Elementary modes analysis allows one to reveal whether a set of known enzymes is sufficient to sustain functionality of the cell. Moreover, it is helpful in detecting missing reactions and predicting which enzymes could fill these gaps. Here, we perform a comprehensive elementary modes analysis and a genomic context analysis of Mycoplasma pneumoniae nucleotide metabolism, and search for new enzyme activities. The purine and pyrimidine networks are reconstructed by assembling enzymes annotated in the genome or found experimentally. We show that these reaction sets are sufficient for enabling synthesis of DNA and RNA in M. pneumoniae. Special focus is on the key modes for growth. Moreover, we make an educated guess on the nutritional requirements of this micro-organism. For the case that M. pneumoniae does not require adenine as a substrate, we suggest adenylosuccinate synthetase (EC 6.3.4.4), adenylosuccinate lyase (EC 4.3.2.2) and GMP reductase (EC 1.7.1.7) to be operative. GMP reductase activity is putatively assigned to the NRDI_MYCPN gene on the basis of the genomic context analysis. For the pyrimidine network, we suggest CTP synthase (EC 6.3.4.2) to be active. Further experiments on the nutritional requirements are needed to make a decision. Pyrimidine metabolism appears to be more appropriate as a drug target than purine metabolism since it shows lower plasticity.

Protein complexes perform many important functions in the cell. Large-scale studies of protein-protein interactions have not only revealed new complexes but have also placed many proteins into multiple complexes. Whilst the advocates of hypothesis-free research touted the discovery of these shared components as new links between diverse cellular processes, critical commentators denounced many of the findings as artefacts, thus questioning the usefulness of large-scale approaches. Here, we survey proteins known to be shared between complexes, as established in the literature, and compare them to shared components found in high-throughput screens. We discuss the various challenges to the identification and functional interpretation of bona fide shared components, namely contaminants, variant and megacomplexes, and transient interactions, and suggest that many of the novel shared components found in high-throughput screens are neither the results of contamination nor central components, but appear to be primarily regulatory links in cellular processes.

The evolution of enzymes and pathways is under debate. Recent studies show that recruitment of single enzymes from different pathways could be the driving force for pathway evolution. Other mechanisms of evolution, such as pathway duplication, enzyme specialization, de novo invention of pathways or retro-evolution of pathways, appear to be less abundant. Twenty percent of enzyme superfamilies are quite variable, not only in changing reaction chemistry or metabolite type but in changing both at the same time. These variable superfamilies account for nearly half of all known reactions. The most frequently occurring metabolites provide a helping hand for such changes because they can be accommodated by many enzyme superfamilies. Thus, a picture is emerging in which new pathways are evolving from central metabolites by preference, thereby keeping the overall topology of the metabolic network.

The WEB tool "AnDom" assigns to a given protein sequence all experimentally determined structural domains contained within it, including multidomain and large proteins. The server uses profile specific matrices from custom generated multiple sequence alignments of all known SCOP domains (SCOP version 1.50). Prediction time is short allowing numerous applications for structural genomics including investigation of complex eucaryotic protein families. The WWW server is at http://www.bork.embl-heidelberg.de/AnDom, and profiles can be downloaded at ftp.bork.embl-heidelberg.de/pub/users/schmidt/AnDom.

By exploiting the rapid increase in available sequence data, the definition of medically relevant protein targets has been improved by a combination of: (i) differential genome analysis (target list): and (ii) analysis of individual proteins (target analysis). Fast sequence comparisons, data mining, and genetic algorithms further promote these procedures. Mycobacterium tuberculosis proteins were chosen as applied examples.

Genetic distance measurements are an important tool to differentiate field populations of disease vectors such as the mosquito vectors of malaria. Here, we have measured the genetic differentiation between Anopheles arabiensis and Anopheles gambiae, as well as between proposed emerging species of the latter taxon, in whole genome scans by using 23-25 microsatellite loci. In doing so, we have reviewed and evaluated the advantages and disadvantages of standard parameters of genetic distance, F(ST), R(ST), (delta mu)(2), and D. Further, we have introduced new parameters, D' and D(K), which have well defined statistical significance tests and complement the standard parameters to advantage. D' is a modification of D, whereas D(K) is a measure of covariance based on Pearson's correlation coefficient. We find that A. gambiae and A. arabiensis are closely related at most autosomal loci but appear to be distantly related on the basis of X-linked chromosomal loci within the chromosomal Xag inversion. The M and S molecular forms of A. gambiae are practically indistinguishable but differ significantly at two microsatellite loci from the proximal region of the X, outside the Xag inversion. At one of these loci, both M and S molecular forms differ significantly from A. arabiensis, but remarkably, at the other locus, A. arabiensis is indistinguishable from the M molecular form of A. gambiae. These data support the recent proposal of genetically differentiated M and S molecular forms of A. gambiae.

