The annotation of short-reads metagenomes is an essential process to understand the functional potential of sequenced microbial communities. Annotation techniques based solely on the identification of local matches tend to confound local sequence similarity and overall protein homology and thus don’t mirror the complex multidomain architecture and the shuffling of functional domains in many protein families. Here, we present MetaGeneHunt to identify specific protein domains and to normalize the hit-counts based on the domain length. We used MetaGeneHunt to investigate the potential for carbohydrate processing in the mouse gastrointestinal tract. We sampled, sequenced, and analyzed the microbial communities associated with the bolus in the stomach, intestine, cecum, and colon of five captive mice. Focusing on Glycoside Hydrolases (GHs) we found that, across samples, 58.3% of the 4,726,023 short-read sequences matching with a GH domain-containing protein were located outside the domain of interest. Next, before comparing the samples, the counts of localized hits matching the domains of interest were normalized to account for the corresponding domain length. Microbial communities in the intestine and cecum displayed characteristic GH profiles matching distinct microbial assemblages. Conversely, the stomach and colon were associated with structurally and functionally more diverse and variable microbial communities. Across samples, despite fluctuations, changes in the functional potential for carbohydrate processing correlated with changes in community composition. Overall MetaGeneHunt is a new way to quickly and precisely identify discrete protein domains in sequenced metagenomes processed with MG-RAST. In addition, using the sister program “GeneHunt” to create custom Reference Annotation Table, MetaGeneHunt provides an unprecedented way to (re)investigate the precise distribution of any protein domain in short-reads metagenomes. Although metagenomes have been assembled7, the vast majority of published metagenomic data still consists of unassembled short-read DNA sequences. For example ~94% of the ~15,000 datasets listed in the TerrestrialMetagenomeDB are short-read metagenomes8. The annotation of these short-reads generally involved the identification of local similarities with sequences in a pre-annotated reference database 9 or specialized database 2. Generally, the processing of these short-reads requires specific computational approaches and infrastructures5 however the development of publicly accessible annotation platforms 10, sometimes used for repository, has somewhat addressed the computational challenge5. For example, as of December 2019, MG-RAST hosted ~400,000 publicly accessible annotated datasets9. Nevertheless, although short-reads annotation enables one to investigate communities’ structure and functional potential , this approach has multiple caveats5. Among others, identifying local sequence similarity between short-read queries and target sequences in database does not guarantee global sequence similarity/homology over the entire sequence. Thus, confounding local matches with global/functional similarity introduces biases as many proteins consist of complex and variable assemblages of distinct functional protein domains13. In this context, a domain-centric annotation system is needed to discriminate the catalytic domain supporting the function of interest from their accessory domains and thus to better investigate the functional potential of sequenced microbial communities. However, to date, the direct identification of protein domains  in short-reads datasets is not reliable and poorly developed5.Over the past decades, high throughput DNA sequencing of metagenomic DNA has generated a huge amount of sequences whose characterization has provided unprecedented insights into the structure and function of microbiomesIdentifying the domains, rather than the proteins, will also allow for the normalization of the hit to account for the domain length. Indeed, the proportion of short-reads matching a domain of interest (or an entire protein) is affected by the length of the domain as the raw number of sequences matching specific domains is expected to reflect both the abundance and the length of the domains. Not accounting for the length of the domain (or protein) of interest during the data processing introduces two major systematic biases. First, the longer the domain of interest; the more their frequency is overestimated. Second, if the data processing involves rarefaction, short, less abundant domains, although important, might be discarded.14 protein domain identification in the M5nr database15 to identify protein domains in publicly accessible datasets. Here, we focused on microbial glycoside hydrolases (GHs) as these enzymes are essential16 for the processing of carbohydrates in mammals’ gut2 and across environments3 where they support the carbon cycling. Like other carbohydrate active enzymes (CAZymes) and many other protein families, GHs are known for their complex multidomain architecture15. Although most biochemically characterized GH-enzymes consist of single-domain GHs (SDGHs), the bioinformatic analysis of sequenced genomes and assembled metagenomes has highlighted the complexity and abundance of multi-domain GHs (MDGHs)17. For example, ~60% of identified α-amylases from GH13 contain multiple domains. Similarly up to ~25% of known enzymes active on cellulose, xylan, and chitin are MDGHs15. In addition, many “non-CAZy” domains, including some α/β-hydrolases and many domains of unknown function (DUFs), are linked to CAZy domains. Focusing on local alignment to identify the functional potential of sequenced microbial communities  overestimate the frequency of the trait of interest whereas identifying matches between short-reads and GH-domains , GH sub-domains , accessory domains , or a DUF should only count toward this specific domain.To address these problems, we designed MetaGeneHunt to perform precise annotation of protein domains in short-reads metagenomes retrieved from MG-RAST. MetaGeneHunt combines short-read local alignment, provided by MG-RAST, with precise PFam-based1 and the Twin Gut Microflora study2, to evaluate the frequency of short-reads matching with GH-containing protein and the frequency of matches located in the GH domains. We next used MetaGeneHunt to investigate how the functional potential for carbohydrate processing in microbial communities associated with food fluctuates as the bolus moved through the gastrointestinal tract (GIT) of five captive mice (Mus musculus). We sampled the bolus along the GIT, rather than feces, because the GIT consists of a series of interconnected environments hosting distinct microbial communities supporting specific processes21. Although distinct microbial communities inhabit the mucosal and luminal space in the GIT23, we decided to investigate the precise distribution of GHs, and their associated domains, in microbial communities associated with the bolus (in the lumen) as these communities interact directly with the substrate. More precisely, we investigated communities in the stomach, intestine, cecum, and colon.We hypothesized that the community structure along the GIT will be similar for all the mice and that the sampled sections of the GIT will be associated with distinct conserved microbial communities across individuals. Indeed, these healthy mice shared the same genetic background, were maintained in the same conditions, and were fed ad libitum with the same plant-based diet. In addition, the sampled sections of the GIT corresponded to anatomically discrete regions, separated from each other by the cardiac, the pyloric, and the ileocecal sphincters, all sections where the bolus is incubated for prolonged time24. In these regions, the bolus is exposed to various pH, salts, and enzymes, among other conditions27. These conditions support the host’s endogenous digestive process and produce discrete environments where microbial communities develop and contribute to the overall digestion process by releasing additional enzymes.We used MetaGeneHunt on publicly accessible metagenomes from MG-RAST including the Mammal Microbiome28, we predicted that changes in microbial community composition along the GIT would correlate with variation in the potential for carbohydrate processing based on identified sequences for GHs. Alternatively, as polysaccharide deconstruction is an essential process supported by multiple lineages28, one could hypothesize that structurally distinct communities would converge to similar functional potential3. In this case the functional potential would be more conserved than the microbial community structure. Finally, we investigated the correlation between structure and functional potential for carbohydrate processing across microbial communities derived from the same location.As GHs are not evenly distributed across microbial lineagesCombining a method for the rapid domain-specific annotation of short-reads datasets with the possibility to (re)analyze publicly accessible from MG-RAST provides an unprecedented opportunity to precisely investigate the functional potential within and across sequenced microbial communities.1 and the Twin Gut Microflora2 studies and found that 43.8 and 40.4% of the short-read sequences matching with a GH domain-containing sequence were localized in GH domains, respectively. Next, among the 4,726,023 short-read sequences matching with a GH domain-containing sequence identified in the Mouse GIT (this study), only 1,973,805(41.7%) of the hits were actually localized in a domain of interest , thus suggesting that unnormalized raw hit counts systematically overestimated the frequency of the longer domains of interest.Next, although GHs were the most abundant domain of interest identified in the mouse GIT, the numbers of hits per GH family were not evenly distributed. Across samples, the total number of GH and CBM domains hits correlated with the size of the domain expressed in amino acids  and thus better mirrored the distribution of the functional domains in the GIT. Next, normalized and localized hit counts were rarefied , divided by their length Fig. , 2. ThisRegarding enzymes targeting structural polysaccharides, domains from GH5, GH8, and GH9, accounting for 2.89, 0.55, and 0.12% of the normalized hits respectively, were the most abundant cellulase domains. No fungal cellulase from GH7, nor GH6, GH44, GH45 and GH48 were detected. We identified sequences encoding potential xylanases from GH10 (~1.3%), GH30 (~0.4%), and few xylanases from GH11. Chitinases from GH18 accounted for 0.91% of the identified sequences whereas a few GH19 were identified but only in some samples. Carbohydrate binding modules (CBMs), together accounting for ~0.7% of the normalized hits, were from the CBM families 48, 4_9, 6, 26, 20, 9_1, and 35  matched the frequency of the domain GH3C (4.00%). Similarly, regarding the GH2 β-galactosidases, GH2N and GH2C accounted for 3.17 and 2.78% of the hits, respectively , with P-values < 0.001 for many domains including GH8 (cellulase/chitosanase), GH42 , GH68 (levansucrases), GH18 (chitinases), GH38 (α-mannosidases), and GH25 (lysozyme) among