Engineered polyethylene terephthalate hydrolases: perspectives and limits.

Polyethylene terephthalate (PET) is a major component of plastic waste. Enzymatic PET hydrolysis is the most ecofriendly recycling technology. The biorecycling of PET waste requires the complete depolymerization of PET to terephthalate and ethylene glycol. The history of enzymatic PET depolymerization has revealed two critical issues for the industrial depolymerization of PET: industrially available PET hydrolases and pretreatment of PET waste to make it susceptible to full enzymatic hydrolysis. As none of the wild-type enzymes can satisfy the requirements for industrialization, various mutational improvements have been performed, through classical technology to state-of-the-art computational/machine-learning technology. Recent engineering studies on PET hydrolases have brought a new insight that flexibility of the substrate-binding groove may improve the efficiency of PET hydrolysis while maintaining sufficient thermostability, although the previous studies focused only on enzymatic thermostability above the glass transition temperature of PET. Industrial biorecycling of PET waste is scheduled to be implemented, using micronized amorphous PET. Next stage must be the development of PET hydrolases that can efficiently degrade crystalline parts of PET and expansion of target PET materials, not only bottles but also textiles, packages, and microplastics. This review discusses the current status of PET hydrolases, their potential applications, and their profespectal goals. KEY POINTS: • PET hydrolases must be thermophilic, but their operation must be below 70 °C • Classical and state-of-the-art engineering approaches are useful for PET hydrolases • Enzyme activity on crystalline PET is most expected for future PET biorecycling.

Structural Perspective of NR4A Nuclear Receptor Family and Their Potential Endogenous Ligands.

There are 48 nuclear receptors in the human genome, and many members of this superfamily have been implicated in human diseases. The NR4A nuclear receptor family consisting of three members, NR4A1, NR4A2, and NR4A3 (formerly annotated as Nur77, Nurr1, and NOR1, respectively), are still orphan receptors but exert pathological effects on immune-related and neurological diseases. We previously reported that prostaglandin A1 (PGA1) and prostaglandin A2 (PGA2) are potent activators of NR4A3, which bind directly to the ligand-binding domain (LBD) of the receptor. Recently, the co-crystallographic structures of NR4A2-LBD bound to PGA1 and PGA2 were reported, followed by reports of the neuroprotective effects of these possible endogenous ligands in mouse models of Parkinson's disease. Based on these structures, we modeled the binding structures of the other two members (NR4A1 and NR4A3) with these potential endogenous ligands using a template-based modeling method, and reviewed the similarity and diversity of ligand-binding mechanisms in the nuclear receptor family.

Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder.

Several large-scale whole-exome sequencing studies in patients with schizophrenia (SCZ) and autism spectrum disorder (ASD) have identified rare variants with modest or strong effect size as genetic risk factors. Dysregulation of cellular calcium homeostasis might be involved in SCZ/ASD pathogenesis, and genes encoding L-type voltage-gated calcium channel (VGCC) subunits Cav1.1 (CACNA1S), Cav1.2 (CACNA1C), Cav1.3 (CACNA1D), and T-type VGCC subunit Cav3.3 (CACNA1I) recently were identified as risk loci for psychiatric disorders. We performed a screening study, using the Ion Torrent Personal Genome Machine (PGM), of exon regions of these four candidate genes (CACNA1C, CACNA1D, CACNA1S, CACNA1I) in 370 Japanese patients with SCZ and 192 with ASD. Variant filtering was applied to identify biologically relevant mutations that were not registered in the dbSNP database or that have a minor allele frequency of less than 1% in East-Asian samples from databases; and are potentially disruptive, including nonsense, frameshift, canonical splicing site single nucleotide variants (SNVs), and non-synonymous SNVs predicted as damaging by five different in silico analyses. Each of these filtered mutations were confirmed by Sanger sequencing. If parental samples were available, segregation analysis was employed for measuring the inheritance pattern. Using our filter, we discovered one nonsense SNV (p.C1451* in CACNA1D), one de novo SNV (p.A36V in CACNA1C), one rare short deletion (p.E1675del in CACNA1D), and 14 NSstrict SNVs (non-synonymous SNV predicted as damaging by all of five in silico analyses). Neither p.A36V in CACNA1C nor p.C1451* in CACNA1D were found in 1871 SCZ cases, 380 ASD cases, or 1916 healthy controls in the independent sample set, suggesting that these SNVs might be ultra-rare SNVs in the Japanese population. The neuronal splicing isoform of Cav1.2 with the p.A36V mutation, discovered in the present study, showed reduced Ca2+-dependent inhibition, resulting in excessive Ca2+ entry through the mutant channel. These results suggested that this de novo SNV in CACNA1C might predispose to SCZ by affecting Ca2+ homeostasis. Thus, our analysis successfully identified several ultra-rare and potentially disruptive gene variants, lending partial support to the hypothesis that VGCC-encoding genes may contribute to the risk of SCZ/ASD.