Different gene context methods are reviewed and summarized and an example of implementing several of these strategies into a publicly accessible web server is given.

Four years after the original sequence submission, we have re-annotated the genome of Mycoplasma pneumoniae to incorporate novel data. The total number of ORFss has been increased from 677 to 688 (10 new proteins were predicted in intergenic regions, two further were newly identified by mass spectrometry and one protein ORF was dismissed) and the number of RNAs from 39 to 42 genes. For 19 of the now 35 tRNAs and for six other functional RNAs the exact genome positions were re-annotated and two new tRNA(Leu) and a small 200 nt RNA were identified. Sixteen protein reading frames were extended and eight shortened. For each ORF a consistent annotation vocabulary has been introduced. Annotation reasoning, annotation categories and comparisons to other published data on M.pneumoniae functional assignments are given. Experimental evidence includes 2-dimensional gel electrophoresis in combination with mass spectrometry as well as gene expression data from this study. Compared to the original annotation, we increased the number of proteins with predicted functional features from 349 to 458. The increase includes 36 new predictions and 73 protein assignments confirmed by the published literature. Furthermore, there are 23 reductions and 30 additions with respect to the previous annotation. mRNA expression data support transcription of 184 of the functionally unassigned reading frames.

Human estrogen receptor-alpha messenger RNA (hERalpha mRNA) has a relatively short half-life, which was determined to be approximately 5 h in MCF-7 cell line after actinomycin D treatment. The 3'-untranslated region (3'UTR) of hERalpha mRNA was previously shown to completely down-regulate chloramphenicol acetyltransferase activity when present at the 3'-end of chloramphenicol acetyltransferase transcripts, suggesting a destabilizing function of the hERalpha 3'UTR sequence. Chimeric genes composed of a serum-inducible Fos promoter, GH-coding sequences, and different segments of the hERalpha complementary DNA 3'UTR sequence were used to confirm this hypothesis and to localize the RNA region responsible for the destabilizing effect. The presence of the complete hERalpha 3'UTR reduced the half-life of the reporter mRNA from more than 24 to 3 h. When the hERalpha 3'UTR was subdivided into four fragments (UTR1-4), one fragment, UTR2, retained the most ability to down-regulate the reporter mRNA (t1/2 = 4 h). A stretch of four AUUUA motifs within UTR2 was shown not to mediate mRNA destabilization. In contrast, further subdivision of the UTR2 into three parts (UTR2a-c) resulted in the loss of the destabilizing activity. Finally, recombination of two UTR2 subfragments (UTR2a and -b) partially restored this function, indicating a cooperative role among the three UTR2a-c subfragments in the process that leads to destabilization of the hERalpha transcript.

A set of linear pathways often does not capture the full range of behaviors of a metabolic network. The concept of 'elementary flux modes' provides a mathematical tool to define and comprehensively describe all metabolic routes that are both stoichiometrically and thermodynamically feasible for a group of enzymes. We have used this concept to analyze the interplay between the pentose phosphate pathway (PPP) and glycolysis. The set of elementary modes for this system involves conventional glycolysis, a futile cycle, all the modes of PPP function described in biochemistry textbooks, and additional modes that are a priori equally entitled to pathway status. Applications include maximizing product yield in amino acid and antibiotic synthesis, reconstruction and consistency checks of metabolism from genome data, analysis of enzyme deficiencies, and drug target identification in metabolic networks.