others , GH28 , GH29 (α-L-fucosidases), and GH97  were more characteristic of the intestine. In addition, some less abundant sequences from GH67 (α-glucuronidases), GH106 (α-L-rhamnosidases), GH26 (β-mannanases), and GH35  were also systematically enriched in the intestine. Conversely, compared to other locations, the intestine contained fewer sequences encoding members of GH13 (α-amylases), GH73 (peptidoglycan hydrolases), GH4 , GH8 (cellulases), and GH18 (chitinases). Next, most samples from the cecum (C) clustered together and were enriched in many GH families underrepresented in the intestine and including GH13, GH73, GH4, GH8, and GH18 among others. Finally, most samples from the large intestine (L), like samples from the stomach, formed a poorly resolved cluster associated with no specifically enriched GHs.Next, we investigated the samples clustering using the traits most affected by the sample origin  and less abundant lineages from the Proteobacteria phylum  and some Actinobacteria  became more characteristic  whereas the sex, the individual, and their interaction had no significant effect.Pearson = 0.02) , variation of the community structure, identified at the genus-level, correlated with change of the functional potential for polysaccharide deconstruction with all the P-values below 0.001 except for samples from the large intestine (P02) Fig. . Communi02) Fig. .30. This new approach is based on, and complements, the GeneHunt annotation approach15 and is designed to analyze short-read metagenomes from MG-RAST9. As such, it doesn’t require large computer infrastructure.MetaGeneHuntis a new way to perform domain specific identification of GHs, and associated domains, in short-read metagenomes. Identifying domains, rather than protein, is essential as GH domains are associated with many variable domains31 and as the protein modularity varies33 bypassing the normalization results in unknown bias. Although MetaGeneHuntcan identify local hits matching with accessory domains , it does provide a comprehensive identification only for the domains listed in the reference annotation table (RAT). In order to further investigate the distribution of these newly identified accessory domains, not targeted at first, a new RAT should be constructed using GeneHunt15.According to the proposed approach, identifying domains (i.e. GHs) rather than entire proteins addresses two major problems: (i) it discriminates local matches inside or outside the functional domains of interest, (ii) it allows the normalization of the raw hit count by accounting for the domain length which directly affects the likelihood of matching hits. Systematically considering the local hits in protein containing GH domains and not accounting for the domain length together result in a systematic bias towards the longer domains and more complex proteins (with multiple domains). When investigating the functional potential and the clustering of samples derived from the same environment, this bias is minimized, and the sample clustering remains essentially unchanged, as the same systematic bias is applied repeatedly. However across environments, as the distribution of GH domains is highly variable36. In herbivores, the GIT microbiome is enriched in carbohydrate active enzyme targeting cellulose and the hemicellulose, among others38. This plant material represents a major source of carbon and energy for the host and its associated microbial communities. In the mouse, beside a set of core traits not affected by the sample origin, the distribution of several GH families mirrors the GIT compartmentalization. This reflects the broad distribution of traits involved in the processing of short oligosaccharide  in sequenced microbial lineages, whereas traits targeting more complex structural substrates tend to be restricted to specific lineages39.In the mouse GIT, as the bolus transits from the stomach to the colon, it exhibits a series of structurally and functionally distinct microbial communities. Although microbes associated with the bolus come directly with the food or from the preceding sections of the GIT along with the bolus and transfer between the microbial community associated with the mucosa, even sequential parts of the GIT  display significantly distinct microbial communities. This suggests that the distinct physiology of sequential section of the GIT cause abrupt modification of the microbial community composition. Being in contact with bolus, these microbial lineages directly contribute to its degradation by releasing enzymes into the lumen40, the bolus is associated with the most diverse DNA. As the stomach is the first section of the GIT where the bolus is incubated24, the corresponding inferred microbial community likely reflects both the active microbes in the stomach and the DNA in and on the food. Regarding the functional potential for polysaccharide deconstruction, although containing many GH sequences, the stomach is not specifically enriched in any GH family. In addition to core traits, the stomach, like the other section of the GIT, contained a high proportion of sequences encoding amylases from GH13 and GH57, cellulases from GH5, GH8, and GH9, and many enzymes targeting short oligosaccharides . Together this suggests that, although enriched in enzymes for carbohydrate processing, there is no selection for specific enzymes in the stomach, relative to the other sections of the GIT.In the stomach of mice, although initially perceived as a mostly sterile environment due to the low pHBacteroidaceae, Prevotellaceae, and Porphyromonadaceae dominate and account for more than 70% of the reads in the intestine. The systematic investigation of sequenced Bacteroidetes lineages reveals their increased potential for polysaccharide processing41. Indeed, at large Bacteroidetes are enriched in enzymes targeting short oligosaccharides , starch (GH13), cellulose (mostly GH5), xylan , and other polysaccharides 39. Consistently, when focusing on the normalized distribution of identified GHs, the bolus in the intestine, the high frequency of GH2, GH5, GH28, GH29, GH97, GH67, GH106, GH26, GH35, and GH57 among others, was identified.In the intestine, the somewhat variable bolus-associated community coming from the stomach is filtered and a more conserved microbial community emerges. Across individuals, this community is characterized by the high frequency of Bacteroidetes. More specifically, 42. As identified here, the microbial community in the cecum, is dominated by lineages of Butyvibrio, Ruminococcus, and Clostridium, all taxa known or predicted to have high potential for polysaccharide deconstruction12. Sequenced genomes from these lineages of Firmicutes are enriched in the GH domains identified independently in the short-read metagenome from the cecum. Finally, in the colon, the microbial community is characterized by a mix of microbial lineages with no characteristic taxon, as depicted for the stomach. Accordingly, the GH content, although abundant was not specifically enriched in any function. Interestingly, communities from the colon and stomach, although being the most distal communities characterized here, displayed a high degree of structural and functional similarity. This supported the murine practice of coprophagy where material released from the colon get re-ingested as a way to retain bacterial proteins43, to further process some partially digested material44, and to re-colonize the GIT46.Next, from the intestine, some of the bolus goes directly to the colon whereas some goes to the cecum before being transferred to the colon. To date, the exact function of the cecum isn’t fully understood although it might be a reservoir of bacteria to colonize the colon. Also the cecum is the primary site of colonic fiber fermentation supporting the release of short chain fatty acids (SCFA) absorbed in the colon15, combined with the power and convenience of MG-RAST9, provides an unprecedented way to quickly and precisely annotate newly sequenced short-readsmetagenomes and to reanalyze publicly accessible datasets . Finally, although the GeneHunt15 and MetaGeneHunt approaches were designed for the precise identification of GHs in sequenced genomes and metagenomes, many other proteins such as receptors, display complex and variable multidomain architectures. In the future, the creation of new dedicated RAT using GeneHunt combined with MetaGeneHunt will provide new efficient ways to investigate the functional potential of sequenced metagenomes.In conclusion, over the past decades, the development of metagenomics and the associated bioinformatics techniques has profoundly affected our understanding of the microbial diversity and its contribution to environmental processes. The functional annotation of short-reads is an essential aspect of this metagenomic revolution. However, a metagenome annotation can only be as good as the annotation of the reference database. Hence, as demonstrated here, the careful domain specific annotation of the M5nr database9  annotation of the Glycoside Hydrolases and accessory domains  in the M5nr database47 created using GeneHunt15 as a reference annotation table (RAT). First, MetaGeneHunt retrieves the M5nr annotated metagenomes from MG-RAST  using the MG-RAST APIs48. Next, sequences matching potential GHs are identified in file “650”, using the MD5id of the annotated hits from the RAT. Next, for these local matches, the precise alignment position is compared to the domain-specific annotation in the RAT. If >20AAs from the query align with a specific protein domain (considering the HMM-envelope position in the RAT)49 then this domain annotation is transferred to the query. Conversely, the annotation is considered negative if >20AAs of the query match outside the domain of interest . The cutoff for the overlapping can be modified at will by the user. Next, the actual sequence count for each identified hit is retrieved from the sequence agglomeration file . Finally, in the subsequent data processing and normalization, hit counts, per protein domain, are normalized according to the size of the protein domain in the Pfam database14.MetaGeneHunt was designed to work with MG-RAST annotated datasetsad libitum with fiber rich diet . Individuals were sacrificed via rapid decapitation (IACUC #349), then during dissection each region of the gut  was sutured in the abdomen to ensure the bolus remained in place. Then the regions were opened aseptically, and the bolus was collected. All experimental procedures were approved by CSULB IACUC and performed according to AAALAC guidelines.Samples of bolus were collected from 42-day old, male (n = 3) and female (n = 2), C57BL/6 J mice  housed in the California State University Long Beach Animal Care Facility. Mice were maintained under 12h:12h light:dark cycle and fed 9.Total DNA was extracted using PowerFecal DNA Isolation Kit , sheared using a focused ultrasonicator , tagged using Multiplex TruSeq DNA Nano , QCed on Agilent Bioanalyzer, and sequenced on Illumina HiSeq.2500 (PE100) at the UCI Genomics High-Throughput Facility . DNA sequences were uploaded to MG-RAST for repository and taxonomic annotationSupplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.


