Perfect Complementarity Research Articles

Deracemization of Carbohelicenes by a Chiral Perylene Bisimide Cyclophane Template Catalyst.

Deracemization describes the conversion of a racemic mixture of a chiral molecule into an enantioenriched mixture or an enantiopure compound without structural modifications. Herein, we report an inherently chiral perylene bisimide (PBI) cyclophane whose chiral pocket is capable of transforming a racemic mixture of [5]‐helicene into an enantioenriched mixture with an enantiomeric excess of 66 %. UV/Vis and fluorescence titration studies reveal this cyclophane host composed of two helically twisted PBI dyes has high binding affinities for the respective homochiral carbohelicene guests, with outstanding binding constants of up to 3.9×1010 m −1 for [4]‐helicene. 2D NMR studies and single‐crystal X‐ray analysis demonstrate that the observed strong and enantioselective binding of homochiral carbohelicenes and the successful template‐catalyzed deracemization of [5]‐helicene can be explained by the enzyme‐like perfect shape complementarity of the macrocyclic supramolecular host.

Deracemization of Carbohelicenes by a Chiral Perylene Bisimide Cyclophane Template Catalyst

AbstractDeracemization describes the conversion of a racemic mixture of a chiral molecule into an enantioenriched mixture or an enantiopure compound without structural modifications. Herein, we report an inherently chiral perylene bisimide (PBI) cyclophane whose chiral pocket is capable of transforming a racemic mixture of [5]‐helicene into an enantioenriched mixture with an enantiomeric excess of 66 %. UV/Vis and fluorescence titration studies reveal this cyclophane host composed of two helically twisted PBI dyes has high binding affinities for the respective homochiral carbohelicene guests, with outstanding binding constants of up to 3.9×1010 m−1 for [4]‐helicene. 2D NMR studies and single‐crystal X‐ray analysis demonstrate that the observed strong and enantioselective binding of homochiral carbohelicenes and the successful template‐catalyzed deracemization of [5]‐helicene can be explained by the enzyme‐like perfect shape complementarity of the macrocyclic supramolecular host.

TRNA: Splicing and Intron Turnover and Possible Functions

A subset of tRNA genes in eukaryotes contains an intron. tRNAs introns are short sequences located 1 nt. 3′ of the anticodon. Introns are removed from precursor tRNAs (pre-tRNA) by the conserved heterotetrameric tRNA splicing endonuclease (SEN), that recognizes pre-tRNA structure rather than sequence motifs at splice junctions. In vertebrate cells intron removal from pre-tRNAs occurs in the nucleoplasm; in contrast, pre-tRNA splicing in budding yeast and fission yeast occurs on the mitochondrial surface (Yoshihisa et al. 2003; Wan & Hopper, 2018); therefore, pre-tRNA splicing on the mitochondrial surface has been conserved for greater than 500 million years. Free tRNA introns are rarely detected in cells because they are rapidly and efficiently destroyed. We identified at least 5 separate, family-specific mechanisms that function in tRNA intron destruction (Wu & Hopper, 2014; Bao, Metcalf & Hopper, unpublished). Interestingly, under particular stress conditions, specific tRNA introns accumulate to high levels, possibly indicative of their functions in stress responses (Peltier, Metcalf, & Hopper, unpublished). Moreover, we discovered that tRNA introns possess long stretches of perfect complementarity to particular budding yeast mRNA ORFs (Bao, Nostramo, & Hopper, unpublished). Deletion of the introns from the 2 copies of the genes encoding tRNAIleUAU results in elevated levels of mRNAs with complementarity, but not mRNAs lacking complementarity; further, transformation of the cells lacking the tRNAIleUAU introns with extrachromosomal intron-containing tRNAIleUAU suppresses the mRNA enhanced levels. These results support the hypothesis that tRNA introns may function as a novel type of noncoding regulatory RNA, perhaps functioning in stress response (Nostramo & Hopper, unpublished).

