Class C ß-Lactamases: Molecular Characteristics.

Class C ß-lactamases or cephalosporinases can be classified into two functional groups (1, 1e) with considerable molecular variability (≤20% sequence identity). These enzymes are mostly encoded by chromosomal and inducible genes and are widespread among bacteria, including Proteobacteria in particular. Molecular identification is based principally on three catalytic motifs (64SXSK, 150YXN, 315KTG), but more than 70 conserved amino-acid residues (≥90%) have been identified, many close to these catalytic motifs. Nevertheless, the identification of a tiny, phylogenetically distant cluster (including enzymes from the genera Legionella, Bradyrhizobium, and Parachlamydia) has raised questions about the possible existence of a C2 subclass of ß-lactamases, previously identified as serine hydrolases. In a context of the clinical emergence of extended-spectrum AmpC ß-lactamases (ESACs), the genetic modifications observed in vivo and in vitro (point mutations, insertions, or deletions) during the evolution of these enzymes have mostly involved the Ω- and H-10/R2-loops, which vary considerably between genera, and, in some cases, the conserved triplet 150YXN. Furthermore, the conserved deletion of several amino-acid residues in opportunistic pathogenic species of Acinetobacter, such as A. baumannii, A. calcoaceticus, A. pittii and A. nosocomialis (deletion of residues 304-306), and in Hafnia alvei and H. paralvei (deletion of residues 289-290), provides support for the notion of natural ESACs. The emergence of higher levels of resistance to ß-lactams, including carbapenems, and to inhibitors such as avibactam is a reality, as the enzymes responsible are subject to complex regulation encompassing several other genes (ampR, ampD, ampG, etc.). Combinations of resistance mechanisms may therefore be at work, including overproduction or change in permeability, with the loss of porins and/or activation of efflux systems.

Diversity of Cationic Antimicrobial Peptides in Black Cumin (Nigella sativa L.) Seeds.

Black cumin (Nigella sativa L.) is known to possess a wide variety of antimicrobial peptides belonging to different structural families. Three novel antimicrobial peptides have been isolated from black cumin seeds. Two of them were attributed as members of the non-specific lipid transfer proteins family, and one as a defensin. We have made an attempt of using the proteomic approach for novel antimicrobial peptides search in N. sativa seeds as well. The use of a well-established approach that includes extraction and fractionation stages remains relevant even in the case of novel peptides search because of the lacking N. sativa genome data. Novel peptides demonstrate a spectrum of antimicrobial activity against plant pathogenic organisms that may cause economically important crop diseases. These results obtained allow considering these molecules as candidates to be applied in "next-generation" biopesticides development for agricultural use.

A thermostable trypsin from freshwater fish Japanese dace (Tribolodon hakonensis): a comparison of the primary structures among fish trypsins.

Trypsin from Japanese dace (Tribolodon hakonensis) (JD-T) living in freshwater (2-18 °C) was purified. JD-T represented typical fish trypsin characteristics regarding the effects of protease inhibitor, calcium-ion, and pH. For the effect of temperature, JD-T quite resembled to the trypsins from tropical-zone marine fish and freshwater fish (the catfish cultured in Thailand), i.e., the optimum temperature was 60 °C, and it was stable below 60 °C at pH 8.0 for 15 min incubation. From the data, it seemed that the trypsin from freshwater fish is thermostable in spite of the fact that their habitat temperatures are low. So, we determined the primary structure of JD-T to discuss its thermostability-structure relationship. JD-T possessed basic structural features of fish trypsin such as the catalytic triad, the Asp189 residue for substrate specificity, 12 Cys residues forming six disulfide-bridges, and the calcium-ion-binding loop. On the other hand, the contents of charged amino acid residues in whole JD-T molecule (16.2%) and N-terminal region (13.8%) were similar to those of tropical-zone marine fish and other freshwater fish trypsins. Then, JD-T conserved the five amino acid residues (Glu70, Asn72, Val75, Glu77, and Glu80) coordinate with calcium-ion, and the proportion of negatively charged amino acids to charged amino acids in the calcium-ion-binding region of JD-T (75.0%) was equivalent to those of tropical-zone marine fish and freshwater fish trypsins. Therefore, it was suggested that the high thermostability of JD-T are stemmed from these structural specificities.

Large-scale identification and visualization of human liver N-glycome enriched from LO2 cells.

