Comprehensive metabolomics of Philippine Stichopus cf. horrens reveals diverse classes of valuable small molecules for biomedical applications.

Stichopus cf. horrens is an economically important sea cucumber species in Southeast Asia due to their presumed nutritional and medicinal benefits. However, compared to other sea cucumbers such as Apostichopus japonicus, there are no biochemical studies on which compounds contribute to the purported bioactivities of S. cf. horrens. To address this, a high-throughput characterization of the global metabolite profile of the species was performed through LC-MS/MS experiments and utilizing open-access platforms such as GNPS, XCMS, and metaboAnalyst. Bioinformatics-based molecular networking and chemometrics revealed the abundance of phospholipids such as phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), and phosphatidylserines (PSs) in the crude samples. Body wall extracts were observed to have higher levels of structural, diacylated PCs, while the viscera have higher relative abundance of single-tail PCs and PEs that could be involved in digestion via nutrient absorption and transport for sea cucumbers. PEs and sphingolipids could also be implicated in the ecological response and morphological transformations of S. cf. horrens in the presence of predatory and other environmental stress. Interestingly, terpenoid glycosides and saponins with reported anti-cancer benefits were significantly localized in the body wall. The sulfated alkanes and sterols present in S. cf. horrens bear similarity to known kairomones and other signaling molecules. All in all, the results provide a baseline metabolomic profile of S. cf. horrens that may further be used for comparative and exploratory studies and suggest the untapped potential of S. cf. horrens as a source of bioactive molecules.

Physicochemical and Biochemical Characterization of Collagen from Stichopus cf. horrens Tissues for Use as Stimuli-Responsive Thin Films.

The mutable collagenous tissue (MCT) of sea cucumber, with its ability to rapidly change its stiffness and extensibility in response to different environmental stress conditions, serves as inspiration for the design of new smart functional biomaterials. Collagen, extracted from the body wall of Stichopus cf. horrens, a species commonly found in the Philippines, was characterized for its suitability as stimuli-responsive films. Protein BLAST search showed the presence of sequences commonly found in type VII and IX collagen, suggesting that Stichopus horrens collagen is heterotypic. The maximum transition temperature recorded was 56.0 ± 2 °C, which is higher than those of other known sources of marine collagen. This suggests that S. horrens collagen has better thermal stability and durability. Collagen-based thin films were then prepared, and atomic force microscopy (AFM) imaging showed the visible collagen network comprising the films. The thin films were subjected to thermomechanical analysis with degradation starting at >175 °C. At 100-150 °C, the collagen-based films apparently lose their translucency due to the removal of moisture. Upon exposure to ambient temperature, instead of degrading, the films were able to revert to the original state due to the readsorption of moisture. This study is a demonstration of a smart biomaterial developed from S. cf. horrens collagen with potential applications in food, pharmaceutical, biomedical, and other collagen-based research.

Specificity of Monoterpene Interactions with Insect Octopamine and Tyramine Receptors: Insights from in Silico Sequence and Structure Comparison.

Octopamine and tyramine receptors (OARs/TARs) are interesting targets for new insecticide development due to their unique roles in insects' physiological and cellular response and their specificity to invertebrates. Monoterpene compounds that bear resemblance to the natural ligands have been shown to bind to the OARs/TARs but elicit varied responses in different insect species. Using in silico methods, we attempt to investigate the molecular basis of monoterpene interactions and their specificity in different OARs and TARs of damaging or beneficial insects. Sequence and structure comparison revealed that the OARs/TARs studied generally have more similarities in terms of structure rather than sequence identity. Together with clustering and network analyses, we also revealed that the role of IL3 might be crucial in the identification of OAR and TAR and their unique function. Among the 35 monoterpenes subjected to ensemble docking, carvacrol had the most negative average binding energies with the target insect OARs and TARs. The differences in the key interacting residues of carvacrol with insect OARs and TARs could be the origin of variation in the responses of insect species to this monoterpene. Results suggest that carvacrol may be a potential natural-product-based insecticide, targeting multiple insect pests while being nonharmful to honeybees and Asian swallowtail butterflies. This work could provide insights into the development of effective species-specific natural-product-based insecticides that are more environmentally friendly than conventional insecticides.

Bacterial Community Assembly, Succession, and Metabolic Function during Outdoor Cultivation of Microchloropsis salina.