AGO-accessible anticancer siRNAs designed with synergistic miRNA-like activity

Small interfering RNAs (siRNAs) therapeutically induce RNA interference (RNAi) of disease-causing genes, but they also silence hundreds of seed-matched off-targets as behaving similar to microRNAs (miRNAs). miRNAs control the pathophysiology of tumors, wherein their accessible binding sites can be sequenced by Argonaute crosslinking immunoprecipitation (AGO CLIP). Herein, based on AGO CLIP, we develop potent anticancer siRNAs utilizing miRNA-like activity (mi/siRNAs). The mi/siRNAs contain seed sequences (positions 2–7) of tumor-suppressive miRNAs while maintaining perfect sequence complementarity to the AGO-accessible tumor target sites. Initially, host miRNA interactions with human papillomavirus 18 (HPV18) were identified in cervical cancer by AGO CLIP, revealing tumor-suppressive activity of miR-1/206 and miR-218. Based on the AGO-miRNA binding sites, mi/siRNAs were designed to target E6 and E7 (E6/E7) transcript with seed sequences of miR-1/206 (206/E7) and miR-218 (218/E7). Synergistic anticancer activity of 206/E7 and 218/E7 was functionally validated and confirmed via RNA sequencing and in vivo xenograft models (206/E7). Other mi/siRNA sequences were additionally designed for cervical, ovarian, and breast cancer, and available as an online tool (http://ago.korea.ac.kr/misiRNA); some of the mi/siRNAs were validated for their augmented anticancer activity (206/EphA2 and 206/Her2). mi/siRNAs could coordinate miRNA-like activity with robust siRNA function, demonstrating the potential of AGO CLIP analysis for RNAi therapeutics.

Selection of Chemical Modifications in the siRNA Seed Region That Repress Off-Target Effect.

RNA interference mediated by small interfering RNA (siRNA) has been widely used as aprocedure to knock down the expression of an intended target gene with perfect sequence complementarity. However, siRNA often exhibits off-target effects on genes with partial sequence complementarities. Such off-target effect is an undesirable adverse effect for knocking down a target gene specifically. Here we describe the powerful strategy to avoid off-target effects without affecting the RNAi activity by the introduction of DNA or 2'-O-methyl modifications in the siRNA seed region. These two types of chemical modifications repress off-target effects through different molecular mechanisms.

Genome-wide detection and analysis of CRISPR-Cas off-targets.

The clustered, regularly interspersed, short palindromic repeats (CRISPR) technology is revolutionizing biological studies and holds tremendous promise for treating human diseases. However, a significant limitation of this technology is that modifications can occur on off-target sites lacking perfect complementarity to the single guide RNA (sgRNA) or canonical protospacer-adjacent motif (PAM) sequence. Several in vivo and in vitro genome-wide off-target profiling approaches have been developed to inform on the fidelity of gene editing. Of these, GUIDE-seq has become one of the most widely adopted and reproducible methods. To allow users to easily analyze GUIDE-seq data generated on any sequencing platform, we developed an open-source pipeline, GS-Preprocess, that takes standard base-call output in bcl format and generate all required input data for off-target identification using bioconductor package GUIDEseq for off-target identification. Furthermore, we created a Docker image with GS-Proprocess, GUIDE-seq, and all its R and system dependencies already installed. The bundled pipeline will empower end users to streamline the analysis of GUIDE-seq data and motivate their use of higher throughput sequencing with increased multiplexing for GUIDE-seq experiments.

An Infrared and Visible Image Fusion Method Guided by Saliency and Gradient Information

Infrared and visible image fusion is a hot topic due to the perfect complementarity of their information. There are two key problems in infrared and visible image fusion. One is how to extract significant target areas and rich texture details from the source images, and the other is how to integrate them to produce satisfactory fused images. To tackle these problems, we propose a novel fusion framework in this paper. A multi-level image decomposition method is used to obtain the base layer and detail layer of the source image. For the fusion of base layer, an ingenious fusion strategy guided by the saliency map of source image is designed to improve the intensity of salient targets and the visual quality of the fused image. For the fusion of detail layer, an efficient approach by introducing the enhanced gradient information is presented to boost the detail features and sharpen the edges of the fused image. Experimental results demonstrate that, compared with fifteen classical and advanced fusion methods, the proposed image fusion framework has better performance in both subjective and objective evaluation.

Identification of the Functional Binding Site for the Convulsant Tetramethylenedisulfotetramine in the Pore of the α 2 β 3 γ 2 GABAA Receptor.