Aberrant glycosylation has been commonly observed in various physiological and pathological disorders (including cancers), and quite a few glycoproteins have been approved by the US Food and Drug Administration (FDA) as markers for early diagnosis. Each glycoprotein may have multiple glycoforms, and cancer-related ones can be only some specific glycoforms which have much higher sensitivity and specificity; for example, AFP glycoform AFP-L3 with N-glycan of 01Y(61F)41Y41M(31M41Y41L41S61M41Y41L41S is of bigger diagnostic value for hepatocellular carcinoma than total AFP (i.e., combination of all glycoforms). Mass spectrometry-based glycomics is currently the state-of-the-art instrumental analytical pipeline for high-throughput characterization of various glycoforms, where not only monosaccharide composition but also comprehensive structural information (sequence and linkage) of N-glycans are now reported thanks to our recently developed N-glycan database search engine GlySeeker. With this new capability, here, we report our large-scale characterization of human liver N-glycome with primary structures; 214 unique N-glycans with unique primary structures were identified and visualized with spectrum-level false discovery rate ≤ 1% and number of best hits of 1. The LO2 N-glycans reported here serve as a basic reference for future liver N-glycome study, and further quantitative analysis will enable characterization of differentially expressed N-glycans and discovery of more effective markers for liver and other diseases. Data are available via ProteomeXchange with identifier PXD008158.

Identification of the membrane-spanning domain of glycoprotein 45 in bovine immunodeficiency virus.

The membrane-spanning domain (MSD) of the transmembrane subunit (TM) anchors the envelope glycoprotein (Env) on the lipid bilayer of the host cell membrane and virions. Its functions include membrane fusion efficiency and intracellular trafficking of the lentivirus envelope protein. Our study aimed to determine the MSD of bovine immunodeficiency virus (BIV) glycoprotein 45 (gp45) and reveal structural characteristics of the BIV Env protein. We have predicted the region of the BIV MSD and obtained the sequence using bioinformatics software. Various kinds of assays, including analogy analysis, fluorescence microscopy, and dye-transfer-based assays, were carried out to validate the prediction. The results, for the first time, show that the BIV MSD is located at the D170 to M191 amino acids of gp45, and the identified MSD divides gp45 into the extracellular domain (ED), MSD and cytoplasmic domain (CT). We further found that the BIV MSD had a similar structure and function as the HIV MSD using amino acid sequence alignment and fluorescence microscopy. Additionally, the dye-transfer-based assay demonstrates that deletion of the BIV MSD efficiently decreases cell-cell fusion. Based on the identification of the MSD, a "snorkeling" model, in which the flanking charged amino acid residues are buried in the lipid bilayer while their side chains interact with polar head groups, was proposed for the BIV MSD. Ultimately, we further improved the primary structure of the BIV envelope glycoprotein.

Correlation Between Protein Primary Structure and Soluble Expression Level of HSA dAb in Escherichia coli.

It is widely accepted that features such as pI, length, molecular mass and amino acid (AA) sequence have a significant influence on protein solubility. Here, we mainly focused on AA composition and explored those that most affected the soluble expression level of human serum albumin (HSA) domain antibody (dAb). The soluble expression and sequence of 65 dAb variants were analysed using clustering and linear modelling. Certain AAs significantly affected the soluble expression level of dAb, with the specific AA combinations being (S, R, N, D, Q), (G, R, C, N, S) and (R, S, G); these combinations respectively affected the dAb expression level in the broth supernatant, the level in the pellet lysate and total soluble dAb. Among the 20 AAs, R displayed a negative influence on the soluble expression level, whereas G and S showed positive effects. A linear model was built to predict the soluble expression level from the sequence; this model had a prediction accuracy of 80%. In summary, increasing the content of polar AAs, especially G and S, and decreasing the content of R, was helpful to improve the soluble expression level of HSA dAb.

NMR strategies to support medicinal chemistry workflows for primary structure determination.