Outdoor cultivation of microalgae has promising potential for renewable bioenergy, but there is a knowledge gap on the structure and function of the algal microbiome that coinhabits these ecosystems. Here, we describe the assembly mechanisms, taxonomic structure, and metabolic potential of bacteria associated with Microchloropsis salina cultivated outdoors. Open mesocosms were inoculated with algal cultures that were either free of bacteria or coincubated with one of two different strains of alga-associated bacteria and were sampled across five time points taken over multiple harvesting rounds of a 40-day experiment. Using quantitative analyses of metagenome-assembled genomes (MAGs), we tracked bacterial community compositional abundance and taxon-specific functional capacity involved in algal-bacterial interactions. One of the inoculated bacteria (Alteromonas sp.) persisted and dispersed across mesocosms, whereas the other inoculated strain (Phaeobacter gallaeciensis) disappeared by day 17 while a taxonomically similar but functionally distinct Phaeobacter strain became established. The inoculated strains were less abundant than 6 numerically dominant newly recruited taxa with functional capacities for mutualistic or saprophytic lifestyles, suggesting a generalist approach to persistence. This includes a highly abundant unclassified Rhodobacteraceae species that fluctuated between 25% and 77% of the total community. Overall, we did not find evidence for priority effects exerted by the distinct inoculum conditions; all mesocosms converged with similar microbial community compositions by the end of the experiment. Instead, we infer that the 15 total populations were retained due to host selection, as they showed high metabolic potential for algal-bacterial interactions such as recycling alga-produced carbon and nitrogen and production of vitamins and secondary metabolites associated with algal growth and senescence, including B vitamins, tropodithietic acid, and roseobacticides. IMPORTANCE Bacteria proliferate in nutrient-rich aquatic environments, including engineered algal biofuel systems, where they remineralize photosynthates, exchange secondary metabolites with algae, and can influence system output of biomass or oil. Despite this, knowledge on the microbial ecology of algal cultivation systems is lacking, and the subject is worthy of investigation. Here, we used metagenomics to characterize the metabolic capacities of the predominant bacteria associated with the biofuel-relevant microalga Microchloropsis salina and to predict testable metabolic interactions between algae and manipulated communities of bacteria. We identified a previously undescribed and uncultivated organism that dominated the community. Collectively, the microbial community may interact with the alga in cultivation via exchange of secondary metabolites which could affect algal success, which we demonstrate as a possible outcome from controlled experiments with metabolically analogous isolates. These findings address the scalability of lab-based algal-bacterial interactions through to cultivation systems and more broadly provide a framework for empirical testing of genome-based metabolic predictions.

Diaporthe/Phomopsis longicolla degrades an array of bisphenol analogues with secreted laccase.

With recent initiatives to ban bisphenol A (BPA) in certain commercial products, manufacturers shifted to the production and use of BPA analogues. However, some of these BPA alternatives still possess endocrine disruptive activities. Many fungal enzymes are known to biodegrade phenolic compounds, such as BPA. However, the activity of these enzymes on BPA analogues remains unexplored. This study reports a secreted laccase from the endophytic fungus Diaporthe longicolla capable of degrading an impressive range of bisphenol analogues. The secreted crude enzymes are optimally active at pH 5 from 39 °C to 60 °C, efficiently degrading BPA as well as BPA analogues BPB, BPC, BPE and BPF. A purified form of laccase was identified from the crude fungal extract using FPLC and peptide sequencing. Furthermore, BPA induced the expression of this D. longicolla laccase gene. Overall, this paper demonstrated that the crude laccase enzyme from D. longicolla metabolizes BPA and select analogues, implicating the potential role of this fungus to remove environmental bisphenols.

Identification of Conomarphin Variants in the Conus eburneus Venom and the Effect of Sequence and PTM Variations on Conomarphin Conformations.

Marine cone snails belonging to the Conidae family make use of neuroactive peptides in their venom to capture prey. Here we report the proteome profile of the venom duct of Conus eburneus, a cone snail belonging to the Tesseliconus clade. Through tandem mass spectrometry and database searching against the C. eburneus transcriptome and the ConoServer database, we identified 24 unique conopeptide sequences in the venom duct. The majority of these peptides belong to the T and M gene superfamilies and are disulfide-bonded, with cysteine frameworks V, XIV, VI/VII, and III being the most abundant. All seven of the Cys-free peptides are conomarphin variants belonging to the M superfamily that eluted out as dominant peaks in the chromatogram. These conomarphins vary not only in amino acid residues in select positions along the backbone but also have one or more post-translational modifications (PTMs) such as proline hydroxylation, C-term amidation, and γ-carboxylation of glutamic acid. Using molecular dynamics simulations, the conomarphin variants were predicted to predominantly have hairpin-like or elongated structures in acidic pH. These two structures were found to have significant differences in electrostatic properties and the inclusion of PTMs seems to complement this disparity. The presence of polar PTMs (hydroxyproline and γ-carboxyglutamic acid) also appear to stabilize hydrogen bond networks in these conformations. Furthermore, these predicted structures are pH sensitive, becoming more spherical and compact at higher pH. The subtle conformational variations observed here might play an important role in the selection and binding of the peptides to their molecular targets.

