarity of
18% is comparatively low (Figure 2 c).
The domain ranked at position three, classified in SCOP
as domain d1pixa1, matches residues 4–504 of the query
sequence. The respective protein, the carboxyltransferase
subunit of the bacterial ion pump glutaconyl-coenzyme
A decarboxylase(Acidaminococcus
solved to 2.20A˚ resolution. The PDB entry release date
Collaboratory of
resolution.
fermentans) was
is 5 August 2003. The respective domain is twice as long
as the top ranking domains. Although the e-value of
2:55 ? 10?25is considerably higher as compared to the top
hits, it may be regarded as significant and the correspond-
ing sequence identity is 24%. On this level of sequence
identity, it is likely that the query and the hit have similar
structures but one has to expect a considerable variation
of structural details.
The definition of the SCOP domain d1pixa1 is confus-
ing in several aspects. First, the terminal letter, ‘1’, of the
domain name d1pixa1 corresponding to the domain
number indicates that the chain contains more than one
domain. However, d1pixa1 corresponds to the complete
chain. Second, d1pixa1, i.e. the complete A chain of 1pix,
in fact consists of two structural domains. This is rather
difficult to see, and this difficulty may be the reason why
the chain is not chopped into domains in SCOP, although
the two domains are clearly identified in the original
determination report (13). But the domain pattern is
clearly recognized when the structure of the A chain of
1pix is superimposed with the QSCOP domains c1vrgA1
and c1vrgA2 (Figure 2 a and b).
To summarize the results obtained in this example,
we find that the QSCOP-BLAST clearly indicates
that the query sequence consists of two structural domains
that have considerable sequence and structure similarity to
the
b
subunit ofpropionyl-CoA
Thermotoga maritima (1vrg). Moreover, we find that the
two domains are related in structure although the
corresponding sequences have a low percentage of
sequence identity (19%). The result indicates that the
structure determination of this target most likely will
reveal a fold consisting of two domains that are closely
related in structure to the corresponding domains of the
A chain of 1vrg.
carboxylaseof
CONCLUSION
The QSCOP-BLAST service retrieves structural informa-
tion on a given target sequence reliably and fast. The
amount of information contained in the hit lists returned
by QSCOP-BLAST is, in fact, remarkable. Provided that
BLAST is able to detect sequence similarities the entries
in the hit list carry information on the domain structure,
the structural similarity, and the diversity of known
folds related to the query sequence. Database searches
involving structure comparison and domain decomposi-
tion are in general time-consuming and require consider-
able computing resources. In contrast, the QSCOP search
engine is efficient due to the hierarchical organization
of domains in the QSCOP classification which is based
on quantitative structural relationships.
ACKNOWLEDGEMENTS
The structure superposition programs ProHit/ProSup
and TopMatch used to construct QSCOP and the
QSCOP database used by the QSCOP-BLAST server
are provided by Proceryon Science for Life GmbH
(http://www.proceryon.com) under an academic license
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Figure 1. QSCOP-BLAST result obtained for the structural genomics target Pfu-683389-001. The figure shows part of the web page returned
by a QSCOP-BLAST search. The sequence is pasted into the widget on top of the figure. The QSCOP-BLAST server returns the respective hit list,
whose redundancy in terms of structural similarity among the hits may be controlled by selecting the appropriate QSCOP layer. In addition,
the BLAST alignment for individual hits may be displayed (not shown).
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agreement which is gratefully acknowledged. All super-
position figures were prepared using PyMol (http://
www.pymol.org). This work was supported by FWF
Austria, grant number P13710-MOB. Funding to pay the
Open Access publication charges for this article was
provided by the University of Salzburg.
Conflict of interest statement. None declared.
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Figure 2. Superposition of various structures found in the hit list
shown in Figure 1. For any pair of superimposed structures, the first
structure is shown in blue and the second in green. In regions where the
structures are equivalent the first structure is shown in red and the
second structure in orange. (a) d1pixa1 (green/orange) superimposed on
c1vrgA2 (blue/red). The structures share 201 residues which occupy
equivalent positions in the two structures (red and orange). The Ca
atoms of these residues superimpose to an root mean square (rms) error
of 1.4A˚. The sequence identity in this region is 27%. (b) d1pixa1
superimposedon c1vrgA1(211
23% sequence identity), (c) c1vrgA1 superimposed on c1vrgA2
(181 equivalent residues, 1.9A˚rms, 18% sequence identity) (d) the
structural domains 61–285 and 321–558 of d1pixa1 (168 equivalent
residues, 2.3A˚ rms, 15% sequence identity).
equivalentresidues,1.6A˚rms,
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