Mammalian mitochondrial inner membrane fusion is mediated by optic atrophy 1 (OPA1). Under physiological conditions, OPA1 undergoes proteolytic processing to form a membrane-anchored long isoform (L-OPA1) and a soluble short isoform (S-OPA1). A combination of L-OPA1 and S-OPA1 is essential for efficient membrane fusion; however, the relevant mechanism is not well understood. In this study, we investigate the cryo-electron microscopic structures of S-OPA1–coated liposomes in nucleotide-free and GTPγS-bound states. S-OPA1 exhibits a general dynamin-like structure and can assemble onto membranes in a helical array with a dimer building block. We reveal that hydrophobic residues in its extended membrane-binding domain are critical for its tubulation activity. The binding of GTPγS triggers a conformational change and results in a rearrangement of the helical lattice and tube expansion similar to that of S-Mgm1. These observations indicate that S-OPA1 adopts a dynamin-like power stroke membrane remodeling mechanism during mitochondrial inner membrane fusion. In eukaryotic cells, series of discrete membranous compartments separate different biochemical reactions, and the membrane fission and fusion mechanisms accomplish communication between and within these compartments . A familOPA1 contains an N-terminal mitochondrial targeting sequence (MTS), a following transmembrane domain (TM), a coiled-coil domain, a highly conserved GTPase domain, a middle domain, and a C-terminal GTPase effector domain (GED). OPA1 has eight spliced variations in the region between the TM and the coiled-coil domain . After bChaetomium thermophilum  together with subvolume averaging (SVA) on the same sample . The final averaged cryo-ET map revealed a consistent architecture compared with that of the helical reconstruction . To elimC. thermophilum S-Mgm1 (PDB ID 6QL4)   into ourID 6QL4) .GDP.AlF4- (PDB ID 2X2E)   failed (ID 2X2E) . FurtherID 2X2E) . This suThe stalk region of S-Mgm1 could also be well docked into the stick-like middle density layer of the map . The whoThe leg density of the S-OPA1 tube could accommodate the S-Mgm1 paddle domain . SequencThe helical reconstruction processing procedure indicated that S-OPA1–coated tube contains six helical starts. By fitting S-Mgm1 crystal structures into the map, we observed that the asymmetric unit of the S-OPA1 packing array contains two S-OPA1 molecules . They foWe then investigated how the S-OPA1 dimers assemble on the liposomal tube . By follHowever, when considering the helical assembly of S-Mgm1, which uses interface-1 and interface-2 to form a helical rung, we could also model the S-OPA1 dimer assembly in a right-handed manner , where iThe aforementioned structural analysis indicated that the S-OPA1 EMB domain is presumably responsible for membrane binding and deformation. According to sequence analysis, such an EMB domain corresponded to the residues from 738 to 853 in S-OPA1 . Among tWe constructed five S-OPA1 mutants by mutating regions 794–800 to all alanine residues (794-800A) or by inducing double- and triple-point mutations . Compared with the WT S-OPA1, all five mutants exhibited moderate suppression of membrane binding, suggesting that the residues of membrane binding were at least partially involved . HoweverOur observation that S-OPA1 can induce tubulation of liposomes without the addition of nucleotide suggests that the tubulation activity of S-OPA1 is independent of its GTP hydrolysis activity. To validate this assumption, we performed mutagenesis. The high sequence conservation of G domains among dynamin proteins enabled us to identify the locations (a.a. 297–302 and 319–327) of the key catalytic residues of S-OPA1  at its PThe GTPase activity of S-OPA1 is indispensable for promoting mitochondrial inner membrane fusion . HoweverWe incubated excess GTP or its non-hydrolyzable or slowly hydrolyzable analogs  with the WT- and truncated S-OPA1–coated tubes for 30 min before examination using cryo-EM . ContrarTo further confirm that the S-OPA1 tube expansion was caused by nucleotide binding, we used the aforementioned mutants with defective GTPase activity  to investigate their tubulation behaviors after incubation with GTP. Studies have indicated that the mutants Q297E and S298A maintain their GTP binding activity  whereas The S-OPA1 coated liposomal tubes became unstable after incubation with GTP, most likely as a result of unsynchronized GTP hydrolysis. Thus, we selected the most stable and homogenous GTPγS binding state for structural study. The helical reconstruction technique failed because of the variable diameters. We therefore utilized cryo-ET and SVA to analyze the assembly of GTPγS-bound S-OPA1 on the membrane .Similar to the nucleotide-free state, the 23 Å–resolution cryo-EM map of S-OPA1–coated tube bound with GTPγS also revealed four density layers: an outer head region, middle stalk region, inner leg region, and innermost membrane region . HoweverSubsequently, we docked the previously generated nucleotide-free state S-OPA1 model into this cryo-EM density . The modBy investigating the packing array of S-OPA1, we observed that the asymmetric unit of S-OPA1 nucleotide–coated tube in the GTPγS binding state also contained an S-OPA1 dimer . This diSubsequently, we further analyzed the conformational changes of S-OPA1 upon nucleotide binding by comparing the models generated from the nucleotide-free and GTPγS binding states. The superposition of two monomers indicated a slight (~4°) G domain/BSE swing after GTPγS binding . In the After GTPγS binding, interface-1 and interface-2 in S-OPA1 assembly generally maintained their original positions, with only a slight change in location of interface-2. However, interface-3 and interface P1 in the nucleotide-free state were broken, and a new inter-rung interface P2 formed between the EMB domains. As a result, for the left-handed model of S-OPA1 dimer assembly, GTPγS binding broke the original helical rungs and formed new rungs through combination of the two nearest pieces . By contOverall, although the S-OPA1 assembly in the nucleotide-free state appeared more compact, its building unit adopted a more relaxed conformation with each S-OPA1 molecule in a closed state. After GTPγS binding, S-OPA1 monomers adopted an open conformation, which constrained the spread conformation of building units (dimers and tetramers) and finally resulted in a loosened helical rung and an expansion of the helical tube.C. thermophilum S-Mgm1 (CtMgm1) (S. cerevisiae S-Mgm1 (ScMgm1)   and S. c(ScMgm1) , which aIn the present study, we investigated the interactions between S-OPA1 and liposomes that have the phospholipid composition of the mitochondrial inner membrane. Similar to other dynamin proteins, S-OPA1 can bind to membranes and induce membrane tubulation by forming a helical array. We revealed that such tubulation processes are independent of GTP binding and hydrolysis activity; however, S-OPA1 can exhibit considerably enhanced GTPase activity after binding to the membrane. We used the cryo-EM approach to solve the structures of S-OPA1 coated on membranes in both nucleotide-free and GTPγS binding states. Docking of an S-Mgm1 crystal structure into cryo-EM maps revealed that S-OPA1 has a classic dynamin-like structure that contains a G/BSE domain, a stalk domain, and an EMB domain. The EMB domain has a similar size and shape as the paddle domain of CtMgm1 and the LIS domain of ScMgm1.Structural analysis revealed four interaction interfaces that contribute to S-OPA1 assembly on the membrane in the nucleotide-free state. Three interfaces  are involved in stalk packing. The fourth interface (P1) from the EMB domain interaction constitutes another factor influencing the stability of S-OPA1 assembly. In contrast to the tube formed by CtMgm1, which was left-handed with four starts, the tube formed by S-OPA1 in the absence of nucleotides was also left-handed but with six starts. In addition, CtMgm1 only used interface-1 and interface-2 to form the helical filament in both the nucleotide-free and binding states, whereas the helical assembly of S-OPA1 used additional interface-3 and interface P1 in the nucleotide-free state. We further identified a key region (794–800) of the EMB domain and observed that the hydrophobic residues of this region are instrumental to membrane tubulation activity. The observed critical role of the stalk and EMB domains for S-OPA1 assembly and membrane interaction is consistent with the findings of other studies that have highlighted the relationship between diseases and mutations in the stalk and EMB regions . The G dWe observed substantial changes in S-OPA1 assembly after GTPγS binding, including motion between the G domain and the stalk, global rotation of the stalk region, and rearrangement of the S-OPA1 helical lattice. Here we defined the conformation of S-OPA1 in its nucleotide-free state as closed conformation and that in its GTPγS-bound state as open conformation. In contrast to conventional dynamin proteins that induce more crowded packing and a constricted tube with a smaller diameter and higher curvature (ready for fission) after nucleotide binding , the lefA mechanochemical mechanism of the protein-induced membrane remodeling adopted by Dyn1 has been hypothesized . The proPrevious studies have revealed that S-OPA1 alone cannot trigger the fusion of the mitochondrial inner membrane but rather must collaborate with L-OPA1 . During Although our structural and functional analyses demonstrated the assembly of S-OPA1 on membranes in nucleotide-free and GTPγS binding states and provided models of how the rearrangement of this assembly affects membrane remodeling, the precise molecular mechanism of S-OPA1–induced membrane fusion and the cooperation between L-OPA1 and S-OPA1 require further study. High-resolution structural information regarding S-OPA1 and L-OPA1 in different GTP hydrolysis states could help to clarify the mechanisms of mitochondrial inner membrane fusion.cDNA corresponding to the short S1 isoform of OPA1 was sub-cloned into the pET32M-3C expression vector  with a N-terminal Trx tag and a followed His6 tag. A PreScission protease cleavage site exists between the His tag and the coding sequence. Mutants were constructed in pET32M-3C/OPA1-S1 via PCR. All proteins were expressed in Transetta (DE3) bacteria cells (Transgene) and purified under the following procedure. Cultures were grown at 37°C until OD at 600 nm reached 0.8. Protein expression was induced with 0.2 mM IPTG for 18 hr at 16°C. The cells were collected by centrifugation. Bacteria pellets were resuspended in lysis buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl and protease inhibitors cocktail (Roche) and disrupted with ultra-sonication. Lysates were incubated with Ni-NTA beads (Roche). After washing with the buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT, and 10 mM imidazole, protein was cleaved by prescission protease at 4°C overnight. After cleavage, protein was eluted with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT, 20 mM imidazole. The eluted protein fraction was further purified by gel filtration chromatography using a Superdex 200 10/300 GL column  in the buffer of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM DTT. The elution volume of the column was pre-calibrated using standard protein molecular weight markers. Purified proteins were frozen in liquid nitrogen and stored at −80°C.2 to a final concentration of 4 mg/ml. The resulting multi-lamellar liposomes were put through five freeze/thaw cycles to make unilamellar liposomes. S-OPA1 protein was then mixed with unilamellar liposomes 1:1 (m:m) at a final concentration of 1 mg/ml in the buffer containing 20 mM HEPES (pH 8.0), 1 mM EGTA, and 1 mM MgCl2. The mixture was incubated at 16°C for 30 min before preparing cryo-EM samples. For tubes incubate with GTP and GTP non-hydrolyzed analogous, 10 mM nucleotide was then added with a final concentration of 1 mM, and the mixture was incubated for another 30 min.The lipids (Avanti Polar Lipids) were mixed in the following ratio: 45% palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 22% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 8% L-α-lysophosphatidylinositol (PI), and 25% 1',3'-bis-sn-glycerol (cardiolipin). The indicated ratios of lipids were mixed in a chloroform solution, evaporated for 4 hr in a vacuum desiccator and rehydrated in the buffer of 20 mM Tris–HCl (pH 8.0), 1 mM EGTA, and 1 mM MgClFor negative staining EM, 5 μl of protein-lipid tubes was applied to glow-discharged continuous carbon films and stained with uranyl acetate (2% w/v) for 1 min. Samples were visualized using a Tecnai Spirit electron microscope (ThermoFisher Scientific) operating at 120 kV and image were recorded with an Eagle camera.Cryo-EM grids were prepared with Vitrobot Mark IV (ThermoFisher Scientific) under 100% humidity. 