Protein Data Bank Japan: Celebrating our 20th anniversary during a global pandemic as the Asian hub of three dimensional macromolecular structural data.

Protein Data Bank Japan (PDBj), a founding member of the worldwide Protein Data Bank (wwPDB) has accepted, processed and distributed experimentally determined biological macromolecular structures for 20 years. During that time, we have continuously made major improvements to our query search interface of PDBj Mine 2, the BMRBj web interface, and EM Navigator for PDB/BMRB/EMDB entries. PDBj also serves PDB-related secondary database data, original web-based modeling services such as Homology modeling of complex structure (HOMCOS), visualization services and utility tools, which we have continuously enhanced and expanded throughout the years. In addition, we have recently developed several unique archives, BSM-Arc for computational structure models, and XRDa for raw X-ray diffraction images, both of which promote open science in the structural biology community. During the COVID-19 pandemic, PDBj has also started to provide feature pages for COVID-19 related entries across all available archives at PDBj from raw experimental data and PDB structural data to computationally predicted models, while also providing COVID-19 outreach content for high school students and teachers.

Difference of binding modes among three ligands to a receptor mSin3B corresponding to their inhibitory activities.

A preceding experiment suggested that a compound, which inhibits binding of the REST/NRSF segment to the cleft of a receptor protein mSin3B, can be a potential drug candidate to ameliorate many neuropathies. We have recently developed an enhanced conformational sampling method, genetic-algorithm-guided multi-dimensional virtual-system-coupled canonical molecular dynamics, and in the present study, applied it to three systems consisting of mSin3B and one of three compounds, sertraline, YN3, and acitretin. Other preceding experiments showed that only sertraline inhibits the binding of REST/NRSF to mSin3B. The current simulation study produced the spatial distribution of the compounds around mSin3B, and showed that sertraline and YN3 bound to the cleft of mSin3B with a high propensity, although acitretin did not. Further analyses of the simulation data indicated that only the sertraline-mSin3B complex produced a hydrophobic core similar to that observed in the molecular interface of the REST/NRSF-mSin3B complex: An aromatic ring of sertraline sunk deeply in the mSin3B's cleft forming a hydrophobic core contacting to hydrophobic amino-acid residues located at the bottom of the cleft. The present study proposes a step to design a compound that inhibits competitively the binding of a ligand to its receptor.

Functional characterization of rare NRXN1 variants identified in autism spectrum disorders and schizophrenia.

BACKGROUND: Rare genetic variants contribute to the etiology of both autism spectrum disorder (ASD) and schizophrenia (SCZ). Most genetic studies limit their focus to likely gene-disrupting mutations because they are relatively easier to interpret their effects on the gene product. Interpretation of missense variants is also informative to some pathophysiological mechanisms of these neurodevelopmental disorders; however, their contribution has not been elucidated because of relatively small effects. Therefore, we characterized missense variants detected in NRXN1, a well-known neurodevelopmental disease-causing gene, from individuals with ASD and SCZ. METHODS: To discover rare variants with large effect size and to evaluate their role in the shared etiopathophysiology of ASD and SCZ, we sequenced NRXN1 coding exons with a sample comprising 562 Japanese ASD and SCZ patients, followed by a genetic association analysis in 4273 unrelated individuals. Impact of each missense variant detected here on cell surface expression, interaction with NLGN1, and synaptogenic activity was analyzed using an in vitro functional assay and in silico three-dimensional (3D) structural modeling. RESULTS: Through mutation screening, we regarded three ultra-rare missense variants (T737M, D772G, and R856W), all of which affected the LNS4 domain of NRXN1α isoform, as disease-associated variants. Diagnosis of individuals with T737M, D772G, and R856W was 1ASD and 1SCZ, 1ASD, and 1SCZ, respectively. We observed the following phenotypic and functional burden caused by each variant. (i) D772G and R856W carriers had more serious social disabilities than T737M carriers. (ii) In vitro assay showed reduced cell surface expression of NRXN1α by D772G and R856W mutations. In vitro functional analysis showed decreased NRXN1α-NLGN1 interaction of T737M and D772G mutants. (iii) In silico 3D structural modeling indicated that T737M and D772G mutations could destabilize the rod-shaped structure of LNS2-LNS5 domains, and D772G and R856W could disturb N-glycan conformations for the transport signal. CONCLUSIONS: The combined data suggest that missense variants in NRXN1 could be associated with phenotypes of neurodevelopmental disorders beyond the diagnosis of ASD and/or SCZ.

