Nucleic Acids Research, 2003, Vol. 31, No. 1 406-409
© 2003 Oxford University Press
TMPDB: a database of experimentally-characterized transmembrane topologies
1 Department of Electronic Information System Engineering, Faculty of Science and Technology, Hirosaki University, Hirosaki 036-8561, Japan 2 Science of Bioresources Program, The United Graduate School of Agricultural Sciences, Iwate University, Morioka 020-8550, Japan
*To whom correspondence should be addressed. Tel: +81 172393638; Fax: +81 172393638; Email: slsimi{at}si.hirosaki-u.ac.jp
Received August 1, 2002; Revised September 9, 2002. Accepted September 20, 2002
ABSTRACT
TMPDB is a database of experimentally-characterized transmembrane (TM) topologies. TMPDB release 6.2 contains a total of 302 TM protein sequences, in which 276 are 
-helical sequences, 17 ß-stranded, and 9 -helical sequences with short pore-forming helices buried in the membrane. The TM topologies in TMPDB were determined experimentally by means of X-ray crystallography, NMR, gene fusion technique, substituted cysteine accessibility method, N-linked glycosylation experiment and other biochemical methods. TMPDB would be useful as a test and/or training dataset in improving the proposed TM topology prediction methods or developing novel methods with higher performance, and as a guide for both the bioinformaticians and biologists to better understand TM proteins. TMPDB and its subsets are freely available at the following web site: http://bioinfo.si.hirosaki-u.ac.jp/~TMPDB/.
INTRODUCTION
Transmembrane (TM) proteins serve extremely important functions in life as pump, channel, receptor, energy transducer etc., and have been reported recently to share 
20–30% of genes in a whole genome (1–4). Nevertheless, the number of three-dimensional (3D) structures with high-resolution is far below one hundred at present, in contrast to more than 18 000 3D structures for soluble proteins registered in PDB (5). It is because TM protein molecules are difficult to crystallize due to their amphiphilic characteristics—hydrophobic TM segments (TMSs) and hydrophilic loops. The functions of TM proteins, however, can be inferred rather easily from their TM topology (i.e., the number of TMSs, TMS position and orientation of TMS to the membrane lipid bilayer) without knowing their 3D structures because of rather simple structural characteristics (6).
In this context, a number of TM topology prediction methods have been developed to determine the structure and function of TM proteins from their amino acid sequences (2,7–22). However, the proposed prediction methods have not attained the desired accuracies for this purpose. The recent reports of evaluating prediction performance by using experimentally-characterized TM topology datasets have revealed that even the best methods predict the TM topology with accuracies of only around 60% (23–25). This could be attributed mainly to the lack of well-characterized topology data to be used for training or tuning TM topology prediction methods. Thus, more high-quality TM topology data are required to evaluate the existing prediction methods more precisely.
For this reason, we have constructed a transmembrane protein database, TMPDB (19,24,26) which is a collection of TM proteins with topologies based on definite experimental evidence such as X-ray crystallography, NMR, gene fusion technique, substituted cysteine accessibility method, Asp (N)-linked glycosylation experiment and other biochemical methods. TMPDB would serve the requirements of both bioinformaticians and biologists, as a test and/or training dataset, for improving the existing TM topology prediction methods and developing novel prediction methods with higher performance as well as for gaining better understanding of TM proteins.
CONSTRUCTION OF TMPDB
We have collected 1074 articles reporting TM topology, by using MEDLINE (27) search with the keywords, ‘transmembrane’ and ‘topology’ (895 articles), by searching directly without using MEDLINE (46 articles), and by referring to the reference position line (RP) of the entries with the following annotations: ‘X-RAY CRYSTALLOGRAPHY’, ‘STRUCTURE BY NEUTRON DIFFRACTION’, ‘STRUCTURE BY ELECTRON CRYO-MICROSCOPY’, ‘STRUCTURE BY NMR’ or ‘TOPOLOGY’ in SWISS-PROT and TrEMBL (28) (133 articles). By checking the content of each collected article, we extracted the experimentally-characterized 302 TM topology models. To obtain the complete sequence annotation that the articles often lack, we crosschecked the sequences in question to public databases such as DDBJ (29), SWISS-PROT, PIR (30) and PDB (5), using the protein name or the partial sequence as a clue. By combining the information contained in the articles and other information of the cross-referenced public databases, we constructed TMPDB in the SWISS-PROT format.
There are 21 cases in total in TMPDB in which two or more articles report topology models for a single sequence, which are almost the same as each other with only a small TMS-position difference (at most 5 amino acids). For these cases, we selected the topology model based on the highest-quality experiment among the reported ones.