We have probed the association of Flp recombinase with its DNA target using protein footprinting assays. The results are consistent with the domain organization of the Flp protein and with the general features of the protein-DNA interactions revealed by the crystal structures of the recombination intermediates formed by Cre, the Flp-related recombinase. The similarity in the organization of the Flp and Cre target sites and in their recognition by the respective recombinases implies that the overall DNA-protein geometry during strand cleavage in the two systems must also be similar. Within the functional recombinase dimer, it is the interaction between two recombinase monomers bound on either side of the strand exchange region (or spacer) that provides the allosteric activation of a single active site. Whereas Cre utilizes the cleavage nucleophile (the active site tyrosine) in cis, Flp utilizes it in trans (one monomer donating the tyrosine to its partner). By using synthetic Cre and Flp DNA substrates that are geometrically restricted in similar ways, we have mapped the positioning of the active and inactive tyrosine residues during cis and trans cleavage events. We find that, for a fixed substrate geometry, Flp and Cre cleave the labile phosphodiester bond at the same spacer end, not at opposite ends. Our results provide a model that accommodates local heterogeneities in peptide orientations in the two systems while preserving the global functional architecture of the reaction complex.

Comparative analysis of metabolic pathways in different genomes yields important information on their evolution, on pharmacological targets and on biotechnological applications. In this study on glycolysis, three alternative ways of comparing biochemical pathways are combined: (1) analysis and comparison of biochemical data, (2) pathway analysis based on the concept of elementary modes, and (3) a comparative genome analysis of 17 completely sequenced genomes. The analysis reveals a surprising plasticity of the glycolytic pathway. Isoenzymes in different species are identified and compared; deviations from the textbook standard are detailed. Several potential pharmacological targets and by-passes (such as the Entner-Doudoroff pathway) to glycolysis are examined and compared in the different species. Archaean, bacterial and parasite specific adaptations are identified and described.

The presence of genes encoding enzymes involved in the citric-acid cycle has been studied in 19 completely sequenced genomes. In the majority of species, the cycle appears to be incomplete or absent. Several distinct, incomplete cycles reflect adaptations to different environments. Their distribution over the phylogenetic tree hints at precursors in the evolution of the citric-acid cycle.

The flavoenzyme glutathione reductase catalyses electron transfer reactions between two major intracellular redox buffers, namely the NADPH/NADP+ couple and the 2 glutathione/glutathione disulfide couple. On this account, microcrystals of the enzyme were tested as redox probes of intracellular compartments. For introducing protein crystals into human fibroblasts, different methods (microinjection, particle bombardment and optical tweezers) were explored and compared. When glutathione reductase crystals are present in a cytosolic environment, the transition of the yellow Eox form to the orange-red 2-electron reduced charge transfer form, EH2, is observed. Taking into account the midpoint potential of the Eox/EH2 couple, the redox potential of the cytosol was found to be < -270 mV at pH 7.4 and 37 degrees C. As a general conclusion, competent proteins in crystalline--that is signal-amplifying--form are promising probes for studying intracellular events.

To improve protein folding simulations, we investigated a new search strategy in combination with the simple genetic algorithm on a two-dimensional lattice model. This search strategy, we called systematic crossover, couples the best individuals, tests every possible crossover point, and takes the two best individuals for the next generation. We compared the standard genetic algorithm with and without this new implementation for various chain lengths and showed that this strategy finds local minima with better energy values and is significantly faster in identifying the global minimum than the standard genetic algorithm.

A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site-specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha-helical structure for the main contact area.

Amoebapore, a 77-residue peptide with pore-forming activity from the human pathogen Entamoeba histolytica, is implicated in the killing of phagocytosed bacteria and in the cytolytic reaction of the amoeba against host cells. Previously, we structurally and functionally characterized three amoebapore isoforms in E. histolytica but recognized only one homolog in the closely related but non-pathogenic species Entamoeba dispar. Here, we identified two novel amoebapore homologs from E. dispar by molecular cloning. Despite strong resemblance of the primary structures of the homologs, molecular modeling predicts a species-specific variance between the peptide structures. Parallel isolation from trophozoite extracts of the two species revealed a lower amount of pore-forming peptides in E. dispar and substantially higher activity of the major isoform from E. histolytica towards natural membranes than that from E. dispar. Differences in abundance and activity of the lytic polypeptides may have an impact on the pathogenicity of amoebae.