Molecular Interaction Mechanism of a 14-3-3 Protein with a Phosphorylated Peptide Elucidated by Enhanced Conformational Sampling.

Enhanced conformational sampling, a genetic-algorithm-guided multidimensional virtual-system coupled molecular dynamics, can provide equilibrated conformational distributions of a receptor protein and a flexible ligand at room temperature. The distributions provide not only the most stable but also semistable complex structures and propose a ligand-receptor binding process. This method was applied to a system consisting of a receptor protein, 14-3-3ε, and a flexible peptide, phosphorylated myeloid leukemia factor 1 (pMLF1). The results present comprehensive binding pathways of pMLF1 to 14-3-3ε. We identified four thermodynamically stable clusters of MLF1 on the 14-3-3ε surface and free-energy barriers among some clusters. The most stable cluster includes two high-density spots connected by a narrow corridor. When pMLF1 passes the corridor, a salt-bridge relay (switching) related to the phosphorylated residue of pMLF1 occurs. Conformations in one high-density spot are similar to the experimentally determined complex structure. Three-dimensional distributions of residues in the intermolecular interface rationally explain the binding constant changes resulting from the alanine mutation experiment for the residues. We also performed a simulation of nonphosphorylated peptide and 14-3-3ε, which demonstrated that the complex structure was unstable, suggesting that phosphorylation of the peptide is crucially important for binding to 14-3-3ε.

The Biological Structure Model Archive (BSM-Arc): an archive for in silico models and simulations.

We present the Biological Structure Model Archive (BSM-Arc, https://bsma.pdbj.org), which aims to collect raw data obtained via in silico methods related to structural biology, such as computationally modeled 3D structures and molecular dynamics trajectories. Since BSM-Arc does not enforce a specific data format for the raw data, depositors are free to upload their data without any prior conversion. Besides uploading raw data, BSM-Arc enables depositors to annotate their data with additional explanations and figures. Furthermore, via our WebGL-based molecular viewer Molmil, it is possible to recreate 3D scenes as shown in the corresponding scientific article in an interactive manner. To submit a new entry, depositors require an ORCID ID to login, and to finally publish the data, an accompanying peer-reviewed paper describing the work must be associated with the entry. Submitting their data enables researchers to not only have an external backup but also provide an opportunity to promote their work via an interactive platform and to provide third-party researchers access to their raw data.

Current knowledge on enzymatic PET degradation and its possible application to waste stream management and other fields.

Enzymatic hydrolysis of polyethylene terephthalate (PET) has been the subject of extensive previous research that can be grouped into two categories, viz. enzymatic surface modification of polyester fibers and management of PET waste by enzymatic hydrolysis. Different enzymes with rather specific properties are required for these two processes. Enzymatic surface modification is possible with several hydrolases, such as lipases, carboxylesterases, cutinases, and proteases. These enzymes should be designated as PET surface-modifying enzymes and should not degrade the building blocks of PET but should hydrolyze the surface polymer chain so that the intensity of PET is not weakened. Conversely, management of PET waste requires substantial degradation of the building blocks of PET; therefore, only a limited number of cutinases have been recognized as PET hydrolases since the first PET hydrolase was discovered by Müller et al. (Macromol Rapid Commun 26:1400-1405, 2005). Here, we introduce current knowledge on enzymatic degradation of PET with a focus on the key class of enzymes, PET hydrolases, pertaining to the definition of enzymatic requirements for PET hydrolysis, structural analyses of PET hydrolases, and the reaction mechanisms. This review gives a deep insight into the structural basis and dynamics of PET hydrolases based on the recent progress in X-ray crystallography. Based on the knowledge accumulated to date, we discuss the potential for PET hydrolysis applications, such as in designing waste stream management.