TMPDB CURRENT HOLDINGS
The latest release of TMPDB contains 302 TM protein sequences: 276 
-helical sequences (TMPDB_alpha dataset), 17 ß-stranded sequences (TMPDB_beta dataset) and 9 -helical sequences with short pore-forming -helices buried in the membrane (e.g., aquaporin 1) (TMPDB_alpha-buried dataset). The dataset of TMPDB_alpha comprises 165 prokaryotic and 111 eukaryotic sequences while the TMPDB_beta dataset includes only prokaryotic sequences with topologies determined by X-ray diffraction. The TMPDB_alpha-buried dataset includes 6 prokaryotic and 3 eukaryotic sequences, in which topologies are given by X-ray diffraction (7 entries), N-linked glycosylation and protease-protection assays (1 entry each, respectively). The distributions of the number of TMSs included in TMPDB_alpha, TMPDB_alpha-buried and TMPDB_beta datasets are summarized in Table 1. We note that TMPDB widely covers a variety of numbers of TMSs.
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Furthermore, we subjected TMPDB_alpha, TMPDB_beta and TMPDB_alpha-buried datasets to a sequence similarity check (<30%) using CLUSTALW version 1.81 (31), and finally obtained non-redundant datasets—TMPDB_alpha_non-redundant with 231 entries (138 prokaryotic and 93 eukaryotic), TMPDB_beta_non-redundant with 15 entries, and TMPDB_alpha-buried_non-redundant with 7 entries (4 prokaryotic and 3 eukaryotic). Among the TMPDB_alpha_non-redundant entries, 112 topology models are determined by gene fusion experiment, 47 by X-ray diffraction, 5 by NMR, 2 by substituted cysteine accessibility method, 11 by Asp (N)-linked glycosylation and 54 by other biochemical experiments.
The results of comparing TMPDB with other published TM topology datasets, i.e., MEMSAT 1.5 (9), HTP (11), PHDhtm (12), DAS (13), SOSUI (16), HMMTOP 1.1 (17), TMHMM 1.0 (18), PRED-TMR (20), Moeller's (32) and MPtopo (33) are shown in Table 2. We can see, for example, that 122 sequences are common in both TMPDB_alpha_non-redundant and Moeller's non-redundant datasets, and 109 sequences are unique in the former while only 26 in the latter.
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By applying TMPDB_alpha_non-redundant dataset TMPDB, we have evaluated 10 proposed TM topology prediction methods: KKD (7), TMpred (8), TopPred II (10), DAS (13), TMAP (14), MEMSAT 2 (15), SOSUI (16), PRED-TMR2+OrienTM (20,21), TMHMM 2.0 (2) and HMMTOP 2.0 (22) (see 24 for the details). The result shows that even the methods with the highest performance could predict the number of TMSs, number of TMSs+position, and N-tail location with accuracies of only 69.6%, 66.7% and 79.7% for prokaryotic sequences, and 68.8%, 64.5% and 72.0% for eukaryotic ones, respectively. Furthermore, by combining several methods out of the 10 and employing a simple majority-voting approach, we have improved the prediction accuracies to 79.7%, 76.8% and 89.1% for prokaryotic sequences, and 73.1%, 69.9% and 80.6% for eukaryotic ones, respectively (ConPred, 24). The detailed results of prediction performance evaluation are posted in our web site: http://bioinfo.si.hirosaki-u.ac.jp/~ConPred/table_accuracy.html.
ConPred is available for use at the site—http://bioinfo.si.hirosaki-u.ac.jp/~ConPred which is linked to the methods involved in the consensus prediction, where users have the individual methods run, and manually copy individual results and paste them in the input field of the ConPred web page. More detailed information on how to use ConPred can be obtained from the site, http://bioinfo.si.hirosaki-u.ac.jp/~ConPred/help.html.
SEARCHING TMPDB
In the TMPDB web page (http://bioinfo.si.hirosaki-u.ac.jp/~TMPDB/), the ‘Database Search’ is available with the use of gene name, PubMed (27) identifier (PMID), accession numbers of DDBJ (29), SWISS-PROT (28) and PIR (30), PDB (5) identifier, the number of TMS(s), or any combinations of those, over TMPDB or selected subset among the 6 TMPDB subsets. A returned search result will be displayed as a list of retrieved entry(ies) with a link to the complete database entry(ies).
FUTURE DIRECTIONS
TMPDB will be updated continuously at least once every year to increase the number of entries as well to add other detailed experimental information from the referred articles, such as gene-fusion points, the accessibility for fused proteins, etc. Also, the next release of TMPDB on the web will support the display of the graphical image of TM topology and employ an SQL-based engine for more efficient and rapid database search.
CITING AND ACCESSING TMPDB
TMPDB should be cited with the present publication as a reference. TMPDB and its subsets are available for anonymous ftp download as a plain text file from http://bioinfo.si.hirosaki-u.ac.jp/~TMPDB/. We appreciate feedback from users concerning new experimentally-characterized TM topology models for submissions, additions and corrections.
ACKNOWLEDGEMENTS
We appreciate Dr Kenta Nakai for taking time to begin this study, and also Takumi Watanabe for his technical support. This research was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (C) ‘Genome Information Science’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant 14015203).
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