Ligand-Induced Conformational Dynamics of A Tyramine Receptor from Sitophilus oryzae.

Tyramine receptor (TyrR) is a biogenic amine G protein-coupled receptor (GPCR) associated with many important physiological functions in insect locomotion, reproduction, and pheromone response. Binding of specific ligands to the TyrR triggers conformational changes, relays the signal to G proteins, and initiates an appropriate cellular response. Here, we monitor the binding effect of agonist compounds, tyramine and amitraz, to a Sitophilus oryzae tyramine receptor (SoTyrR) homology model and their elicited conformational changes. All-atom molecular dynamics (MD) simulations of SoTyrR-ligand complexes have shown varying dynamic behavior, especially at the intracellular loop 3 (IL3) region. Moreover, in contrast to SoTyrR-tyramine, SoTyrR-amitraz and non-liganded SoTyrR shows greater flexibility at IL3 residues and were found to be coupled to the most dominant motion in the receptor. Our results suggest that the conformational changes induced by amitraz are different from the natural ligand tyramine, albeit being both agonists of SoTyrR. This is the first attempt to understand the biophysical implication of amitraz and tyramine binding to the intracellular domains of TyrR. Our data may provide insights into the early effects of ligand binding to the activation process of SoTyrR.

Conomarphins cause paralysis in mollusk: Critical and tunable structural elements for bioactivity.

Two conomarphins were purified as the major component of the venom of Conus eburneus. Conomarphins Eb1 and Eb2 showed biological activity in the mollusk Pomacea padulosa, causing sluggishness and retraction of siphon, foot, and cephalic tentacles. To further probe the effects of conserved amino acids and posttranslational modifications in conomarphins, we prepared four synthetic analogues: conomarphin Eb1 Hyp10Pro, Hyp10Ala, d-Phe13Ala, and l-Phe13 variants. Structure-activity relationship analysis indicated that d-Phe13 is critical to the biological activity of conomarphins. In contrast, amino acid changes at position 10 and removal of posttranslational modification in Hyp10Pro can be tolerated. The high expression level and observed mollusk activity of conomarphins may suggest their potential role as defensive arsenal of Conoidean snails against other predatory gastropods.

Changes in the Structure of the Microbial Community Associated with Nannochloropsis salina following Treatments with Antibiotics and Bioactive Compounds.

Open microalgae cultures host a myriad of bacteria, creating a complex system of interacting species that influence algal growth and health. Many algal microbiota studies have been conducted to determine the relative importance of bacterial taxa to algal culture health and physiological states, but these studies have not characterized the interspecies relationships in the microbial communities. We subjected Nanochroloropsis salina cultures to multiple chemical treatments (antibiotics and quorum sensing compounds) and obtained dense time-series data on changes to the microbial community using 16S gene amplicon metagenomic sequencing (21,029,577 reads for 23 samples) to measure microbial taxa-taxa abundance correlations. Short-term treatment with antibiotics resulted in substantially larger shifts in the microbiota structure compared to changes observed following treatment with signaling compounds and glucose. We also calculated operational taxonomic unit (OTU) associations and generated OTU correlation networks to provide an overview of possible bacterial OTU interactions. This analysis identified five major cohesive modules of microbiota with similar co-abundance profiles across different chemical treatments. The Eigengenes of OTU modules were examined for correlation with different external treatment factors. This correlation-based analysis revealed that culture age (time) and treatment types have primary effects on forming network modules and shaping the community structure. Additional network analysis detected Alteromonadeles and Alphaproteobacteria as having the highest centrality, suggesting these species are "keystone" OTUs in the microbial community. Furthermore, we illustrated that the chemical tropodithietic acid, which is secreted by several species in the Alphaproteobacteria taxon, is able to drastically change the structure of the microbiota within 3 h. Taken together, these results provide valuable insights into the structure of the microbiota associated with N. salina cultures and how these structures change in response to chemical perturbations.