Tetramethylenedisulfotetramine (TETS) is a so-called “caged” convulsant that is responsible for thousands of accidental and malicious poisonings. Similar to the widely used GABA receptor type A (GABAA) antagonist picrotoxinin, TETS has been proposed to bind to the noncompetitive antagonist (NCA) site in the pore of the receptor channel. However, the TETS binding site has never been experimentally mapped, and we here set out to gain atomistic level insights into how TETS inhibits the human α2β3γ2 GABAA receptor. Using the Rosetta molecular modeling suite, we generated three homology models of the α2β3γ2 receptor in the open, desensitized, and closed/resting state. Three different ligand-docking algorithms (RosettaLigand, Glide, and Swissdock) identified two possible TETS binding sites in the channel pore. Using a combination of site-directed mutagenesis, electrophysiology, and modeling to probe both sites, we demonstrate that TETS binds at the T6′ ring in the closed/resting-state model, in which it shows perfect space complementarity and forms hydrogen bonds or makes hydrophobic interactions with all five pore-lining threonine residues of the pentameric receptor. Mutating T6′ in either the α2 or β3 subunit reduces the IC50 of TETS by ∼700-fold in whole-cell patch-clamp experiments. TETS is thus interacting at the NCA site in the pore of the GABAA receptor at a location that is overlapping but not identical to the picrotoxinin binding site.SIGNIFICANCE STATEMENTOur study identifies the binding site of the highly toxic convulsant tetramethylenedisulfotetramine (TETS), which is classified as a threat agent by the World Health Organization. Using a combination of homology protein modeling, ligand docking, site-directed mutagenesis, and electrophysiology, we show that TETS is binding in the pore of the α2β3γ2 GABA receptor type A receptor at the so-called T6′ ring, wherein five threonine residues line the permeation pathway of the pentameric receptor channel.

Format chain exchange (FORCE) for high-throughput generation of bispecific antibodies in combinatorial binder-format matrices

Generation of bispecific antibodies (bsAbs) requires a combination of compatible binders in formats that support desired functionalities. Here, we report that bsAb-matrices can be generated by Format Chain Exchange (FORCE), enabling screening of combinatorial binder/format spaces. Input molecules for generation of bi/multi-valent bsAbs are monospecific entities similar to knob-into-hole half-antibodies, yet with complementary CH3-interface-modulated and affinity-tagged dummy-chains. These contain mutations that lead to limited interface repulsions without compromising expression or biophysical properties of educts. Mild reduction of combinations of educts triggers spontaneous chain-exchange reactions driven by partially flawed CH3-educt interfaces resolving to perfect complementarity. This generates large bsAb matrices harboring different binders in multiple formats. Benign biophysical properties and good expression yields of educts, combined with simplicity of purification enables process automation. Examples that demonstrate the relevance of screening binder/format combinations are provided as a matrix of bsAbs that simultaneously bind Her1/Her2 and DR5 without encountering binder or format-inflicted interferences.

TRNA: Splicing and Intron Turnover

In most eukaryotic genomes a subset of tRNA genes contains introns. Introns in tRNAs genes are short sequences located 1 nt. 3′ of the anticodon. Introns are removed from precursor tRNAs (pre‐tRNA) by a conserved heterotetrameric protein enzyme, tRNA splicing endonuclease (SEN), that recognizes pre‐tRNA structure rather than sequence motifs at splice junctions. In vertebrate cells intron removal from pre‐tRNAs occurs in the nucleoplasm; in contrast, pre‐tRNA splicing in budding yeast was surprisingly discovered to occur on the mitochondrial surface (Yoshihisa et al. 2003). We recently reported that the SEN complex also locates to the mitochondrial surface in fission yeast (Wan & Hopper 2018) and, therefore, pre‐tRNA splicing on the mitochondrial surface has been conserved for at least 500 million years. The mitochondrial outer membrane protein, Tom70, is required for efficient SEN subunit location to mitochondria in both budding and fission yeast (Wan & Hopper 2018). The freed introns are rarely detected in cells and until recently it was unknown how are they are turned over. We identified at least 5 separate, species‐specific mechanisms that function in tRNA intron destruction (Wu & Hopper, 2014; Bao, unpublished). Interestingly, under particular stress conditions, specific tRNA introns accumulate, possibly indicating their functions in stress response (Peltier, unpublished). Moreover, we discovered that tRNA introns possess long stretches of perfect complementarity to particular budding yeast mRNAs (Bao, unpublished), also indicative of their possible roles in gene regulation. Our initial experiments support the hypothesis that tRNA introns may function as a novel type of noncoding regulatory RNA (Nostramo, unpublished).Support or Funding InformationNIH GMS 122884

Expressions of miR-155 and miR-181 and predictions of their structures and targets in pigs (Sus scrofa).