3 μl of protein-lipid tubes was applied to glow-discharged Quantifoil R2/1 holy carbon grids, blotted, and plunged into liquid ethane. For grids using for tomography data collection, homemade protein A coated colloidal gold was added as a fiducial marker.2 was applied with each exposure. Images were collected at 2–4 μm underfocus.Images for helical reconstruction were recorded on a cryo-electron microscope Titan Krios (ThermoFisher Scientific) operating at 300kV using SerialEM software . A Falco2 was applied over the entire tilt series.Tilt series data were collected on a cryo-electron microscope Titan Krios G2 (ThermoFisher Scientific) using SerialEM software , with a 2 was divided for the final CTF correction of the map reconstructed by IHRSR. SPIDER  for 20 min at 4°C. The supernatant and pellet were analyzed by SDS–PAGE.Sedimentation assay was carried on as previous study . Protein2. GTP hydrolysis was quantified by monitoring the free phosphate concentration using a malachite green assay , 1 mM EGTA, and 1 mM MgCl2. After 1 ~ 2 hr incubation, which was set based on the activity of different mutants, at 37°C, the reactions were quenched with 10 μl 0.1 M EDTA. The phosphate was quantified using malachite green assay. 150 μl malachite green solution was added and the absorbance at 650 nm is monitored in a 96-well plate reader (EnSpire 2300). All experiments were repeated at least three times.The size of S-OPA1 in solution is determined by static multiangle light scattering (MALS) coupled with gel filtration. The size-exclusion chromatography column  is equilibrated with 20 mM Tris (pH 8.0), 150 mM NaCl, and 100 μl of 1 mg/ml purified S-OPA1 was applied. The detector DAWN HELEOS II (Wyatt) was used to measure the mass distribution. Data were analyzed using the provided ASTRA software.S-OPA1 and Δ196–252 was diluted to 0.3 mg/ml in 50 mM HEPES (pH 7.4), 300 mM NaCl and 1 mM DTT. The amine-reactive crosslinker bis(sulfosuccinimidyl) suberate  was added to a final concentration of 50 μM. After a 15 min incubation at room temperature, the crosslinking reaction was quenched with 50 mM Tris (pH 8.0). Crosslinked products were analyzed using a 3–8% Tris–acetate PAGE. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:This study reveals two cryo-EM structures of truncated human OPA1 helical polymers on tubulated liposomes in both nucleotide-free and GTP-γ-S-bound forms. In combination with biochemical approaches, this study provides important information on our understanding of how OPA1 remodels the inner mitochondrial membrane.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Hitoshi Nakatogawa as the Reviewing Editor and Olga Boudker as the Senior Editor.Thank you for submitting your article "Cryo-1 EM structures reveal interactions of S-OPA1 with membrane and changes upon nucleotide binding" for consideration by The reviewers have discussed the reviews with one another, and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this manuscript, Zhang et al. report two cryo-EM structures of truncated human OPA1 helical polymers on tubulated liposomes in both nucleotide-free and GTP-γ-S-bound forms. They also investigated the liposome binding and tubulation ability of Opa1 by biochemical approaches. Major findings in this study are the assembly of Opa1 polymer, and its conformational change upon its GTP-γ-S binding. As properly cited by the authors of this manuscript, recently the crystal and cyro-EM structures of yeast Opa1 homolog Mgm1 were reported. Nonetheless, the reviewers found that the structure characterization presented in this paper, with considerable technical merit, still holds sufficient conceptual advance in a mammalian homolog, and provides important information on our understanding of how OPA1 remodels the inner mitochondrial membrane. However, the reviewers have also raised a number of critical issues which the authors should address to improve the integrity of the current manuscript and strengthen their conclusions.Essential revisions:As essential revisions, we request the authors to address the following issues.1) Figure 2: handedness, fit of the nucleotide-free map. There is a mismatch of the statement of a "left-handed helical map" with the stalk filament fitted in a right-handed manner . To avoid confusion, the authors should consistently state the handedness of their maps based on the assembly of the stalk filament.Nevertheless, it is doubtful that the right-handed stalk assembly fitted into the nucleotide-free map, is correct, for the following reasons:i) None of the interfaces observed in this filament has been described before for any dynamin family member.ii) The map is only of low resolution and an unambiguous fitting of the domains is not straightforward.iii) The new 'interfaces' have not been confirmed by any mutation.iv) The suggested rotation of the OPA1 dimer upon nucleotide-binding would lead to steric clashes with neighboring molecules of the same filament and with molecules from the adjacent filament .Looking at the density map and the fitted models , there appears to be a second option for fitting of the nucleotide-free map. Such an assembly would indeed constitute a left-handed stalk filament. It would also contain the same oligomerisation interfaces as in the GTP-γ-S bound OPA1 oligomer, and these interfaces would be similar to the previously described assembly interfaces 1 and 2 of dynamin , MxA/B , and Mgm1 . Furthermore, such left-handed helical stalk assembly may explain why GTP-γ-S binding leads to expansion of the membrane tube and not constriction, unlike in dynamin (see the recent Mgm1 structure paper for discussion of helix handedness and the effect of the powerstroke). Finally, such assembly would explain the transition between the nucleotide-free and nucleotide-bound OPA1 assembly.For both the nucleotide-free and the nucleotide-bound maps, the stalk has to be rotated for 180 degree around its longest axis that interface-1 and interface-2 can form in a similar manner compared to dynamin and Mgm1.This should be illustrated in a detailed comparison in Supplementary Figure 8. For example, the OPA1 assembly mode contrasts with dynamin, MxA and Drp1, which use three assembly interfaces, but is similar to Mgm1, that requires only two interfaces.Taken together, the current fitting of the nucleotide-free map is ambiguous and the resulting stalk assembly is unseen in the dynamin family and thus appears highly speculative. The authors should consider alternative fittings for both maps.2) As now the structures of the yeast Opa1 homolog Mgm1 are available, the authors are suggested to use the Mgm1 model to fit the cryo-EM density and make corresponding analysis. Considering the substantial difference between the Mgm1 paddle domain and dynamin PH domain, as well as the special kink of the Mgm1 stalk domain, more accurate models could be obtained by this analysis. In this case, the authors may add also the Mgm1 sequence into the alignment in Supplementary Figure 5A.3) As the structural difference between the nucleotide-free and GTP-γ-S bound states of Opa1 polymer is a major finding of this manuscript, the author should perform an ITC analysis to confirm and quantify the association between Opa1 and GTP-γ-S.4) Supplementary Figure 5: GMB comparison with PH domain in dynamin. A sequence alignment of dynamin and OPA1 shows a very low amino acid identity in the region of the PH and GMB domain. A reliable alignment is not possible in this region. The authors speculate that amino acids 794-800 form an amphipathic helix which may bind to the membrane. However, the envisaged sequence is less than two turns in length, which would constitute only a small membrane anchoring point. Second, the proposed OPA1 amino acids are aligned to an amino acid stretch in the PH domain of dynamin, which forms two β-strands . In fact, the aligned β-strand region in the PH domain contains a known membrane binding loop. These observations do not support the conclusion that membrane binding in OPA1 is mediated by an amphipathic helix. The investigation of charged and hydrophobic residues in the GMB domain in order to detect the membrane binding region is sufficient to explain the experiments in Figure 5 without invoking an amphipathic helix.5) Figures 1E and 4C: It should be clearly stated which nucleotide was used in which experiment and whether GTP-γ-S-induced structural changes were visible in negative stain EM. Representative negative-stain images should be provided for both conditions. The experiments with G interface mutants in Figure 4C make only sense in the presence of GTP-γ-S, since no structural changes can be expected for these mutants in the absence of nucleotide.eLife. Your revised article has been assessed by two reviewers who evaluated the original manuscript, and the evaluation has been overseen by the Reviewing Editor Hitoshi Nakatogawa and the Senior Editor Olga Boudker.Thank you for resubmitting your work entitled "Cryo-EM structures reveal interactions of S-OPA1 with membrane and changes upon nucleotide binding" for further consideration by The reviewers found that the manuscript has been improved significantly but they raised some remaining issues that need to be addressed before final acceptance, as outlined below:Essential revisions:1) The assembly of the stalks in the nucleotide-free structure is still confusing. The new fittings, depiction of stalk interfaces and the schematic Figure 7E show that both the nucleotide-free and GTP-γ-S bound sOPA1 use interface-1 and 2 to form a filament. These interfaces are also used in Mgm1 and have been shown by mutagenesis to contribute to assembly. In contrast, the newly observed interface-3 is unique to the nucleotide-free form, is opposite of all previously known stalk interfaces, and due to missing mutagenesis, its significance for assembly remains unknown. To avoid confusion, the authors should use interface-1 and 2 to define the OPA1 filament also in the nucleotide-free form, e.g. in Figure 3A and E, Figure 3—figure supplement 2 and Figure 7E left. In Figure 7E left, not the blue and green dimers, but the blue striped and blue dimers should form the filament, as in Figure 7E right. In this case, I1 and I2 are present within a filament, and I3 is in between filaments. It would be helpful for the reader if one such turn is shown in a unique color in Figure 2A or Figure 3A (similar as it is now with the 'I3 filament') to indicate whether such filament is right- or left-handed. How do the G domains in this case connect? One might expect that the putative G domain interface is then in between different rungs.2) Figure 4C: The new data on the GTP-bound sOPA1 mutants are nicely contributing to the manuscript. However, tube diameters have to be quantified for the mutants in the absence and presence of GTP  to make a convincing claim. It also appears from Figure 1F that the representative tubes of the Δ196-252 variant are somehow smaller compared to sOPA1. Could the authors provide also some quantification of these?3) Nomenclature GMB domain: The fitted paddle is anything but a globular domain (unlike the previously assumed PH domain). Maybe 'Extended Membrane Binding Domain' (EMB) instead?4) Figure 1D legend should contain the used GTP concentration. It appears from Figure 4B that the stimulation of wt is about 10-fold, not 70-fold? Essential revisions:As essential revisions, we request the authors to address the following issues.1) Figure 2: handedness, fit of the nucleotide-free map. There is a mismatch of the statement of a "left-handed helical map" with the stalk filament fitted in a right-handed manner . To avoid confusion, the authors should consistently state the handedness of their maps based on the assembly of the stalk filament.Nevertheless, it is doubtful that the right-handed stalk assembly fitted into the nucleotide-free map, is correct, for the following reasons: […] The authors should consider alternative fittings for both maps.We would like to thank the reviewers very much for this important critical points. Yes, the helical map of S-OPA1 coated tube is left-handed. The model in the previous Figure 7C is misleading. The apparent right-handed stalk region is just due to a visual effect from the compact packing of S-OPA1 on membrane in nucleotide-free state, in which the interaction between neighboring helical rung is a key factor for the stability of assembly.As suggested by reviewers, we utilized the recent published crystal structure of S-Mgm1 (PDB code 6QL4) to re-perform structural fitting of both nucleotide-free and GTP-γ-S bound maps. With a better structural fitting, we updated our structural interpretation according to the suggestions from reviewers. In the revision , the building block of S-OPA1 assembly in both nucleotide-free and GTP-γ-S bound states has been assigned as a new dimer, which shares the similar interface 1 and interface 2 in comparison with S-Mgm1 and Dyn1. However, in the nucleotide-free state, the unique