Central to drug discovery is the correct characterization of the primary structures of compounds. In general, medicinal chemists make great synthetic and characterization efforts to deliver the intended compounds. However, there are occasions which incorrect compounds are presented, such as those reported for Bosutinib and TIC10. This may be due to a variety of reasons such as uncontrolled reaction schemes, reliance on limited characterization techniques (LC-MS and/or 1D 1H NMR spectra), or even the lack of availability or knowledge of characterization strategies. Here, we present practical NMR approaches that support medicinal chemist workflows for addressing compound characterization issues and allow for reliable primary structure determinations. These strategies serve to differentiate between regioisomers and geometric isomers, distinguish between N- versus O-alkyl analogues, and identify rotamers and atropisomers. Overall, awareness and application of these available NMR methods (e.g. HMBC/HSQC, ROESY and VT experiments, to name only a few) should help practicing chemists to reveal chemical phenomena and avoid mis-assignment of the primary structures of compounds.

Structural analysis of Centrolobium tomentosum seed lectin with inflammatory activity.

A glycosylated lectin (CTL) with specificity for mannose and glucose has been detected and purified from seeds of Centrolobium tomentosum, a legume plant from Dalbergieae tribe. It was isolated by mannose-sepharose affinity chromatography. The primary structure was determined by tandem mass spectrometry and consists of 245 amino acids, similar to other Dalbergieae lectins. CTL structures were solved from two crystal forms, a monoclinic and a tetragonal, diffracted at 2.25 and 1.9 Å, respectively. The carbohydrate recognition domain (CRD), metal-binding site and glycosylation site were characterized, and the structural basis for mannose/glucose-binding was elucidated. The lectin adopts the canonical dimeric organization of legume lectins. CTL showed acute inflammatory effect in paw edema model. The protein was subjected to ligand screening (dimannosides and trimannoside) by molecular docking, and interactions were compared with similar lectins possessing the same ligand specificity. This is the first crystal structure of mannose/glucose native seed lectin with proinflammatory activity isolated from the Centrolobium genus.

Angiotensin I Converting Enzyme Inhibitory Peptides Derived from Phycobiliproteins of Dulse Palmaria palmata.

We examined the inhibitory activity of angiotensin I converting enzyme (ACE) in protein hydrolysates from dulse, Palmaria palmata. The proteins extracted from dulse were mainly composed of phycoerythrin (PE) followed by phycocyanin (PC) and allophycocyanin (APC). The dulse proteins showed slight ACE inhibitory activity, whereas the inhibitory activity was extremely enhanced by thermolysin hydrolysis. The ACE inhibitory activity of hydrolysates was hardly affected by additional pepsin, trypsin and chymotrypsin treatments. Nine ACE inhibitory peptides (YRD, AGGEY, VYRT, VDHY, IKGHY, LKNPG, LDY, LRY, FEQDWAS) were isolated from the hydrolysates by reversed-phase high-performance liquid chromatography (HPLC), and it was demonstrated that the synthetic peptide LRY (IC50: 0.044 µmol) has remarkably high ACE inhibitory activity. Then, we investigated the structural properties of dulse phycobiliproteins to discuss the origin of dulse ACE inhibitory peptides. Each dulse phycobiliprotein possesses α-subunit (Mw: 17,477-17,638) and ß-subunit (Mw: 17,455-18,407). The sequences of YRD, AGGEY, VYRT, VDHY, LKNPG and LDY were detected in the primary structure of PE α-subunit, and the LDY also exists in the APC α- and ß-subunits. In addition, the LRY sequence was found in the ß-subunits of PE, PC and APC. From these results, it was suggested that the dulse ACE inhibitory peptides were derived from phycobiliproteins, especially PE. To make sure the deduction, we carried out additional experiment by using recombinant PE. We expressed the recombinant α- and ß-subunits of PE (rPEα and rPEß, respectively), and then prepared their peptides by thermolysin hydrolysis. As a result, these peptides showed high ACE inhibitory activities (rPEα: 94.4%; rPEß: 87.0%). Therefore, we concluded that the original proteins of dulse ACE inhibitory peptides were phycobiliproteins.

Different disulfide bridge connectivity drives alternative folds in highly homologous Brassicaceae trypsin inhibitors.