Background and Aim:MicroRNAs (miRNAs) are responsible for gene expression control at the post-transcription level in many species. Several miRNAs are required in the regulation of immune responses, such as B-cell differentiation, T-cell receptor signaling pathway, CD4+ T cell selection, and so on. Studies on miRNAs have been extensively conducted in humans and mice; however, reports relevant to miRNAs, especially miR-155 and miR-181, in pigs are limited. Consequently, the present study aimed to investigate the structures, target genes, and expressions of miR-155 and miR-181 in various porcine cells and tissues.Materials and Methods:Five healthy male pigs from a porcine reproductive and respiratory syndrome virus-negative farm were studied. Before slaughter, blood samples were collected for peripheral blood mononuclear cell isolation. After slaughter, samples of spleen, lymph nodes, and forelimb muscles were collected. Both miR-155 and miR-181 were investigated for their structures with RNAfold web server, for their target genes from three online web servers, and for their expressions using polymerase chain reaction (PCR).Results:The structures of miR-155 and miR-181 contained hairpins with free energies of −35.27 and −35.29 kcal/mole, respectively. Target gene prediction revealed that miR-155 had perfect complementarity with Socs1 and Mapk3k14, while miR-181 had perfect complementarity with Ddx3x, Nfat5, Foxp1, and Mpp5. PCR showed that both miRNAs were detectable from all investigated cells and tissues. Moreover, the highest expression of both miRNAs was found from the lymph node of the pigs.Conclusion:Both miR-155 and miR-181 might be involved with the regulation of porcine immune functions as both miRNAs were detected in several cells and tissues of the pigs. In addition, they had very high complementarities with the seed regions of several immune-related genes.

All Two-Dimensional Pseudocapacitive Sheet Materials for Flexible Asymmetric Solid-State Planar Microsupercapacitors with High Energy Density.

With the rapid development of portable devices and wireless protocols, miniaturized energy storage units have become an important prerequisite. Although in-plane microsupercapacitors are emerging as competitive candidate devices, their practical applications have been severely hindered by their low energy density. Here, employing pseudocapacitive active materials working in complementary voltage windows, namely, manganese oxide (MnO2) and titanium carbide (Ti3C2), both in the two-dimensional sheet morphology, a flexible asymmetric interdigitated solid-state microsupercapacitor was assembled. Profiting from the perfect voltage complementarity of the two types of sheets, the high exposure of electrochemically active sites and the maximized utilization of the sheets due to the planar ion transport, the designed device achieved excellent electrochemical performance even when using a gel electrolyte. In particular, the device obtained a high specific capacitance of 106 F g-1 (295 mF cm-2), a wide potential window (2 V), an ultrahigh rate performance (retaining 83% even with a 20-fold in current density to 20 A g-1), an excellent cycling stability (87% retention after 104 cycles at 10 A g-1), and a competitive energy density of 58 W h kg-1 (162 μW h cm-2) that are even comparable to those of some microbatteries, while maintaining a high power density of 985 W kg-1 (2.7 mW cm-2). Importantly, this outstanding electrochemical performance was also stably maintained under various bending conditions. These results indicate that two-dimensional pseudocapacitive sheet materials have a plethora of possibilities for constructing flexible and wearable devices.

Identification of human miR-1839-5p by small RNA-seq, a miRNA enriched in neoplastic tissues.