Longitudinal Analysis of Microbiota in Microalga Nannochloropsis salina Cultures.

Large-scale open microalgae cultivation has tremendous potential to make a significant contribution to replacing petroleum-based fuels with biofuels. Open algal cultures are unavoidably inhabited with a diversity of microbes that live on, influence, and shape the fate of these ecosystems. However, there is little understanding of the resilience and stability of the microbial communities in engineered semicontinuous algal systems. To evaluate the dynamics and resilience of the microbial communities in microalgae biofuel cultures, we conducted a longitudinal study on open systems to compare the temporal profiles of the microbiota from two multigenerational algal cohorts, which include one seeded with the microbiota from an in-house culture and the other exogenously seeded with a natural-occurring consortia of bacterial species harvested from the Pacific Ocean. From these month-long, semicontinuous open microalga Nannochloropsis salina cultures, we sequenced a time-series of 46 samples, yielding 8804 operational taxonomic units derived from 9,160,076 high-quality partial 16S rRNA sequences. We provide quantitative evidence that clearly illustrates the development of microbial community is associated with microbiota ancestry. In addition, N. salina growth phases were linked with distinct changes in microbial phylotypes. Alteromonadeles dominated the community in the N. salina exponential phase whereas Alphaproteobacteria and Flavobacteriia were more prevalent in the stationary phase. We also demonstrate that the N. salina-associated microbial community in open cultures is diverse, resilient, and dynamic in response to environmental perturbations. This knowledge has general implications for developing and testing design principles of cultivated algal systems.

Bioinformatic processing to identify single nucleotide polymorphism that potentially affect Ape1 function.

Inactivation of DNA damage response mechanisms is associated with several disease syndromes, including cancer, aging and neurodegeneration. A major corrective pathway for alkylation or oxidative DNA damage is base excision repair (BER). As part of an effort to identify variation in DNA repair genes, we used the expressed sequence tag (EST) database to identify amino acid variation in Ape1, an essential gene in the BER repair pathway. Nucleotide substitutions were considered valid only if the amino acid changes were observed in at least two independent EST sequencing runs (i.e. two independent EST reports). In total eighty amino acid variants were identified for the Ape1 gene. Using software tools SIFT and PolyPhen, which predict impacts of amino acid substitutions on protein structure and function, twenty-six variants were predicted by both algorithms to be deleterious to protein function. Majority of these intolerant mutations such as V206C and F240S, lie within the core of the protein and may affect the stability and folding of Ape1, or in the case of N212H, N212K, and Y171N, are close to the enzyme's active site and could drastically affect its function. A few of the intolerant mutations, i.e., G178V and E217R, are surface residues and are far from the active site, and as such, the predicted effect on Ape1 stability or function is not evident. These variants are reagents for further protein function studies and molecular epidemiology studies of cancer susceptibility.

Elucidating the higher-order structure of biopolymers by structural probing and mass spectrometry: MS3D.

Chemical probing represents a very versatile alternative for studying the structure and dynamics of substrates that are intractable by established high-resolution techniques. The implementation of MS-based strategies for the characterization of probing products has not only extended the range of applicability to virtually all types of biopolymers but has also paved the way for the introduction of new reagents that would not have been viable with traditional analytical platforms. As the availability of probing data is steadily increasing on the wings of the development of dedicated interpretation aids, powerful computational approaches have been explored to enable the effective utilization of such information to generate valid molecular models. This combination of factors has contributed to making the possibility of obtaining actual 3D structures by MS-based technologies (MS3D) a reality. Although approaches for achieving structure determination of unknown targets or assessing the dynamics of known structures may share similar reagents and development trajectories, they clearly involve distinctive experimental strategies, analytical concerns and interpretation paradigms. This Perspective offers a commentary on methods aimed at obtaining distance constraints for the modeling of full-fledged structures while highlighting common elements, salient distinctions and complementary capabilities exhibited by methods used in dynamics studies. We discuss critical factors to be addressed for completing effective structural determinations and expose possible pitfalls of chemical methods. We survey programs developed for facilitating the interpretation of experimental data and discuss possible computational strategies for translating sparse spatial constraints into all-atom models. Examples are provided to illustrate how the concerted application of very diverse probing techniques can lead to the solution of actual biological systems.

The collaboratory for MS3D: a new cyberinfrastructure for the structural elucidation of biological macromolecules and their assemblies using mass spectrometry-based approaches.