Multimodal Structural Distribution of the p53 C-Terminal Domain upon Binding to S100B via a Generalized Ensemble Method: From Disorder to Extradisorder.

Intrinsically disordered regions (IDRs) of a protein employ a flexible binding manner when recognizing a partner molecule. Moreover, it is recognized that binding of IDRs to a partner molecule is accompanied by folding, with a variety of bound conformations often being allowed in formation of the complex. In this study, we investigated a fragment of the disordered p53 C-terminal domain (CTDf) that interacts with one of its partner molecules, S100B, as a representative IDR. Although the 3D structure of CTDf in complex with S100B has been previously reported, the specific interactions remained controversial. To clarify these interactions, we performed generalized ensemble molecular dynamics (MD) simulations (virtual-system coupled multicanonical MD, termed V-McMD), which enable effective conformational sampling beyond that provided by conventional MD. These simulations generated a multimodal structural distribution for our system including CTDf and S100B, indicating that CTDf forms a variety of complex structures upon binding to S100B. We confirmed that our results are consistent with chemical shift perturbations and nuclear Overhauser effects that were observed in previous studies. Furthermore, we calculated the conformational entropy of CTDf in bound and isolated (free) states. Comparison of these CTDf entropies indicated that the disordered CTDf shows further increase in conformational diversity upon binding to S100B. Such entropy gain by binding may comprise an important feature of complex formation for IDRs.

Detection of cave pockets in large molecules: Spaces into which internal probes can enter, but external probes from outside cannot.

Geometric features of macromolecular shapes are important for binding with other molecules. Kawabata, T. and Go, N. (2007) defined a pocket as a space into which a small probe can enter, but a large probe cannot. In 2010, mathematical morphology (MM) was introduced to provide a more rigorous definition, and the program GHECOM was developed using the grid-based representation of molecules. This method was simple, but effective in finding the binding sites of small compounds on protein surfaces. Recently, many 3D structures of large macromolecules have been determined to contain large internal hollow spaces. Identification and size estimation of these spaces is important for characterizing their function and stability. Therefore, we employ the MM definition of pocket proposed by Manak, M. (2019)-a space into which an internal probe can enter, but an external probe cannot enter from outside of the macromolecules. This type of space is called a "cave pocket", and is identified through molecular grid-representation. We define a "cavity" as a space into which a probe can enter, but cannot escape to the outside. Three types of spaces: cavity, pocket, and cave pocket were compared both theoretically and numerically. We proved that a cave pocket includes a pocket, and it is equal to a pocket if no cavity is found. We compared the three types of spaces for a variety of molecules with different-sized spherical probes; cave pockets were more sensitive than pockets for finding almost closed internal holes, allowing for more detailed representations of internal surfaces than cavities provide.

Gaussian-input Gaussian mixture model for representing density maps and atomic models.

A new Gaussian mixture model (GMM) has been developed for better representations of both atomic models and electron microscopy 3D density maps. The standard GMM algorithm employs an EM algorithm to determine the parameters. It accepted a set of 3D points with weights, corresponding to voxel or atomic centers. Although the standard algorithm worked reasonably well; however, it had three problems. First, it ignored the size (voxel width or atomic radius) of the input, and thus it could lead to a GMM with a smaller spread than the input. Second, the algorithm had a singularity problem, as it sometimes stopped the iterative procedure due to a Gaussian function with almost zero variance. Third, a map with a large number of voxels required a long computation time for conversion to a GMM. To solve these problems, we have introduced a Gaussian-input GMM algorithm, which considers the input atoms or voxels as a set of Gaussian functions. The standard EM algorithm of GMM was extended to optimize the new GMM. The new GMM has identical radius of gyration to the input, and does not suddenly stop due to the singularity problem. For fast computation, we have introduced a down-sampled Gaussian functions (DSG) by merging neighboring voxels into an anisotropic Gaussian function. It provides a GMM with thousands of Gaussian functions in a short computation time. We also have introduced a DSG-input GMM: the Gaussian-input GMM with the DSG as the input. This new algorithm is much faster than the standard algorithm.

New tools and functions in data-out activities at Protein Data Bank Japan (PDBj).