interfaces I3 at the stalk region and P1 at the GMB region are also observed and described. These two unique interfaces disappear after GTP-γ-S binding with a new interface P2 of GMB region appearing, indicating a large assembly change of S-OPA1 after nucleotide binding. In the revision, the assemblies of S-OPA1 on membrane in both nucleotide-free and GTP-γ-S bound states are all left-handed . With this new structural interpretation, the model of how the assembly of S-OPA1 responses to GTP-γ-S binding is also revised . Besides, the comparison of oligomer assembly between S-OPA1, S-Mgm1 and Dyn1 are also re-performed .2) As now the structures of the yeast Opa1 homolog Mgm1 are available, the authors are suggested to use the Mgm1 model to fit the cryo-EM density and make corresponding analysis. Considering the substantial difference between the Mgm1 paddle domain and dynamin PH domain, as well as the special kink of the Mgm1 stalk domain, more accurate models could be obtained by this analysis. In this case, the authors may add also the Mgm1 sequence into the alignment in Supplementary Figure 5A.We would like to thank the reviewers for this important suggestion. In the revision, we have utilized the recent published crystal structure of S-Mgm1 (PDB code 6QL4) to re-perform structural fitting of both nucleotide-free and GTP-γ-S bound maps. In addition, the sequence alignment between S-OPA1 and S-Mgm1 was also performed .3) As the structural difference between the nucleotide-free and GTP-γ-S bound states of Opa1 polymer is a major finding of this manuscript, the author should perform an ITC analysis to confirm and quantify the association between Opa1 and GTP-γ-S.We would like to thank the reviewers for this important suggestion. Yes, we tried to perform ITC analysis to confirm GTP-γ-S binding. However, we found a severe protein precipitation during GTP or GTP-analogs titration, which stopped the possibility of using ITC to quantify the association between SOPA1 and GTP-γ-S.As suggested below by reviewers, we then utilized different mutations of S-OPA1 at its GTPase domain to examine whether it is the GTP binding or hydrolysis to induce assembly change and thus tube expansion . For those mutants that lose GTP hydrolysis activity but keep the ability of GTP binding, we observed the tube expansion effect after adding GTP. This further confirms it is the GTP binding to trigger the assembly change of SOPA1 on membrane.4) Supplementary Figure 5: GMB comparison with PH domain in dynamin. A sequence alignment of dynamin and OPA1 shows a very low amino acid identity in the region of the PH and GMB domain. A reliable alignment is not possible in this region. The authors speculate that amino acids 794-800 form an amphipathic helix which may bind to the membrane. However, the envisaged sequence is less than two turns in length, which would constitute only a small membrane anchoring point. Second, the proposed OPA1 amino acids are aligned to an amino acid stretch in the PH domain of dynamin, which forms two β-strands . In fact, the aligned β-strand region in the PH domain contains a known membrane binding loop. These observations do not support the conclusion that membrane binding in OPA1 is mediated by an amphipathic helix. The investigation of charged and hydrophobic residues in the GMB domain in order to detect the membrane binding region is sufficient to explain the experiments in Figure 5 without invoking an amphipathic helix.We agree with the reviewers. The amphipathic helix assumption and statement have been removed in the revision and we only discussed the characteristic charged and hydrophobic residues of 794-800 region in the GMB domain.5) Figures 1E and 4C: It should be clearly stated which nucleotide was used in which experiment and whether GTP-γ-S-induced structural changes were visible in negative stain EM. Representative negative-stain images should be provided for both conditions. The experiments with G interface mutants in Figure 4C make only sense in the presence of GTP-γ-S, since no structural changes can be expected for these mutants in the absence of nucleotide.We would like to thank the reviewers for this nice suggestion. In the original Figures 1E and 4C, we did not add nucleotide in liposome tubulation experiments. As suggested by reviewers, we further utilized negative stain EM to examine the potential structural changes induced after the addition of GTP for various G domain mutants. The new results have been added into Figure 4C. For those mutants that lose GTP hydrolysis activity but keep the ability of GTP binding, we observed the tube expansion effect after adding GTP. This further confirms it is the GTP binding to trigger the assembly change of SOPA1 on membrane.Essential revisions:1) The assembly of the stalks in the nucleotide-free structure is still confusing. The new fittings, depiction of stalk interfaces and the schematic Figure 7E show that both the nucleotide-free and GTP-γ-S bound sOPA1 use interface-1 and 2 to form a filament. These interfaces are also used in Mgm1 and have been shown by mutagenesis to contribute to assembly. In contrast, the newly observed interface-3 is unique to the nucleotide-free form, is opposite of all previously known stalk interfaces, and due to missing mutagenesis, its significance for assembly remains unknown. To avoid confusion, the authors should use interface-1 and 2 to define the OPA1 filament also in the nucleotide-free form, e.g. in Figure 3A and E, Figure 3—figure supplement 2 and Figure 7E left. In Figure 7E left, not the blue and green dimers, but the blue striped and blue dimers should form the filament, as in Figure 7E right. In this case, I1 and I2 are present within a filament, and I3 is in between filaments. It would be helpful for the reader if one such turn is shown in a unique color in Figure 2A or Figure 3A (similar as it is now with the 'I3 filament') to indicate whether such filament is right- or left-handed. How do the G domains in this case connect? One might expect that the putative G domain interface is then in between different rungs.We appreciate the reviewers so much for this additional important suggestion. We tried to re-define S-OPA1 filament using the common interface-1 and 2 . With this new definition, as the reviewers expected, the G domain dimerization interface in this case is now in between different rungs . However, with this new definition, we found the filament becomes right-handed, which generates a new inconsistency with the left-handed assembly at GTP-γ-S bound state. Therefore, we would like to keep our original definition in the main figures but put this alternative one at the supplements.Besides, we agree with the reviewers that the newly observed interface-3 is unique and its significance for filament assembly is not validated due to lack of mutagenesis. However, based on the fitted model, we calculated the areas of those interfaces, which yielded 1324 Å2 for interface-1, 124 Å2 for interface-2, and 1599 Å2 for interface-3. The area of interface-3 is large enough to play an important role to stabilize the assembly of S-OPA1. Thus, although the future mutagenesis work is needed, the role of interface-3 should not be neglected. To be noted, the computed area of interface-2 might not be accurate due to the existence of unfitted density there .2) Figure 4C: The new data on the GTP-bound sOPA1 mutants are nicely contributing to the manuscript. However, tube diameters have to be quantified for the mutants in the absence and presence of GTP  to make a convincing claim. It also appears from Figure 1F that the representative tubes of the Δ196-252 variant are somehow smaller compared to sOPA1. Could the authors provide also some quantification of these?We would like to thank the reviewers very much for this good suggestion. The diameters of sOPA1 mutants coated tubes before and after adding GTP have been quantified with statistics , which is in consistency with our previous observation. The relevant text has been revised with this additional information.Upon the reviewers’ request, we also added the diameter statistics of Δ196-252 coated tube at the nucleotide-free state . As the reviewers observed, the averaged diameter of Δ196-252 coated tube is a bit smaller than the wild type.3) Nomenclature GMB domain: The fitted paddle is anything but a globular domain (unlike the previously assumed PH domain). Maybe 'Extended Membrane Binding Domain' (EMB) instead?Corrected as the suggestion.4) Figure 1D legend should contain the used GTP concentration. It appears from Figure 4B that the stimulation of wt is about 10-fold, not 70-fold?The legend of Figure 1 has been revised accordingly. For the fold of activity stimulation by adding liposome, the parameter Kcat was used in this study for comparison (see Supplementary file 1 and Supplementary file 1—source data 1). The apparent increase of the activity can be represented by Kcat/Km, which is about 10-fold as mentioned by the reviewers.


This paper investigates the time-domain performance of a switchable filter impulse radio ultra-wideband (IR-UWB) antenna for microwave breast imaging applications. A miniaturized CPW-fed integrated filter antenna with switchable performance in the range of the Worldwide Interoperability for Microwave Access (WiMAX) and Wireless Local Area Network (WLAN) bands could operate well within a 3.0 to 11 GHz frequency range. The time-domain performance of the filter antenna was investigated in comparison to that of the designed reference wideband antenna. By comparing both antennas’ time-domain characteristics, it was seen that the switchable filter antenna had good time-domain resolution along with the frequency-domain operation. Additionally, the time-domain investigation revealed that the switchable filter wide-band antenna performed similarly to the reference wide band antenna. This antenna was also utilized for a tumor detection application, and it was seen that the switchable filter wide-band antenna could detect a miniaturized irregularly shaped tumor easily, which is quite promising. Such an antenna with a good time-domain resolution and tumor detection capability will be a good candidate and will find potential applications in microwave breast imaging. Since 2002, the Federal Communication Commission (FCC) has permitted ultra-wideband (UWB) communication within the frequency range of 3.1–10.6 GHz, which has led to potential applications in the fields of microwave imaging, microwave sensors, surveillance systems, wireless vital sign monitoring, radio-frequency identification (RFID), tumor detection, wireless body area networks, etc. ,5. Due tIn microwave imaging applications, researchers have tried to develop techniques for breast cancer detection, since it is one of the diagnosed cancers in women with the highest chance of incidence. According to a report by the American Cancer Society (ACS), 25% of the cancer diagnoses that occur globally are related to breast cancer. Additionally, the survival rate is almost 80% to 90% at an early stage, which eventually falls to 24% in the corresponding advanced scenarios. Thus, early-stage detection and cost-effective techniques are some of the utmost requirements. Microwave imaging techniques for breast cancer detection have therefore been studied over the past years to meet such requirements .Since we know that the UWB spectrum falls within 3–11 GHz, however, we know that certain other standards fall within this spectrum. Such frequency bands create an unavoidable overlap among UWB communication systems and affect their corresponding applications for utilization such as microwave imaging applications. These frequency bands include WLANs operating at 5 to 6 GHz, with sub-standards of lower WLAN having frequencies ranging from 5.15 to 5.35 GHz and upper WLANs ranging from 5.725 to 5.825 GHz, WiMAX that works in the 3.30–3.60 GHz range, X-band for satellite communication functioning in the 7.15–7.60 GHz range, and the International Telecommunication Union (ITU 8-GHz) band functioning at 7.90–8.60 GHz ,11,12. HIt is well established that the corresponding notched band is generated by introducing slot resonators within the reference antenna such as complementary split-ring resonators, fractal-based different proposed resonators, etc. ,14,15. PAdditionally, different antennas have been tried and developed in this regard, to meet the time-domain requirements, but they only have a stationary notched band ,19,20. UThis paper is focused on the investigation of the time-domain performance of a switchable filter impulse radio ultra-wideband antenna for microwave breast imaging applications. A miniaturized CPW-fed integrated filter antenna with switchable performance in the range of WiMAX and WLAN bands is analyzed, and its corresponding response in the frequency domain is first provided for validation purposes only. The frequency-domain performance shows that the antenna can operate