Low-molecular-mass trypsin inhibitors from Arabidopsis thaliana, Brassica napus var. oleifera, and Sinapis alba L. (ATTI, RTI, and MTI, respectively) display more than 69% amino acid sequence identity. Among others, the amino acid sequence Cys-Ala-Pro-Arg-Ile building up the inhibitor reactive site, and the eight Cys residues forming four disulfide bridges are conserved. However, the disulfide bridge connectivity of RTI and MTI (C1-C3, C2-C4, C5-C6, and C7-C8) is different from that of ATTI Cys (C1-C8, C2-C5, C3-C6, and C4-C7). Despite the different disulfide bridge connectivity, the reactive site loop of ATTI, RTI, and MTI is solvent exposed permitting trypsin recognition. Structural considerations here reported suggest that proteins showing high amino acid sequence identity and common functional properties could display different three-dimensional structures. This may reflect high inhibitor plasticity in relation to plant-pathogen interactions, plant tissue development as well as the different redox potential of cell compartments.

A typical acidic extracellular polysaccharide alludes to algae-bacteria-collaboration in microalgal-bacterial symbiosis.

Microalgal-bacterial symbioses are prevalent in aquatic ecosystems and play a pivotal role in carbon sequestration, significantly contributing to global carbon cycling. The understanding of the contribution of exopolysaccharides (EPSs), a crucial carbon-based component, to the structural integrity of microalgal-bacterial symbioses remains insufficiently elucidated. To address this gap, our study aims to enhance our comprehension of the composition and primary structure of EPSs within a specific type of granular microalgal-bacterial symbiosis named microalgal-bacterial granular sludge (MBGS). Our investigation reveals that the acidic EPSs characteristic of this symbiosis have molecular weights ranging from several hundred thousand to over one million Daltons, including components like glucopyranose, galactopyranose, mannose, and rhamnose. Our elucidation of the backbone linkage of a representative exopolysaccharide revealed a →3)-ß-D-Galp-(1→4)-ß-D-Glcp-(1→ glycosidic linkage. This linear structure closely resembles bacterial xanthan, while the branched chain structure bears similarities to algal EPSs. Our findings highlight the collaborative synthesis of acidic EPSs by both microalgae and bacteria, emphasizing their joint contribution in the production of macromolecules within microalgal-bacterial symbiosis. This collaborative synthesis underscores the intricate molecular interactions contributing to the stability and function of these symbiotic relationships.

N- versus O-alkylation: utilizing NMR methods to establish reliable primary structure determinations for drug discovery.

A classic synthetic issue that remains unresolved is the reaction that involves the control of N- versus O-alkylation of ambident anions. This common chemical transformation is important for medicinal chemists, who require predictable and reliable protocols for the rapid synthesis of inhibitors. The uncertainty of whether the product(s) are N- and/or O-alkylated is common and can be costly if undetermined. Herein, we report an NMR-based strategy that focuses on distinguishing inhibitors and intermediates that are N- or O-alkylated. The NMR strategy involves three independent and complementary methods. However, any combination of two of the methods can be reliable if the third were compromised due to resonance overlap or other issues. The timely nature of these methods (HSQC/HMQC, HMBC. ROESY, and (13)C shift predictions) allows for contemporaneous determination of regioselective alkylation as needed during the optimization of synthetic routes.

Ultrasound assisted wall-breaking extraction and primary structures, bioactivities, rheological properties of novel Exidia yadongensis polysaccharide.

New natural multifunctional polysaccharide and its innovatory extraction technology may be urgently needed for food industries. Our aims were to establish new extraction method and investigate the primary structures, bioactivities and rheological properties of novel E. yadongensis polysaccharide (EYP). Ultrasound assisted mechanical wall-breaking extraction (MAUE) was successfully established for the EYP extraction from a new E. yadongensis. Based on the MAUE with RSM, the polysaccharide yield of 17.92 ± 0.56 % with the optimal parameters of five extraction factors were obtained, and current MAUE was characterized by its high yield, low extraction temperature and short ultrasound time. After the isolation and purification, the EYP as a protein-bound polysaccharide was obtained. FT-IR and NMR analysis showed that the main backbone of the EYP comprised of (1 â†’ 4)-ß-D-glucopyranosyl and (1 â†’ 6)-ɑ-D-mannopyranosyl groups; EYP exhibited significant antioxidant, antibacterial, antitumor, antidiabetic activities, and good viscoelastic properties in low pH solutions (P < 0.05). The EYP may be used as a natural functional and cohesive agent in food industries.

Comparative Analysis of the Immune Response and the Clinical Allergic Reaction to Papain-like Cysteine Proteases from Fig, Kiwifruit, Papaya, Pineapple and Mites in an Italian Population.