The Protein Data Bank Japan (PDBj), a member of the worldwide Protein Data Bank (wwPDB), accepts and processes the deposited data of experimentally determined biological macromolecular structures. In addition to archiving the PDB data in collaboration with the other wwPDB partners, PDBj also provides a wide range of original and unique services and tools, which are continuously improved and updated. Here, we report the new RDB PDBj Mine 2, the WebGL molecular viewer Molmil, the ProMode-Elastic server for normal mode analysis, a virtual reality system for the eF-site protein electrostatic molecular surfaces, the extensions of the Omokage search for molecular shape similarity, and the integration of PDBj and BMRB searches.

Rigid-Body Fitting of Atomic Models on 3D Density Maps of Electron Microscopy.

Cryo electron microscopy has revolutionarily evolved for the determination of the 3D structure of macromolecular complexes. The modeling procedures on the 3D density maps of electron microscopy are roughly classified into three categories: fitting, de novo modeling and refinement. The registered atomic models from the maps have mostly been hand-built and auto-refined. Several programs aiming at automatic modeling have also been developed using various kinds of molecular representations. Among these three classes of the modeling procedures, the rigid body fitting is reviewed here, because it is the most basic modeling process applied before the other steps. The fitting problems are classified as the fittings of single subunit or multiple subunits, and the fittings on global or local parts of maps. A higher resolution map enables more local fitting. Various molecular representations have been employed in the fitting programs. A point and digital image models are generally used to represent molecules, but new representations, such as the Gaussian mixture model, have been applied recently.

Mutational analysis of cutinase-like enzyme, Cut190, based on the 3D docking structure with model compounds of polyethylene terephthalate.

The cutinase-like enzyme, Cut190, from Saccharomonospora viridis AHK190 can degrade the inner block of polyethylene terephthalate (PET) in the presence of Ca2+, and its mutant, S226P/R228S, exhibited increased activity and higher thermostability. The crystal structures of the Cut190 S226P mutant in the absence and presence of Ca2+ were determined, and revealed the large conformational change induced upon Ca2+ binding. However, the substrate-bound 3D structures of Cut190 remained unknown. In this study, to determine the substrate-binding site and improve the enzyme activity, we first built 3D structures of a PET model compound bound to the crystal structures, using the distance restraints between the scissile carbonyl group of the compound and the catalytic site of the enzyme. We then mutated the putative substrate-binding site predicted from the models, and experimentally determined the enzymatic activities of the mutants for the model substrate poly(butylene succinate-co-adipate). The mutated sites with decreased activity were consistent with the putative binding sites predicted by the 3D model from the Ca2+-bound crystal structure, suggesting that the structure of the Ca2+-bound state represents the active state. Notably, we generated two mutants with significantly increased activities.

Protein Data Bank Japan (PDBj): updated user interfaces, resource description framework, analysis tools for large structures.

The Protein Data Bank Japan (PDBj, http://pdbj.org), a member of the worldwide Protein Data Bank (wwPDB), accepts and processes the deposited data of experimentally determined macromolecular structures. While maintaining the archive in collaboration with other wwPDB partners, PDBj also provides a wide range of services and tools for analyzing structures and functions of proteins. We herein outline the updated web user interfaces together with RESTful web services and the backend relational database that support the former. To enhance the interoperability of the PDB data, we have previously developed PDB/RDF, PDB data in the Resource Description Framework (RDF) format, which is now a wwPDB standard called wwPDB/RDF. We have enhanced the connectivity of the wwPDB/RDF data by incorporating various external data resources. Services for searching, comparing and analyzing the ever-increasing large structures determined by hybrid methods are also described.

VaProS: a database-integration approach for protein/genome information retrieval.

Life science research now heavily relies on all sorts of databases for genome sequences, transcription, protein three-dimensional (3D) structures, protein-protein interactions, phenotypes and so forth. The knowledge accumulated by all the omics research is so vast that a computer-aided search of data is now a prerequisite for starting a new study. In addition, a combinatory search throughout these databases has a chance to extract new ideas and new hypotheses that can be examined by wet-lab experiments. By virtually integrating the related databases on the Internet, we have built a new web application that facilitates life science researchers for retrieving experts' knowledge stored in the databases and for building a new hypothesis of the research target. This web application, named VaProS, puts stress on the interconnection between the functional information of genome sequences and protein 3D structures, such as structural effect of the gene mutation. In this manuscript, we present the notion of VaProS, the databases and tools that can be accessed without any knowledge of database locations and data formats, and the power of search exemplified in quest of the molecular mechanisms of lysosomal storage disease. VaProS can be freely accessed at http://p4d-info.nig.ac.jp/vapros/ .