well within a 3.0 to 11 GHz frequency range, having a switchable notched band ranging from 3.3 to 6 GHz upon changing the capacitance of the integrated capacitor within the resonator. The time-domain performance of the filter antenna is investigated in comparison to that of the designed reference wideband antenna. By comparing both antennas, it is seen that the switchable filter antenna has good time-domain resolution along with the frequency domain operation. This antenna is also utilized for tumor detection applications, and it is seen that the switchable filter wide-band antenna can detect a miniaturized irregularly shaped tumor easily, which is quite promising.The arrangement of this manuscript is as follows: The first reference wide-band antenna was developed using a conventional UWB geometry as shown in Secondly, a switchable filter wide-band antenna was developed and optimized as shown in φ = 0°, while the corresponding receiving antenna was rotated at φ = 0°, 90°, 180°, and eventually, the S21 response was measured by means of a vector network analyzer (VNA) in each case, which was connected to the Tx and Rx antennas via radio frequency (RF) cables.A time-domain investigation of both the reference and switchable filter antennas was performed in an anechoic chamber as demonstrated in Various parameters from the frequency domain are presented as well to characterize the antenna performance in the corresponding time domain. The most important parameter that incorporates the frequency, time, and space together is the fidelity factor (FF), which was analyzed based on  and calcφ = 0°, 90°, and 180°. Similarly, the S21 characteristics of the switchable filter antenna were consistent with the reference antenna having a corresponding shift at higher frequencies, which is usual and arises due to differences in the gain of both antennas at higher frequencies and higher-order harmonics. However, the results are valid and acceptable for both antennas, and we can say that both possessed good performance in the time domain as well as in the frequency domain.The magnitude of the S21 of the reference and switchable filter antennas are shown in A is the amplitude factor, n represents the order, and φ = 0°, 90°, and 180°, and it is seen that the difference in each orientation is very small and acceptable. Good agreement is observed among the simulation and measurement results here as well. It must be noted here that the reference antenna will always have a good fidelity factor in comparison to the switchable antenna due to the integration of the filter structure within the reference antenna. However, still, the results show that the selected switchable filter antenna has good performance in the time domain and meets all the requirements. The fidelity factor was calculated from The effectiveness of the antenna was tested using a canonical breast model. The electromagnetic properties of the breast tissue and the tumor utilized here were defined as follows. The skin was modeled by using a conductivity g method ,27,28, tThe proposed imaging setup comprises two antennas, one for transmitting the UWB signal and another for receiving it to achieve a 2D scan in the  plane. For the reference antenna case, we used two similar reference antennas for transmitting and receiving, and for the switchable case, we used two similar switchable antennas for transmitting and receiving. The breast was compressed between these similar antennas in each scenario, and the antennas were made movable by scanning the breast completely. The steps involved in image formation were as follows: First, the phantom was placed between two antennas having no tumor, and the S-parameters were recorded in this case at each position and termed as We investigated the time-domain performance of a switchable filter impulse radio ultra-wideband (IR-UWB) antenna for microwave breast imaging applications. A miniaturized CPW-fed integrated filter antenna with switchable performance in the range of the WiMAX and WLAN bands was studied, and its potential applications were explored. The time-domain performance of the filter antenna was investigated in comparison to that of the designed reference wideband antenna, and it was shown that the switchable antenna had a good time-domain resolution. Both antennas were then utilized for a tumor detection application, and it was seen that the switchable filter wide-band antenna could detect a miniaturized irregularly shaped tumor easily, which is quite promising. This antenna will be one of the promising candidates for microwave breast imaging.


Flavobacterium johnsoniae, which encodes necessary proteins for complete enzymatic depolymerization of crystalline chitin, has recently been characterized but no detailed structural information on the enzymes was provided. Here we present protein structures of the F. johnsoniae chitobiase (FjGH20) and chitinase B (FjChiB). FjGH20 is a multi-domain enzyme with a helical domain not before observed in other chitobiases and a domain organization reminiscent of GH84 (β-N-acetylglucosaminidase) family members. The structure of FjChiB reveals that the protein lacks loops and regions associated with exo-acting activity in other chitinases and instead has a more solvent accessible substrate binding cleft, which is consistent with its endo-chitinase activity. Additionally, small angle X-ray scattering data were collected for the internal 70 kDa region that connects the N- and C-terminal chitinase domains of the unique 158 kDa multi-domain chitinase A (FjChiA). The resulting model of the molecular envelope supports bioinformatic predictions of the region comprising six domains, each with similarities to either Fn3-like or Ig-like domains. Taken together, the results provide insights into chitin utilization by F. johnsoniae and reveal structural diversity in bacterial chitin metabolism.Chitin is one of the most abundant renewable organic materials found on earth. The chitin utilization locus in  N-acetyl-d-glucosamine (GlcNAc) units, share many properties with cellulose; both polysaccharides are completely insoluble and coalesce into crystalline fibers, and their decomposition is highly challenging. Metabolism of chitin and cellulose proceeds through similar systems, where multiple enzymatic activities are required. Endo-acting enzymes cleave amorphous regions of the crystals, while exo-acting processive enzymes depolymerize the chains from either the reducing- or non-reducing ends. In addition, lytic polysaccharide monooxygenases (LPMOs) introduce chain breaks in crystalline regions, whereas β-glycosidases complete the hydrolysis by converting oligosaccharides into monosaccharides2. The chitinolytic machinery of the bacterium Serratia marcescens has served as a model system for enzymatic chitin turnover, and consists of five core enzymes: SmChiA and B (exo-acting processive chitinases), SmChiC (endo-acting non-processive chitinase), CBP21 (LPMO), and a chitobiase converting chitooligosaccharides (CHOs) into GlcNAc2.Chitin is as a key component of arthropod exoskeletons and fungal cell walls, and as such one of the most abundant renewable organic materials found on earth. The long chitin polysaccharides, consisting of β(1 → 4)-linked 3. PULs are discrete gene clusters, comprising all of the proteins needed to bind, transport and enzymatically deconstruct complex polysaccharides, and many PULs targeting both plant- and microbial glycans have been characterized in recent years6. No culturable Bacteroidete has yet conclusively been shown to deconstruct cellulose via the actions of a PUL7, but we recently identified and characterized a chitin utilization locus (ChiUL) from the aerobic soil bacterium Flavobacterium johnsoniae8. An atypical feature of the ChiUL compared to other characterized PULs from gut bacteria is that the main chitinase, FjChiA, is not attached to the outer membrane but is instead solubly secreted into the extracellular milieu by the Type IX secretion system10. This possibly reflects the fact that the environment of F. johnsoniae is less nutrient dense compared to the gastrointestinal tracts of animals and may require both protein secretion and cellular motility for efficient scavenging for resources11.Bacteria belonging to the Bacteroidetes phylum are well-known as efficient metabolizers of complex polysaccharides, and many of these species contain so-called polysaccharide utilization loci (PULs) which target distinct glycansF. johnsoniae ChiUL consists of 11 genes: two SusC/D-like pairs for oligosaccharide capture and transport, an inner membrane transporter, a two-component sensor/regulator pair, and four enzymes. While one of the enzymes of the locus, a predicted glucosamine-6-phosphate deaminase, proved impossible to produce by heterologous expression, the three remaining enzymes directly targeting chitin or CHOs were studied in detail. The large (~ 158 kDa) FjChiA enzyme is essential for efficient chitin depolymerization and growth on chitin10, and consists of two catalytic domains, FjChiA_N  and FjChiA_C , both from glycoside hydrolase family 18 (GH18) and sharing only ~ 20 to 30% sequence identity to previously characterized family members. FjChiA_N and FjChiA_C are connected by a ~ 70 kDa segment, FjChiA_M, which lacks significant similarity in primary structure to any known proteins but was shown to bind crystalline polysaccharides and may as such present a novel carbohydrate-binding module (CBM) motif8. The chitin depolymerization activity of the native full-length FjChiA was dramatically better than a combination of the two terminal catalytic domains, indicating the importance for the FjChiA_M domain in this multi-domain enzyme. The remaining GH18 chitinase of the locus, FjChiB (endo-acting), was active on chitin and CHOs, but to a much lesser extent compared to FjChiA. The fourth enzyme, the FjGH20 chitobiase/N-acetylglucosaminidase, was active only on CHOs.The https://www.cazy.org12), GH18 is a large family comprising over 20,000 members with close to 500 having been biochemically characterized to date. Of these, 93 enzymes are currently listed as structurally determined (April 2020). While this is a small subset compared to the family size, the number of solved structures provides deep insight into the enzymes’ structure–function relationships. The catalytic domains of GH18 enzymes consist of (β/α)8 barrels which are often appended by CBMs or other chitin-binding structures that are important for overall enzyme efficiency15. A notable feature present in some GH18 members is a region/domain inserted between the β-strand 7 and α-helix 7 of the barrel, referred to as the chitinase insertion domain (CID), which is proposed to aid in defining the processivity of the enzymes by forming a  tunnel over a bound polysaccharide chain. Structural information on GH20 enzymes is sparser, with 22 structures currently deposited to the protein data bank (PDB). While several GH20 enzymes have been shown to be multi-modular and the family as a whole contains a range of substrate specificities, the enzymes all comprise a core catalytic domain consisting of a (β/α)8 barrel that carries conserved catalytic residues20.In the carbohydrate-active enzymes database  envelope of the novel FjChiA_M domain. The protein structures reveal features consistent with their enzymatic activities and reveal a novel domain in FjGH20. The ab initio modelling of the FjChiA_M SAXS envelope indicated an elongated protein which, supported by bioinformatic analyses, is suggested to be comprised of distinct modules. Collectively, the results add to our understanding of the structure–function relationships of these important enzymes that are involved in the metabolism of the abundant polysaccharide chitin.In our previous work, we solved the structures of the surface-tethered SusD-like proteins CusDFjGH20 shares 25 to 38% sequence identity with previously characterized GH20 family members, but the overall amino acid sequence identity is as low as 15% compared to certain studied enzymes. Based on these data, we hypothesized that FjGH20 might also contain structural features or domains previously not seen in other family members. Thus, to illuminate these features we pursued structural determination of the FjGH20 by X-ray crystallography and were able to solve the protein structure from diffraction data extending to 1.70 Å resolution. The crystallographic asymmetric unit contained two protein molecules whose interaction gives rise to a buried surface area of ~ 11%, as determined by PISA21. Analysis of the protein in solution by size exclusion chromatography was consistent with a monomer unit being the prominent species (≥ 80%) and thus the dimer observed in the crystal structure is likely not of biological relevance. Each protomer consists of three domains: a central domain (residues 145–502) composed of a (β/α)8 TIM-barrel, an N-terminal domain (residues 1–144) comprising a