Several plant papain-like cysteine proteases are exploited by the food, cosmetic, pharmaceutical and textile industries. However, some of these enzymes can cause allergic reactions. In this context, we investigated the frequency of sensitization and allergic reactions to some fruit and/or latex cysteine proteases, which are used as additives by the food industry to improve and modify the quality of their products. The FABER test was used to analyse the patients' sensitization towards five plants and, for comparison, two homologous mite cysteine proteases. In an Italian population of 341 allergic patients, 133 (39%) had IgE specific for at least one of the seven cysteine proteases under investigation. Most of the patients were IgE positive for Der p 1 and/or Der f 1 (96.38%) reported a clinical history suggestive of respiratory allergy to mites, whereas none of the subjects sensitized to the homologs from papaya, pineapple and fig reported allergy symptoms following ingestion of these foods. Only one patient referred symptoms from ingesting kiwifruit. Therefore, the obtained results showed that sensitization to the fruit enzymes was only rarely concomitant with allergic reactions. These observations, together with the literature reports, suggest that the allergy to plant papain-like cysteine proteases might mainly be an occupational disease.

Structure, antioxidant property and protection on PC12 of a polysaccharide isolated and screened from Abelmoschus esculentus L.Moench (okra).

AeP-P-2, a pectic polysaccharide, was extracted from the fruit pod of okra. It composed of rhamnose (Rha), arabinose (Ara), glucose (Glc), galactose (Gal) and galacturonic acid (GalA) with the ratio of 4.75:2.01:1.00:4.91:7.24. The main structural feature of AeP-P-2 are 1,4-linked galacturonan units (homogalacturonan backbone) and (1 → 2) and (1 → 2,4) linked Rha (rhamnogalacturonan I region). And the other side chains contained →1)-linked Ara, (1 → 5)-linked Ara, (1 → 4)-linked Glc, (1 → 6)-linked Gal, (1 → 4)-linked Rha, (1 → 2,4)-linked Rha, →1)-linked Ara and →1)-linked Gal. When the concentration of AeP-P-2 was 3.2 mg/mL, the scavenging rates on DPPH·, ABTS, O2-· and ·OH reached to 61.88%, 87.10%, 52.17% and 60.32%, respectively. AeP-P-2 also could protect PC12 cells from the damage of H2O2 and reduce apoptosis caused by oxidative damage by decreasing the level of ROS. The findings indicated that okra was a functional vegetable and AeP-P-2 was worth studying and developing into antioxidant component.

Peptides Isolated from Amphibian Skin Secretions with Emphasis on Antimicrobial Peptides.

The skin of amphibians is a tissue with biological functions, such as defense, respiration, and excretion. In recent years, researchers have discovered a large number of peptides in the skin secretions of amphibians, including antimicrobial peptides, antioxidant peptides, bradykinins, insulin-releasing peptides, and other peptides. This review focuses on the origin, primary structure, secondary structure, length, and functions of peptides secreted from amphibians' skin. We hope that this review will provide further information and promote the further study of amphibian skin secretions, in order to provide reference for expanding the research and application of amphibian bioactive peptides.

The effect of hypochlorite- and peroxide-induced oxidation of plasminogen on damage to the structure and biological activity.

In this study, we examined for the first time the effect of the HOCl/OCl-- and H2O2-induced oxidation of Glu-plasminogen on damage to its primary structure and the biological activity of plasmin. The consolidated results obtained with the aid of MS/MS, electrophoresis, and colourimetry, demonstrated that none of the oxidised amino acid residues found in the proenzyme treated with 25 µM HOCl/OCl- or 100 µM H2O2 were functionally significant for plasminogen. However, the treatment of plasminogen with increasing concentrations of HOCl/OCl- from 25 µM to 100 µM or H2O2 from 100 µM to 300 µM promoted a partial loss in the activity of oxidised plasmin. Several methionine residues (Met57, Met182, Met385, Met404, Met585, and Met788) localized in different protein domains have been shown to serve as ROS traps, thus providing an efficient defense mechanism against oxidative stress. Oxidised Trp235, Trp417, Trp427, Trp761, and Tyr672 are most likely responsible for the reduced biological activity of Glu-plasminogen subjected to strong oxidation. The results of the present study, along with those of previous studies, indicate that the structure of Glu-plasminogen is adapted to oxidation to withstand oxidative stress induced by ROS.