Modern biomedical research is evolving with the rapid growth of diverse data types, biophysical characterization methods, computational tools and extensive collaboration among researchers spanning various communities and having complementary backgrounds and expertise. Collaborating researchers are increasingly dependent on shared data and tools made available by other investigators with common interests, thus forming communities that transcend the traditional boundaries of the single research laboratory or institution. Barriers, however, remain to the formation of these virtual communities, usually due to the steep learning curve associated with becoming familiar with new tools, or with the difficulties associated with transferring data between tools. Recognizing the need for shared reference data and analysis tools, we are developing an integrated knowledge environment that supports productive interactions among researchers. Here we report on our current collaborative environment, which focuses on bringing together structural biologists working in the area of mass spectrometric based methods for the analysis of tertiary and quaternary macromolecular structures (MS3D) called the Collaboratory for MS3D (C-MS3D). C-MS3D is a Web-portal designed to provide collaborators with a shared work environment that integrates data storage and management with data analysis tools. Files are stored and archived along with pertinent meta data in such a way as to allow file handling to be tracked (data provenance) and data files to be searched using keywords and modification dates. While at this time the portal is designed around a specific application, the shared work environment is a general approach to building collaborative work groups. The goal of this is to not only provide a common data sharing and archiving system, but also to assist in the building of new collaborations and to spur the development of new tools and technologies.

MS3D structural elucidation of the HIV-1 packaging signal.

The structure of HIV-1 Psi-RNA has been elucidated by a concerted approach combining structural probes with mass spectrometric detection (MS3D), which is not affected by the size and crystallization properties of target biomolecules. Distance constraints from bifunctional cross-linkers provided the information required for assembling an all-atom model from the high-resolution coordinates of separate domains by triangulating their reciprocal placement in 3D space. The resulting structure revealed a compact cloverleaf morphology stabilized by a long-range tertiary interaction between the GNRA tetraloop of stemloop 4 (SL4) and the upper stem of stemloop 1 (SL1). The preservation of discrete stemloop structures ruled out the possibility that major rearrangements might produce a putative supersite with enhanced affinity for the nucleocapsid (NC) domain of the viral Gag polyprotein, which would drive genome recognition and packaging. The steric situation of single-stranded regions exposed on the cloverleaf structure offered a valid explanation for the stoichiometry exhibited by full-length Psi-RNA in the presence of NC. The participation of SL4 in a putative GNRA loop-receptor interaction provided further indications of the plasticity of this region of genomic RNA, which can also anneal with upstream sequences to stabilize alternative conformations of the 5' untranslated region (5'-UTR). Considering the ability to sustain specific NC binding, the multifaceted activities supported by the SL4 sequence suggest a mechanism by which Gag could actively participate in regulating the vital functions mediated by 5'-UTR. Substantiated by the 3D structure of Psi-RNA, the central role played by SL4 in specific RNA-RNA and protein-RNA interactions advances this domain as a primary target for possible therapeutic intervention.

Toward building a database of bifunctional probes for the MS3D investigation of nucleic acids structures.

This report illustrates the approaches employed to investigate critical aspects of the activity of crosslinking reagents toward nucleic acid substrates, which should be evaluated to identify candidate probes for mass spectrometric 3D (MS3D) investigations of biomolecules and macromolecular complexes. Representative members of different classes of bifunctional reagents were taken into consideration, including bikethoxal and phenyl-diglyoxal [bis-(1,2-dicarbonyls)], cisplatin (coordinative binding agents), chlorambucil and nitrogen mustard [bis-(2-chloroethyl)amines], and sym-triazine trichloride (triazines). Nanospray-Fourier transform mass spectrometry (FTMS) was applied without desalting or separation procedures to characterize the covalent products obtained by probing dinucleotide and trinucleotide substrates under a variety of experimental conditions in vitro. The carefully controlled composition of these substrates enabled us to obtain valid comparisons of probe activity toward individual nucleotides and evaluate possible base-specific effects, including the stability of the different adducts in solution under the selected reaction conditions. The gas-phase behavior of the observed products was investigated using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) to obtain valuable information for guiding the design of sequencing experiments and helping the data interpretation. Structured RNA substrates, such as HIV-1 stemloop 1, were finally employed to investigate the structural determinant of adduct formation and highlight the different nature of the spatial information provided by the various candidate probes.

Mass spectrometric investigation of protein alkylation by the RNA footprinting probe kethoxal.