six-stranded β-sheet and two α-helices where the helices are sandwiched between the β-sheet and the central domain, and a C-terminal domain (residues 504–673) consisting of an eight-helix bundle with two short β-strands  and Homo sapiens . The N-terminal domain of GH20 family members is ubiquitous and bears structural resemblance to certain CBMs, but a defined biological role for this domain remains undetermined. The catalytic site for the N-acetylhexosaminidase activity in these enzymes is found in the cleft of the TIM-barrel. Several of the active site residues shown to be important for substrate binding and catalysis in GH20 members are conserved in FjGH20, including the H-x-G-G-D-E motif where a glutamic acid (Glu317) is proposed to act as a general acid/base in the reaction. The aspartic acid in this motif (Asp316), along with a conserved tyrosine residue (Tyr413), are proposed to position and polarize the N-acetyl group for the substrate-assisted catalytic mechanism24. The FjGH20 substrate-binding pocket is distinct in one significant way compared to the archetypical GH20 from S. marcescens (SmGH20)25 in that FjGH20 lacks the extended loop that is located between β-strand 7 and α-helix 7 in SmGH20 , which is involved in sugar binding in the + 1 subsite26 of SmGH20. Interestingly, a portion of a loop extending from the FjGH20 helical bundle domain wraps around, and closes off, one side of the active site cleft in the TIM barrel with a tyrosine residue (Tyr538) that projects its sidechain into a position similar to the Trp685 residue in SmGH20. A similar arrangement occurs in the homologous human β-hexosaminidase, which also has a short loop between β-strand 7 and α-helix 7 of the TIM-barrel and has a tyrosine residue in a position similar to Tyr538 in FjGH20. Interestingly, in the human protein the tyrosine residue originates from a loop in a different protomer27. It seems unlikely that this different feature of the substrate-binding sites significantly affects overall activity since FjGH20, human β-hexosaminidase, and a GH20 enzyme from B. thetaiotaomicron, which lacks an analogous aromatic residue, maintain enzymatic activity toward their target substrates28.The FjGH20 C-terminal helical bundle is a distinct domain amongst structurally determined GH20 family members. The GH20 family is distantly related to GH84, which also contains enzymes with N‐acetyl‐β‐hexosaminidase activity and its members display a similar structural architecture, i.e. an N-terminal β-sheet domain and a (β/α)8 TIM-barrel catalytic domain containing GH20-like catalytic residues. Some GH84 members, such as GH84C (NagJ) from Clostridium perfringens30, contain a C-terminal helical bundle analogous to that observed in FjGH20 , the presence of an analogous C-terminal domain in FjGH20 to GH84 enzymes may suggest a closer relationship between the two GH families than has been previously suggested and/or could be a remnant of their common ancestry.The FjChiB to 1.63 Å resolution. The asymmetric unit contained one protein molecule without contacts indicative of oligomerization. The overall FjChiB structure is similar to other structurally determined GH18 chitinases, having a (β/α)8 TIM-barrel fold and containing a CID between strand 7 and helix 7 of the (β/α)8-barrel , ChiB only shares up to 30% sequence identity to enzymes with solved structures and its structure could therefore provide novel insights into the GH18 family as a whole. To investigate these features, we pursued structural determination by X-ray crystallography and were able to solve the structure of 2, is conserved in FjChiB  common amongst GH18 family members, which supports a substrate-assisted catalytic mechanismSmChiB, a small insertion between β-strand 1 and α-helix 1 leads to a capping of the cleft at the -3 site 14, which likely explains why this enzyme favors exo-binding (at the non-reducing end of a chitin chain), rather than endo-binding31. FjChiB lacks this insertion and instead shows more similarity to the GH18 ChtII from the insect pest Ostrinia furnacalis, which also lacks this insertion and has been shown to be able to bind longer oligosaccharides beyond the -3 site32. Like several GH18 chitinases, FjChiB has a CID inserted between strand 7 and helix 7 of the (β/α)8-barrel which folds into a distinct domain and builds up one face of the active site cleft. In SmChiB, this region is large and folds over one end of the cleft effectively forming a tunnel and shielding the − 1 and + 1 sites from the bulk solvent14. There is significant diversity amongst GH18 chitinase members in the CID region, both in length and sequence, and the CID in FjChiB, while similar in overall structure, is shorter than the one found in SmChiB, leading to a much more open cleft at the + 1 and + 2 sites  with carbohydrate-binding functionality, which lacks close similarity to any previously studied proteins8. Attempts to crystallize either the full-length protein or the middle domain of FjChiA were unsuccessful. However, thanks to high sequence similarity, reliable homology modeling of the FjChiA N- and C-terminal GH18 chitinase domains was possible using PHYRE233. The modelling resulted in high-confidence structure predictions of TIM-barrel proteins consistent with both FjChiA_N and FjChiA_C belonging to the GH18 family  to determine a solution structure of FjChiA_M. Unfortunately, the full-length protein suffered from both aggregation and radiation damage issues, even when utilizing SAXS measurements coupled to size exclusion chromatography, and the data could not be utilized for analysis. The FjChiA_M protein, however, proved to be much more amenable to the technique and allowed for the generation of a low-resolution model of the domain (Table r), indicated that the protein is modular and elongated with some degree of flexibility34  followed by four immunoglobulin (Ig)-like domains . Fn3-like and Ig-like domains are similar to each other, with both comprising seven to nine strands arranged into two β-sheets that pack onto each other35. However, Fn3-like domains tend to have shorter strands and longer intervening loop regions compared to Ig-like domains. The two FjChiA_M Fn3-like domains share 36% identity to each other and are both most closely related  to the Fn3-like domain of the pilin protein BcpA from Bacillus cereus (PDB accession 3kpt). The first two Ig-like domains share 75% sequence identity while the last two shares only 20 to 30% identity with each other and with the first two. Structure predictions of these last four domains are consistent with each comprising an Ig-like domain with each sharing between 20 to 30% sequence identity to Ig-like domains from the antifreeze protein (MpAFP) from Marinomonas primoryensis35. MpAFP is a large (1.5 MDa) protein comprised of > 100 tandem Ig-like domains that are proposed to extend and project the ice-binding domain of the protein away from the cell. Both Fn3-like and Ig-like domains are commonly found in extracellular carbohydrate-active enzymes and, while sometimes displaying weak carbohydrate-binding ability, it has been suggested that they can play a role in loosening and exfoliating chains from fibrous polysaccharides36. In SmChiA, an Fn3-like domain is connected to the catalytic domain in close proximity to the active site and may have a role in the interaction with polysaccharide substrates38, and it is possible that Ig-like modules could also have substrate interaction roles.The FjChiA_M domain is composed of distinct modules which likely fold similarly to Fn3-like and Ig-like domains. Further, the protein is observed as elongated in solution in a fashion similar to “beads on a string” where each bead may sit on crystalline chitin and exfoliate chains for the two terminal chitinase domains. Our previous biochemical characterization8 showed that the FjChiA_M domain adheres to several insoluble and crystalline polysaccharides including α- and β-chitin and cellulose. The elongated conformation of the protein may be a feature that is important not only for adhesion/exfoliation but also for tethering FjChiA_N and FjChiA_C together physically for increased cooperativity between the domains. A tentative model of the FjChiA solution structure together with the entire ChiUL machinery is presented in Fig. Collectively, the solution scattering results support the bioinformatic prediction that the F. johnsoniae ChiUL, including both carbohydrate-binding proteins and enzymes  using an isocratic gradient of 50 mM Tris pH 8.0 with 250 mM NaCl. Molecular weights were determined by standard curve using the Gel Filtration Markers Kit for Protein Molecular Weights 12–200 kDa (Sigma) as standards. Initial crystallization conditions were screened for using a Phoenix crystallization robot (Art Robbins Instruments) by the sitting-drop vapor-diffusion method. About 400 in-house made conditions were screened by using MRC plates with a 60 µl reservoir solution per well, and drop solutions were prepared by mixing 0.25 µl well solution and 0.25 µl protein solution at 20 mg/ml. Further optimization in Hampton 24-well hanging drop plates using 500 µl reservoirs and 1 µl + 1 µl drops, yielded crystals from 14% PEGMME 5 K and 0.1 M Na-Malonate pH 7.5. Diffraction data was collected at BL14.1 at Bessy (2016–04-16), and was integrated and scaled using XDS39 and AIMLESS41, respectively. The structure was solved by molecular replacement with Auto-Rickshaw43, with MoRDa44 identifying and using PDB accession 1now, human lysosomal beta-hexosaminidase45, as the template. An initial model was built using autobuilding in ARP/wARP49. Inspection of electron density maps was done in Coot50 with positional refinement in REFMAC51. The data collection, processing, and refinement statistics for all of the datasets can be found in Table The protein was produced and purified as previously reportedFjChiB was produced and purified as previously reported8 and stored in 50 mM Tris buffer at pH 8.0 containing 50 mM NaCl. Crystallization conditions were screened for with a Mosquito robot (SPT Labtech) using the JCSG + screening kit (Molecular Dimensions) in MRC sitting drop plates. A condition which yielded a crystal hit was optimized in sitting drop plates with a reservoir volume of 40 µl and protein mixed with reservoir solution in a 1:1 ratio in 0.6 µl drop sizes using a protein stock solution at 20 mg/mL. The optimized formulation contained 0.15 M magnesium formate with 15% PEG3550 and yielded cuboid crystals within a week. An initial dataset diffracting to 1.85 Å was collected at the ESRF id23-2 (2014–12-02) which was integrated and scaled using XDS39 and AIMLESS41, after which the structure was determined by molecular replacement with Auto-Rickshaw43, with MoRDa44 identifying and using PDB accession 3fnd, a putative chitinase from Bacteroides thetaiotaomicron, as the search template. An initial model was built using autobuilding in ARP/wARP49. A subsequent data set diffracting to 1.63 Å was collected at the ESRF id30a3 (2017-05-03), processed with XDS39, and the solution defined by rigid body refinement using Phenix Refine52 and the previously determined FjChiB structure. Since the new data set provided an improvement in resolution, only this dataset was pursued for further refinement and deposition. Coot50 and Phenix Refine52 were used in iterative cycles of manual and computational refinement. The data collection, processing, and refinement statistics for all of the datasets can be found in Table 8. X-ray scattering data were obtained at BL4-2 at the Stanford Synchrotron Radiation Lightsource (SSRL) with a Pilatus3 X 1 M detector (Dectris) operated at 11.0 keV. Full-length ChiA and ChiA_M were buffer exchanged into 50 mM Tris pH 8.0 with 250 mM NaCl and 250 μM DTT, using a HiPrep 26/10 Desalting column and an ÄKTA Explorer . The proteins were concentrated to 20 mg/mL by ultrafiltration using a Vivaspin  10 kDa molecular weight cut-off polyethersulfone spin column and a protein dilution series was created using the ultrafiltration filtrate. 10 images with 1 s exposure, taken at a distance of 1802.5 mm, were averaged and, after background subtraction of the buffer, were utilized for analysis in the ATSAS suite version 3.053. PRIMUS54 and GNOM55 were utilized to assess the data. The data for the full-length ChiA showed significant indications of aggregation and radiation damage and were not pursued further. Images of ChiA_M at concentrations of 1.25, 2.5, and 5.0 mg/mL yielded Guinier Rg estimates within 3% of each other with no indication of radiation damage and the data collected at 5 mg/mL were chosen for further analysis. DAMMIF56 was used to generate 100 models that were subsequently clustered by DAMCLUST57 and the top cluster was averaged by DAMAVER58 and then refined by DAMMIN59. The SAXS data collection and analysis parameters can be found in Table The protein was produced and purified as previously reportedSupplementary Information.