Rational metabolic engineering requires powerful theoretical methods such as pathway analysis, in which the topology of metabolic networks is considered. All metabolic capabilities in steady states are composed of elementary flux modes, which are minimal sets of enzymes that can each generate valid steady states. The modes of the fructose-2,6-bisphosphate cycle, the combined tricarboxylic-acid-glyoxylate-shunt system and tryptophan synthesis are used here for illustration. This approach can be used for many biotechnological applications such as increasing the yield of a product, channelling a product into desired pathways and in functional reconstruction from genomic data.

INTRODUCTION: The measurement of quality of life is an increasingly important issue, particularly in regard to treatment of severe and chronic diseases. The aim of this pilot study was to assess potentially divergent profiles of quality of life in persons with two different pathologies: moderate dementia and cancer. METHOD: This pilot study was carried out in the neurology and cancer services of the medical school in Montpellier, France (Hôpital Gui de Chaulliac and CRLC Val d'Aurelle). The cumulative self-reporting test WHOQOL 100 (World Health Organization Quality of Life with 100 questions) was administered in 57 patients with either moderate senile dementia (27 cases with a Mini-Mental State Examination score >15; mean age of 73) or cancer (30 cases, mainly women with breast cancer; mean age of 53). The stability of responses was tested in a 2-week period. RESULTS: Results of the study showed clear and significant differences between the two groups in the domains of mobility and psychology. Further, eight questions and six facets with a significant difference in responses were found. Responses seemed more stable in the domains of autonomy, social relationship, and religion for the cancer group, and in autonomy and psychology for the dementia group. The age difference may be an important factor in the different quality of life measured but did not significantly influence responses to the test questions. CONCLUSION: The WHOQOL 100 seems a powerful instrument to assess quality of life in diseases such as cancer and moderate dementia. In this study, interesting differences in responses to the test questions between the two pathologic conditions were identified. Items that were unreliable on retesting are singled out. These results will be applied and reevaluated in the development of future, illness-specific and shorter versions of the WHOQOL 100.

Predicting function from sequence using computational tools is a highly complicated procedure that is generally done for each gene individually. This review focuses on the added value that is provided by completely sequenced genomes in function prediction. Various levels of sequence annotation and function prediction are discussed, ranging from genomic sequence to that of complex cellular processes. Protein function is currently best described in the context of molecular interactions. In the near future it will be possible to predict protein function in the context of higher order processes such as the regulation of gene expression, metabolic pathways and signalling cascades. The analysis of such higher levels of function description uses, besides the information from completely sequenced genomes, also the additional information from proteomics and expression data. The final goal will be to elucidate the mapping between genotype and phenotype.

MOTIVATION: The untranslated regions (UTRs) of mRNA upstream (5'UTR) and downstream (3'UTR) of the open reading frame, as well as the mRNA precursor, carry important regulatory sequences. To reveal unidentified regulatory signals, we combine information from experiments with computational approaches. Depending on available knowledge, three different strategies are employed. RESULTS: Searching with a consensus template, new RNAs with regulatory RNA elements can be identified in genomic screens. By this approach, we identify new candidate regulatory motifs resembling iron-responsive elements in the 5'UTRs of HemA, FepB and FrdB mRNA from Escherichia coli. If an RNA element is not yet defined, it may be analyzed by combining results from SELEX (selective enrichment of ligands by exponential amplification) and a search of databases from RNA or genomic sequences. A cleavage stimulating factor (CstF) binding element 3 of the polyadenylation site in the mRNA precursor serves as a test example. Alternatively, the regulatory RNA element may be found by studying different RNA foldings and their correlation with simple experimental tests. We delineate a novel instability element in the 3'UTR of the estrogen receptor mRNA in this way. AVAILABILITY: Strategy, methods and programs are available on request from T.Dandekar. CONTACT: dandekar@embl-heidelberg.de

A systematic comparison of nine bacterial and archaeal genomes reveals a low level of gene-order (and operon architecture) conservation. Nevertheless, a number of gene pairs are conserved. The proteins encoded by conserved gene pairs appear to interact physically. This observation can therefore be used to predict functions of, and interactions between, prokaryotic gene products.