MicroRNAs (miRNAs) modulate gene expression through destabilization or translational inhibition of cytoplasmic transcripts or by transcriptional regulation through binding to genomic DNA. Although miRNAs are globally down-regulated in cancer, some are overexpressed in neoplastic tissues, playing key roles in tumorigenesis (oncomiRs), sometimes behaving as effective cancer markers. Using total RNA from human uterus adenocarcinoma and non-neoplastic uterus, we conducted a small RNA-sequencing experiment followed by prediction of novel miRNAs using MirDeep* software. Synteny analysis and whole genome alignments were performed using BLAST. We also evaluated expression by a reverse transcriptase-polymerase chain reaction (RT-PCR) in normal tissues of the FSD2 gene, which spans the human miR-1839-5p gene in the opposite direction. MirDeep* analysis predicted a miRNA not previously annotated in databases, identical to and likely the orthologue of mouse miR-1839-5p. Whole-genome local alignments of this miRNA revealed a single perfect hit that is indeed syntenic to mouse miR-1839-5p. Alignments with other mammalian orthologues showed considerable conservation. We validated the prediction via a stem-loop RT-PCR assay, also employed to screen RNA samples from several additional normal and cancer tissues, showing increased expression in neoplastic tissues compared to their respective non neoplastic counterparts. Human heart tissue expresses both miR-1839-5p and FSD2. Human tissues express an orthologue of mouse miR-1839-5p and, given its expression pattern, we suggest that this miRNA could be explored as a potential oncomiR or cancer marker. Also, according to the genomic organization of miR-1839-5p and FSD2, perfect complementarity exists between the two elements, making possible miRNA-directed cleavage in human cardiac tissue.

Selective Recognition of Phenazine by 2,6‐Dibutoxylnaphthalene‐Based Tetralactam Macrocycle

Summary of main observation and conclusionA 2,6‐dibutoxylnaphthalene‐based tetralactam macrocycle was designed and synthesized. This macrocycle shows highly selective recognition to phenazine – a well‐known secondary metabolite in bacteria and an emerging disinfection byproduct in drinking water. In contrast, the macrocycle shows no binding to the structurally similar dibenzo‐1,4‐dioxin. It was revealed that hydrogen bonding, π‐π and σ‐π interactions are the major driving forces between phenazine and the new tetralactam macrocycle. A perfect complementarity in electrostatic potential surfaces may explain the high selectivity. In addition, the macrocycle shows fluorescent response to phenazine, demonstrating its potential in fluorescent detection of phenazine.

In silico identification of microRNAs and their target genes in watermelon (Citrullus lanatus)

Watermelon (Citrullus lanatus) is an annual herbaceous crop, mainly grown for its fleshy fruit, which is an excellent source of vitamins and therefore fetches a huge market demand all over the world. Yield of watermelon can be improved by increasing the productivity that relies on plant growth, development and stress response. MicroRNAs (miRNAs), a group of short (∼22 nt), noncoding, endogenous and evolutionary conserved post-transcriptional regulatory RNAs, play a major role in various biological and metabolic processes associated with growth, development and stress response. In this study, we deployed a comparative genomics-based approach using expressed sequence tags (ESTs) to identify and characterize potential miRNAs and their target genes in watermelon. Three potential miRNAs (cla-miR-2515, cla-miR482d and cla-miR-8461) were predicted from 17,594 publicly available watermelon EST sequences, according to a series of filtering criteria used in the protocol. Moreover, 21 potential mRNA targets encoding for 19 different proteins linked to various metabolic processes such as inositol-3-phosphate synthase activity, metal ion binding and oxidoreductase activity were identified in watermelon genome, based on perfect or nearly perfect complementarity of miRNAs with their targeted mRNA sequences. Phylogenetic analysis of these miRNAs revealed that cla-miR8461 and cla-miR2515 are unique miRNAs closely related to Schistosoma mansoni and Drosophila pseudoobscura, respectively.

Multiple-gene targeting and mismatch tolerance can confound analysis of genome-wide pooled CRISPR screens

BackgroundGenome-wide loss-of-function screens using the CRISPR/Cas9 system allow the efficient discovery of cancer cell vulnerabilities. While several studies have focused on correcting for DNA cleavage toxicity biases associated with copy number alterations, the effects of sgRNAs co-targeting multiple genomic loci in CRISPR screens have not been discussed.ResultsIn this work, we analyze CRISPR essentiality screen data from 391 cancer cell lines to characterize biases induced by multi-target sgRNAs. We investigate two types of multi-targets: on-targets predicted through perfect sequence complementarity and off-targets predicted through sequence complementarity with up to two nucleotide mismatches. We find that the number of on-targets and off-targets both increase sgRNA activity in a cell line-specific manner and that existing additive models of gene knockout effects fail at capturing genetic interactions that may occur between co-targeted genes. We use synthetic lethality between paralog genes to show that genetic interactions can introduce biases in essentiality scores estimated from multi-target sgRNAs. We further show that single-mismatch tolerant sgRNAs can confound the analysis of gene essentiality and lead to incorrect co-essentiality functional networks. Lastly, we also find that single nucleotide polymorphisms located in protospacer regions can impair on-target activity as a result of mismatch tolerance.ConclusionWe show the impact of multi-target effects on estimating cancer cell dependencies and the impact of off-target effects caused by mismatch tolerance in sgRNA-DNA binding.