The Nav channel α subunit is known to function both with and without associated β subunits. There is increasing evidence that these β subunits have multiple roles that include not only influencing the voltage-dependent gating but also the ability to alter the spatial distribution of the pore-forming α subunit. Recent structural data has shown possible ways in which β1 subunits may interact with the α subunit. However, the position of the β1 subunit would not be compatible with a previous trimer structure of the β3 subunit. Furthermore, little is currently known about the dynamic behavior of the β subunits both as individual monomers and as higher order oligomers. Here, we use multiscale molecular dynamics simulations to assess the dynamics of the β3, and the closely related, β1 subunit. These findings reveal the spatio-temporal dynamics of β subunits and should provide a useful framework for interpreting future low-resolution experiments such as atomic force microscopy.Voltage-gated sodium (Na Additionally, β subunits also function independently as cell-adhesion molecules (CAMs)   and may  Ranvier  to promoin vivo, the effects of the β subunits are increasingly recognized and may well offer alternative therapeutic routes in the long run  show cardiac conduction problems  – like fold with a single transmembrane (TM) helix and unstructured intracellular domain, their binding to the α subunit differs. Both β2 and β4 bind covalently , via a dsubunits . Increassubunits  and can subunits .v channels with β subunits bound all contain β subunit density in this region. The first of the eukaryotic structures with a β subunit bound was that of Nav 1.4 from Electric Eel by v 1.4 was solved by v 1.7 α subunit, not only could the position of β1 be resolved, but also the position of β2 and various toxin molecules (The α subunit itself is constructed from four homologous domains (DI – DIV), each containing six TM helices that make up the voltage sensor domain  and pore-forming domain (helices 5 and 6). The exact location of where β subunits bind to the α subunit is uncertain, additionally the exact ratio of α:β subunit is also not very well characterized and may vary depending on tissue type and the cellular environment . Evidencolecules . These solecules . Interesolecules  althoughDespite the plethora of recent structural information, several aspects regarding the role of the β subunits remain unclear. What is the dynamic behavior of β subunit monomers? Do they oligomerize, and if so, how? How does the trimeric Ig domain structure of β3 relate to the position and orientation of the β subunits observed when in complex with the α subunits? To try and address these questions, we have used multiscale molecular dynamics simulations. We show that although β1 and β3 exhibit a relatively high sequence identity (51%), the behavior of the monomers is quite different, with β3 being more dynamic than β1. We attribute this to distinct residue – lipid contacts in the Ig domains of both subunits. We also demonstrate that the lipid composition is likely to have a key role in controlling the dynamical behavior.v 1.4-β1 complex  a large variety of conformations were visited. To construct the β3 trimer model a frame from the first run was taken at a pitch angle of 44.7° with the long axis of the Ig domain approximately perpendicular to the plane of the membrane. This was overlaid with each chain of the β3 crystal structure containing just the extracellular domain (ECD) (see section “β3 Trimer”). After the model was constructed it was checked for no steric clashes, of which there were none. All overlays were performed in PyMol .−1. All simulations were carried out in the NPT ensemble. The temperature and pressure were set to 300 K and 1 bar using the Nosé-Hoover thermostat  simulations were performed using GROMACS  2019 wit-rescale  temperat-rescale  were useAll simulations performed are summarized in Upper leaflet: POPC(40):POPE(10):Sph(15):GM3(10): CHOL(25)2(10): CHOL(25)Lower leaflet: POPC(10):POPE(40):POPS(15):PIP2, 1-palmitoyl-2-oleoyl-gly cero-3-phosphatidylinositol-4,5-bisphosphate.Where POPE, 1-palmitoyl-2-oleoyl-glycero-3-phosphatidy lethanolamine; Sph, sphingomyelin; GM3, monosialodihexosylg anglioside; CHOL, cholesterol; POPS, 1-palmitoyl-2-oleoyl-glycero-3-phosphatidylserine; PIPex,ey,ez as a reference . In order to calculate the PAs the center of mass was taken as the center of mass of secondary structures contained within the Ig domain . This was chosen to minimize any noise associated with flexible loop movement over the course of the simulation. The PAs p1, p2, and p3 were obtained via the diagonalization of the moment of inertia tensor, I.Assessment of the Ig domain’s favored orientation was achieved by calculating the principal axes (PAs) at each frame and measuring the Tait-Bryan angles using the standard basis U =  and Λ is a diagonal matrix of eigenvalues that correspond to the principal moments of inertia. At every frame, the first, second, and third principal axes were used to define a rotation matrix  and from this the Tait-Bryan angles computed. Using an intrinsic rotation formalism of ZYX the yaw, pitch, and roll angles were defined. In this study we focus on the pitch angle in relation to the Ig domain. All angle analysis was produced from in-house python scripts are available at https://github.com/bigginlab/protein_orientation.Where v α/β subunit complexes revealed the β1 Ig domain to adopt a conformation such that the long axis of the strands sits roughly parallel to the membrane surface  which, when bound to the α subunit, positions the TM helix approximately parallel to the bilayer normal. However, during simulations, both β1 and β3 TM helices adopt a significant tilt angle , leadingFurther analysis of the contacts made between β subunits and the membrane  suggesteIn contrast, the Ig domain of β3 exhibits fewer regions of high contact, as expected from the analysis shown in mut hereafter. A second system was also prepared with two mutations in the linker (K149E and K152E), in addition to ones applied in the Ig domain (β1 Igmut + linkermut). Finally, a system with only the linker mutated (β1 linkermut) was prepared to assess what impact the linker has on β1 Ig domain dynamics.At this point the extent of non-conserved residues was analyzed in both the β1 and β3 sequences, with a particular focus on charged residue differences in the Ig and linker domains between both subunits . There amut, β1 Igmut + linkermut, and β1 linkermut) reveal distinct dynamics  that β3 monomers could aggregate and form higher-order oligomers including dimers and trimers , we set Long, fibril-like structures were formed in all repeats, with the Ig domains often making tip-tip interactions in a manner reminiscent of the interactions between the DIP and Dpr neuronal recognition proteins . Protein2 and cholesterol where the latter two interact with the TMD.We also investigated protein – lipid contact sites. Interactions were counted using the headgroup bead of each lipid type and a cut-off value of 6.5 Å. It can be seen  that theAlthough similar in sequence and underlying fold, the behaviors of the β1 and β3 subunits in the membrane exhibit some striking differences. Our simulations suggest that the ECD of the β3 subunit is much more dynamic than β1. In contrast to the β1 subunit structure from Pan et al. , the Ig In both the β1 and β3 models there is a slight shortening of the TMD as well as the large helical tilt in the membrane. In the cryo-EM structure , this reA major difference between monomeric and trimeric β3 lipid interactions is in the Ig domain. As part of a trimer, the Ig domain is no longer able to sample large pitch states due to the favorable hydrophobic interactions in the N-terminus of each chain. As such, the lipid interaction between the Ig trimer is markedly reduced when compared to the monomer with only a few charged residues  interacting with the membrane surface. Interestingly if the β3 subunit were to interact with the VSDs of the pore-forming α subunit in a similar fashion to β1 there would need to be substantial rearrangement of the Ig domains. This leads to the question of what, if any, the role of the TMD helices could play in α - β and/or β - β interactions? Lipid contact analysis in the TMD reveals that there is little difference between the trimeric and monomeric models. Close to the Ig domain, the restriction imposed via the stable Ig trimer reduces translational motion, whereas at the intracellular end the translational motion is much more dynamic with no clear preference for residue – residue interaction between chains. These results suggest that the TMD of the β3 subunit does not have an overall stabilizing effect on the β3 trimer and in fact may only be required for correct positioning within the membrane. This is in agreement with previous super-resolution microscopy data, where the density function estimated from the C-terminal mEos2-tagged β3 was consistent with a relatively unconstrained transmembrane helix/C terminus. This suggests that any trimerization events are likely to be controlled via the Ig domain. Additionally, when the helices of each β3 chain are in close proximity, the conserved E176 residue appears to be orientated away from the trimer center and preferentially interacts with the POPC membrane (data not shown). These observations are also supported by recent experimental work examining the role of E176 in β3 subunits, and that also concluded that oligomerization was dependent on the extracellular domain but not E176 .It has been previously reported  via the In this work, we have explored the dynamics of the β1 and β3 subunit monomers with a lipid bilayer. The dynamics exhibited a remarkable and unexpected difference in behavior of the ECD, which we attribute to distinct binding patterns within the Ig domain. It will be interesting to investigate the influence of the non-conserved charged residues between both subunits in future experiments. A full-length model of a β3 subunit based on a trimeric structure of the Ig domain only, suggests that the TM helices do not interact particularly strongly. Finally, the CG simulations suggest that higher order oligomerization of monomers may be mediated by “end-on-end” interactions. These results should provide a useful framework on which to interpret low-resolution methods such as AFM that are examining the nature of oligomerization in ion channels. The existing agreement between experiment and simulation is encouraging.All datasets generated for this study can be obtained via request to the corresponding author.WG performed and analyzed all simulations and co-wrote the manuscript. AD performed analysis and gave advice. PB conceived the work and co-wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.