We introduce a simple and rapid strategy to identify genes that are responsible for species-specific phenotypes. The genome of a species that has a specific phenotype is compared with at least one, closely related, species that lacks this phenotype. Homologous genes that are shared among the species compared are identified and discarded from the list of candidates for species-specific genes. The process is automated and rapidly yields a small subset of the genome that likely contains genes responsible for the species-specific features. Functions are assigned to the genes, and dubious annotations are filtered out. Information is extracted not only from the presence of genes, but also from their absence with respect to known phenotypes. We have applied the technique to identify a set of species-specific genes in Helicobacter pylori by comparing it with its closest relatives for which complete genome sequences are available, Haemophilus influenzae and Escherichia coli. Of the genes of this set for which functional features can be obtained, a large fraction (63%, 123 proteins) is (potentially) involved in H. pylori's interaction with its host. We hypothesize that a family of outer membrane proteins is critical for the ability of H. pylori to colonize host cells in highly acidic environments.

Major pathogenic functions of Entamoeba histolytica involved in destruction of host tissues are the degradation of extracellular matrix proteins mediated by secreted cysteine proteinases and contact-dependent killing of host cells via membrane-active factors. A soluble protein with an affinity for membranes was purified from amoebic extracts to apparent homogeneity. N-terminal sequencing and subsequent molecular cloning of the factor revealed that it is a member of the cysteine proteinase family of E. histolytica, which we termed CP5. Further experiments with the purified protein showed that it has potent proteolytic activity that is abrogated in the presence of inhibitors specific for cysteine proteinases. The enzyme firmly associates with membranes retaining its proteolytic activity and it produces cytopathic effects on cultured monolayers. A model of the three-dimensional structure of CP5 revealed the presence of a hydrophobic patch that may account for the potential of the protein to associate with membranes. Immunocytochemical localization of the enzyme to the surface of the amoeba in combination with the recent finding that the gene encoding CP5 is missing in the closely related but non-pathogenic Entamoeba dispar suggests a potential role of the protein in host tissue destruction of E. histolytica.

The genes encoding the small nucleolar RNA (snoRNA) species snR190 and U14 are located close together in the genome of Saccharomyces cerevisiae. Here we report that these two snoRNAs are synthesized by processing of a larger common transcript. In strains mutant for two 5'-->3' exonucleases, Xrn1p and Rat1p, families of 5'-extended forms of snR190 and U14 accumulate; these have 5' extensions of up to 42 and 55 nucleotides, respectively. We conclude that the 5' ends of both snR190 and U14 are generated by exonuclease digestion from upstream processing sites. In contrast to snR190 and U14, the snoRNAs U18 and U24 are excised from the introns of pre-mRNAs which encode proteins in their exonic sequences. Analysis of RNA extracted from a dbr1-delta strain, which lacks intron lariat-debranching activity, shows that U24 can be synthesized only from the debranched lariat. In contrast, a substantial level of U18 can be synthesized in the absence of debranching activity. The 5' ends of these snoRNAs are also generated by Xrn1p and Rat1p. The same exonucleases are responsible for the degradation of several excised fragments of the pre-rRNA spacer regions, in addition to generating the 5' end of the 5.8S rRNA. Processing of the pre-rRNA and both intronic and polycistronic snoRNAs therefore involves common components.

Critical events in 3'-end processing of pre-mRNA are the recognition of the AAUAAA polyadenylation signal by cleavage and polyadenylation specificity factor (CPSF) and the binding of cleavage stimulation factor (CstF) via its 64-kDa subunit to the downstream element. The stability of this CPSF.CstF.RNA complex is thought to determine the efficiency of 3'-end processing. Since downstream elements reveal high sequence variability, in vitro selection experiments with highly purified CstF were performed to investigate the sequence requirements for CstF-RNA interaction. CstF was purified from calf thymus and from HeLa cells. Surprisingly, calf thymus CstF contained an additional, novel form of the 64-kDa subunit with a molecular mass of 70 kDa. RNA ligands selected by HeLa and calf thymus CstF contained three highly conserved sequence elements as follows: element 1 (AUGCGUUCCUCGUCC) and two closely related elements, element 2a (YGUGUYN0-4UUYAYUGYGU) and element 2b (UUGYUN0-4AUUUACU(U/G)N0-2YCU). All selected sequences tested functioned as downstream elements in 3'-end processing in vitro. A computer survey of the EMBL data library revealed significant homologies to all selected elements in naturally occurring 3'-untranslated regions. The majority of element 2a homologies was found downstream of coding sequences. Therefore, we postulate that this element represents a novel consensus sequence for downstream elements in 3'-end processing of pre-mRNA.