The reactivity of the RNA footprinting reagent kethoxal (KT) toward proteins was investigated by electrospray ionization-Fourier transform mass spectrometry. Using standard peptides, KT was shown to selectively modify the guanidino group of arginine side chains at neutral pH, while primary amino groups of lysine and N-terminus were found to be unreactive under these conditions. Gas-phase fragmentation of KT adducts provided evidence for a cyclic 1,2-diol structure. Esterification of the 1,2-diol product was obtained in borate buffer, and its structure was also investigated by tandem mass spectrometry. When model proteins were probed with this RNA footprinting reagent, the adducts proved to be sufficiently stable to allow for the application of different peptide-mapping procedures to identify the location of modified arginines. Probing of proteins under native folding conditions provided modification patterns that very closely matched the structural context of arginines in the global protein structure. A strong correlation was demonstrated between the susceptibility to modification and residue accessibility calculated from the known 3D structure. When the complexes formed by HIV-1 nucleocapsid (NC) protein and RNA stemloops SL2 and SL3 were investigated, KT footprinting provided accurate information regarding the involvement of individual arginines in binding RNA and showed different reactivity according to their mode of interaction.

Atmospheric pressure MALDI-FTMS of normal and chemically modified RNA.

Atmospheric pressure (AP) MALDI has been combined with Fourier transform mass spectrometry (FTMS) to obtain the unambiguous characterization of RNA samples modified by solvent accessibility reagents used in structural studies of RNA and protein-RNA complexes. The formation of cation adducts typical of MS analysis of nucleic acids was effectively reduced by extensive washing of the anionic analytes retained onto the probe surface by strong interactions with a cationic layer of poly(diallyldimethylammonium chloride) (PADMAC). This rapid desalting procedure allowed for the detection of DNA and RNA samples in high femtomole quantities distributed over a 4 x 4 mm sample well. AP MALDI-FTMS was shown to provide high-resolution spectra for analytes as large as approximately 6.4 kDa with little or no evidence of metastable decomposition. The absence of significant metastable decay observed for precursor ions selected for tandem experiments offered a further measure of the low energy content typical of ions generated by AP MALDI. This feature proved to be very beneficial in the characterization of chemically modified RNA samples, which become particularly prone to base losses upon alkylation. The high resolution offered by FTMS enabled the application of a data-reduction algorithm capable of rejecting any signal devoid of plausible isotopic distribution, thus facilitating the analysis of complex analyte mixtures produced by nuclease treatment of RNA substrates. Proper selection of nucleases and digestion conditions can ensure the production of hydrolytic fragments of manageable size, which could extend the range of applicability of this bottom-up strategy to the structural investigation of very large RNA and protein-RNA complexes.

Untying the FIV frameshifting pseudoknot structure by MS3D.

The structure of the putative feline immunodeficiency virus (FIV) ribosomal frameshifting pseudoknot (PK) has been investigated by a mass spectrometric three-dimensional (MS3D) approach, which involves the application of established solvent-accessibility probes and chemical crosslinkers with detection by electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). Regardless of their size, probed substrates can be treated with ribonucleases and analyzed by ESI-FTMS to obtain the correct position of chemically modified nucleotides. Protection maps and distance information can be utilized to generate 3D models using the constraint satisfaction algorithm provided by MC-SYM and the energy minimization modules included in CNS. Control experiments were performed on a mutant of mouse mammary tumor virus pseudoknot (VPK), for which an NMR structure is available. Comparison between the MS3D model and the high-resolution structure provided a approximately 3A root-mean-square deviation calculated from all the atoms present in double-stranded regions. Applied to FIV-PK, the MS3D approach confirmed that the selected sequence could fold into an actual pseudoknot, supporting the sequence alignment predictions. Characteristic features of H-type pseudoknots were recognized immediately, but a putative A13-U30 pair was not observed at the stem junction, making FIV-PK resemble VPK more closely than the initially suggested simian retrovirus type-1 pseudoknot. In our model, the unpaired U30 protrudes into the medium, while the hinging A13 assumes a stacked conformation that enables the stems to form a approximately 60 degrees bend and relieve the strain caused by a short loop 1. The model provided the basis to explain the different alkylation patterns observed in the absence and presence of Mg(2+), suggesting the possible formation of a specific metal-binding site between loop 1 and stem 2. This instance illustrates how the MS3D model of FIV-PK can be utilized effectively to generate hypotheses and support functional observations in the absence of a high-resolution structure.