A Synthetic Methodology Toward Pyrrolo[2,3- b]pyridones for GC Base Pair Recognition.

Flexible synthetic access to a novel biarylic GC binding motif is presented, consisting of a pyridone connected to a fused pyrrolo[2,3- b]pyridone. Extensive molecular modeling led to an optimized design with perfect complementarity to the Hoogsteen site inside DNA's major groove. A wide range of functional elements can be introduced by minor modifications of the synthetic strategy. Our approach relies on mild Pd-catalyzed coupling reactions, featuring a triple heterohalogenated orthogonally addressable pyridine as a key intermediate.

A Flexible Demand Model for Complements Using Household Production Theory

We utilize household production theory to address the problem of micro-level demand estimation across complementary goods. According to this theory, consumers buy inputs and combine them to produce final goods from which they derive utility. We use this idea to build a structural model of demand for complements that can be estimated on purchase data in the presence of corner solutions and indivisible packages. We find that, even when reusing the same functional form as some previous models of demand for substitutes, our model can accommodate very different patterns of consumer preferences over complements and lead to a flexible demand system from perfect complementarity to no complementarity. We estimate the model on purchase data from a panel of consumers and show how it can be used to increase the profitability of couponing strategies by taking into account the spillover effect of coupons on demand for complementary categories. We also show how our model can be used by manufacturers to make decisions regarding the size of packages by taking into account cross-category consumption.

MiR-122, small RNA annealing and sequence mutations alter the predicted structure of the Hepatitis C virus 5' UTR RNA to stabilize and promote viral RNA accumulation.

Annealing of the liver-specific microRNA, miR-122, to the Hepatitis C virus (HCV) 5′ UTR is required for efficient virus replication. By using siRNAs to pressure escape mutations, 30 replication-competent HCV genomes having nucleotide changes in the conserved 5′ untranslated region (UTR) were identified. In silico analysis predicted that miR-122 annealing induces canonical HCV genomic 5′ UTR RNA folding, and mutant 5′ UTR sequences that promoted miR-122-independent HCV replication favored the formation of the canonical RNA structure, even in the absence of miR-122. Additionally, some mutant viruses adapted to use the siRNA as a miR-122-mimic. We further demonstrate that small RNAs that anneal with perfect complementarity to the 5′ UTR stabilize and promote HCV genome accumulation. Thus, HCV genome stabilization and life-cycle promotion does not require the specific annealing pattern demonstrated for miR-122 nor 5′ end annealing or 3′ overhanging nucleotides. Replication promotion by perfect-match siRNAs was observed in Ago2 knockout cells revealing that other Ago isoforms can support HCV replication. At last, we present a model for miR-122 promotion of the HCV life cycle in which miRNA annealing to the 5′ UTR, in conjunction with any Ago isoform, modifies the 5′ UTR structure to stabilize the viral genome and promote HCV RNA accumulation.

Polypentagonal ice-like water networks emerge solely in an activity-improved variant of ice-binding protein

Polypentagonal water networks were recently observed in a protein capable of binding to ice crystals, or ice-binding protein (IBP). To examine such water networks and clarify their role in ice-binding, we determined X-ray crystal structures of a 65-residue defective isoform of a Zoarcidae-derived IBP (wild type, WT) and its five single mutants (A20L, A20G, A20T, A20V, and A20I). Polypentagonal water networks composed of ∼50 semiclathrate waters were observed solely on the strongest A20I mutant, which appeared to include a tetrahedral water cluster exhibiting a perfect position match to the [Formula: see text] first prism plane of a single ice crystal. Inclusion of another symmetrical water cluster in the polypentagonal network showed a perfect complementarity to the waters constructing the [Formula: see text] pyramidal ice plane. The order of ice-binding strength was A20L < A20G < WT < A20T < A20V < A20I, where the top three mutants capable of binding to the first prism and the pyramidal ice planes commonly contained a bifurcated γ-CH3 group. These results suggest that a fine-tuning of the surface of Zoarcidae-derived IBP assisted by a side-chain group regulates the holding property of its polypentagonal water network, the function of which is to freeze the host protein to specific ice planes.