Specific residue interactions as revealed from a few and readily available experiments can be quite important in shaping a protein's tertiary topology by complementing basic and general folding principles. This experimental information is employed in structure prediction (mainchain topology) based on sequence knowledge and the genetic algorithm with its ability to optimize simultaneously many parameters. Examples investigated include the distribution of cysteinyl S-S bonds, protein side-chain ligands to iron-sulfur cages, cofactor-ligands, crosslinks amongst side-chains, and conserved hydrophobic and catalytic residues. Such interactions yield an improvement in the predicted topology (0.4-6.6 A root mean square deviation in the positions of the backbone C alpha-atoms relative to those observed) compared with those resulting from simulations relying only on basic protein folding principles. For several examples the resultant topology depended critically on knowledge of the few and specific interactions such that the relationship between predicted and observed C alpha-positions was near random without their use. The combined methodology (experimental data and the genetic algorithm) should prove helpful in settings where experiment and theory can cooperate in successive steps to elucidate an unknown structure.

BACKGROUND: Amoebapore of the protozoan Entamoeba histolytica and NK-lysin of porcine cytotoxic lymphocytes are effector peptides from organisms separated extremely early in their evolutionary paths. The peptides intrigued us, however, with indications of some functional similarity. We thus wanted to derive and compare predictions for their as yet unknown three-dimensional structures as a guide for and to be tested by further experiments. RESULTS: Molecular models were generated by use of a genetic algorithm that selects according to basic protein structure principles exploiting available information such as the primary structures, secondary structure predictions and positions of disulfide bonds. Topological differences aside, the structural motif of an antiparallel four-alpha-helix bundle with adjacent connections and intramolecular crosslinks is predicted for both types of peptides. It combines the feature of amphipathic alpha-helices with a disulfide-bonded compact structure known from the beta-sheeted defensins and small toxins. CONCLUSIONS: The models presented here strengthen the notion that amoebapore and NK-lysin are particular among cytolytic and antibacterial polypeptides and share a similar function and structural motif. They also allow experimental testing and a better comparison of the two proteins in view of the predicted similarities and differences of their respective folds.

A topological and functional overview of a DNA recognition protein with unknown structure can be achieved by combining three different, but complementary approaches: modeling by the genetic algorithm, functional analysis of mutated variants, and testing the target DNA using non-canonical oligonucleotides. As an example we choose the Flp protein, a site-specific recombinase from Saccharomyces cerevisiae. We derive the topological outline including the DNA binding cleft, examine DNA binding regions by deletional and mutational analysis, and analyze the DNA binding site using 7-deazaadenine, 7-deazaguanine, inosine and 4-O-methylthymine as probes. The combined data offer a comprehensive sketch of a plausible protein architecture for Flp. The structure is detailed enough to verify the prediction accuracy for different peptide regions from pre-existing data and by new experimental design.

Grid-free protein folding simulations based on sequence and secondary structure knowledge (using mostly experimentally determined secondary structure information but also analysing results from secondary structure predictions) were investigated using the genetic algorithm, a backbone representation, and standard dihedral angular conformations. Optimal structures are selected according to basic protein building principles. Having previously applied this approach to proteins with helical topology, we have now developed additional criteria and weights for beta-strand-containing proteins, validated them on four small beta-strand-rich proteins with different topologies, and tested the general performance of the method on many further examples from known protein structures with mixed secondary structural type and less than 100 amino acid residues. Topology predictions close to the observed experimental structures were obtained in four test cases together with fitness values that correlated with the similarity of the predicted topology to the observed structures. Root-mean-square deviation values of C alpha atoms in the superposed predicted and observed structures, the latter of which had different topologies, were between 4.5 and 5.5 A(2.9 to 5.1 A without loops). Including 15 further protein examples with unique folds, root-mean-square deviation values ranged between 1.8 and 6.9 A with loop regions and averaged 5.3 A and 4.3 A, including and excluding loop regions, respectively.