Gene expression and pathway analysis of CTNNB1 in cancer and stem cells.

AIM: To investigate ß-catenin (CTNNB1) signaling in cancer and stem cells, the gene expression and pathway were analyzed using bioinformatics. METHODS: The expression of the catenin ß 1 (CTNNB1) gene, which codes for ß-catenin, was analyzed in mesenchymal stem cells (MSCs) and gastric cancer (GC) cells. Beta-catenin signaling and the mutation of related proteins were also analyzed using the cBioPortal for Cancer Genomics and HOMology modeling of Complex Structure (HOMCOS) databases. RESULTS: The expression of the CTNNB1 gene was up-regulated in GC cells compared to MSCs. The expression of EPH receptor A8 (EPHA8), synovial sarcoma translocation chromosome 18 (SS18), interactor of little elongation complex ELL subunit 1 (ICE1), patched 1 (PTCH1), mutS homolog 3 (MSH3) and caspase recruitment domain family member 11 (CARD11) were also shown to be altered in GC cells in the cBioPortal for Cancer Genomics analysis. 3D complex structures were reported for E-cadherin 1 (CDH1), lymphoid enhancer binding factor 1 (LEF1), transcription factor 7 like 2 (TCF7L2) and adenomatous polyposis coli protein (APC) with ß-catenin. CONCLUSION: The results indicate that the epithelial-mesenchymal transition (EMT)-related gene CTNNB1 plays an important role in the regulation of stem cell pluripotency and cancer signaling.

Homozygous inactivation of CHEK2 is linked to a familial case of multiple primary lung cancer with accompanying cancers in other organs.

In clinical practice, there are a number of cancer patients with clear family histories, but the patients lack mutations in known familial cancer syndrome genes. Recent advances in genomic technologies have enhanced the possibility of identifying causative genes in such cases. Two siblings, an elder sister and a younger brother, were found to have multiple primary lung cancers at the age of 60. The former subsequently developed breast cancer and had a history of uterine myoma. The latter had initially developed prostate cancer at the age of 59 and had a history of colon cancer. Single-nucleotide polymorphism (SNP) genotyping revealed that ∼10% of the genomes were homozygous in both patients. Exome sequencing revealed nonsynonymous mutations in five genes in the runs of homozygosity: CHEK2, FCGRT, INPP5J, MYO18B, and SFI1. Evolutionary conservation of primary protein structures suggested the functional importance of the CHEK2 mutation, p.R474C. This mutation altered the tertiary structure of CHK2 by disrupting the salt bridge between p.R474 and p.E394. No such structural changes were observed with the other mutated genes. Subsequent cell-based transfection analysis revealed that CHK2 p.R474C was unstable and scarcely activated. We concluded that the homozygous CHEK2 variant was contributory in this case of familial cancer. Although homozygous inactivation of CHEK2 in mice led to cancers in multiple organs, accumulation of additional human cases is needed to establish its pathogenic role in humans.

HOMCOS: an updated server to search and model complex 3D structures.

The HOMCOS server ( http://homcos.pdbj.org ) was updated for both searching and modeling the 3D complexes for all molecules in the PDB. As compared to the previous HOMCOS server, the current server targets all of the molecules in the PDB including proteins, nucleic acids, small compounds and metal ions. Their binding relationships are stored in the database. Five services are available for users. For the services "Modeling a Homo Protein Multimer" and "Modeling a Hetero Protein Multimer", a user can input one or two proteins as the queries, while for the service "Protein-Compound Complex", a user can input one chemical compound and one protein. The server searches similar molecules by BLAST and KCOMBU. Based on each similar complex found, a simple sequence-replaced model is quickly generated by replacing the residue names and numbers with those of the query protein. A target compound is flexibly superimposed onto the template compound using the program fkcombu. If monomeric 3D structures are input as the query, then template-based docking can be performed. For the service "Searching Contact Molecules for a Query Protein", a user inputs one protein sequence as the query, and then the server searches for its homologous proteins in PDB and summarizes their contacting molecules as the predicted contacting molecules. The results are summarized in "Summary Bars" or "Site Table"display. The latter shows the results as a one-site-one-row table, which is useful for annotating the effects of mutations. The service "Searching Contact Molecules for a Query Compound" is also available.