Comparison of Fragments in Human Hemostatic Proteins That Mimics Fragments in Proteins of A/H1N1 Viruses and Coronaviruses.

Objective: To compare the repertoire of proteins of the human hemostatic system and fragments mimicking these proteins in the proteins of influenza A/H1N1 viruses and coronaviruses. Material and methods. Influenza viruses A/H1N1 (A/Brevig Mission/1/18), A/St. Petersburg /RII04/2016 (H1N1)pdm09, coronaviruses SARS-CoV and SARS-CoV-2 (strain Wuhan-Hu-1) were used for comparative computer analysis. The sources of the primary structures of proteins of the analyzed viruses and 41 proteins of the human hemostatic system were publicly available Internet databases, respectively, www.ncbi.nlm.nih.gov and www.nextprot.org. The search for homologous sequences in the structure of viral proteins and hemostatic proteins was carried out by comparing fragments of 12 amino acids in length, taking as related those that showed identity at ≥8 positions. Results. Comparative analysis of the repertoire of cellular proteins of the hemostatic system and fragments mimicking these proteins in the structure of proteins of viruses A/H1N1 1918, A(H1N1)pdm09 isolated in 2016, SARS-CoV and SARS-CoV-2, showed a significant difference between SARS-CoV-2 and analyzed viruses. In the protein structure of the SARS-CoV-2 virus, mimicry was revealed for almost all analyzed hemostasis proteins. As for the comparison of viruses A/H1N1 1918, A(H1N1)pdm09 2016 and SARS-CoV, the influenza virus A/H1N1 1918 and SARS-CoV are the closest in the repertoire of hemostatic proteins. Conclusion. Obtained bioinformatic analysis data can serve as a basis for further study of the role of homologous fragments in the regulation of hemostasis of the host organism.

Bioinformatics-Structural Approach to the Search for New D-Amino Acid Oxidases.

D-amino acid oxidase (DAAO, EC 1.2.1.2) plays an important role in the functioning of prokaryotes as well as of lower (yeast and fungi) and higher eukaryotes (mammals). DAAO genes have not yet been found in archaean genomes. D-amino acid oxidase is increasingly used in various fields, which requires the development of new variants of the enzyme with specific properties. However, even within one related group (bacteria, yeasts and fungi, mammals), DAAOs show very low homology between amino acid sequences. In particular, this fact is clearly observed in the case of DAAO from bacteria. The high variability in the primary structures of DAAO severely limits the search for new enzymes in known genomes. As a result, many (if not most) DAAO genes remain either unannotated or incorrectly annotated. We propose an approach that uses bioinformatic methods in combination with general 3D structure and active center structure analysis to confirm that the gene found encodes D-amino acid oxidase and to predict the possible type of its substrate specificity. Using a homology search, we obtained a set of candidate sequences, modelled the tertiary structure of the selected enzymes, and compared them with experimental and model structures of known DAAOs. The effectiveness of the proposed approach for discrimination of DAAOs and glycine oxidases is shown. Using this approach, new DAAO genes were found in the genomes of six strains of extremophilic bacteria, and for the first time in the world, one gene was identified in the genome of halophilic archaea. Preliminary experiments confirmed the predicted specificity of DAAO from Natronosporangium hydrolyticum ACPA39 with D-Leu and D-Phe.

Sequence to structural analysis of ORF5 protein in Norway rat Hepatitis E Virus.

Hepatitis E virus (HEV) is a major causative agent of acute hepatitis in developing countries. The Norway rat HEV genome consists of six open reading frames (ORFs), i.e., ORF1, ORF2, ORF3, ORF4, ORF5 and ORF6. The additional reading frame encoded protein ORF5 is attributed to life cycle of rat HEV. The ORFF5 protein's function remains undetermined. Therefore, it is of interest to analyze the ORF5 protein for its physiochemical properties, primary structure, secondary structure, tertiary structure and functional characteristics using bioinformatics tools. Analysis of the ORF5 protein revealed it as highly unstable, hydrophilic with basic pI. The ORF5 protein consisted mostly of Arg, Pro, Ser, Leu and Gly. The 3D structural homology model of the ORF5 protein generated showed mixed α/ß structural fold with predominance of coils. Structural analysis revealed the presence of clefts, pores and a tunnel. This data will help in the sequence, structure and functional annotation of ORF5.