Biology subjects

Wakatsuki, S.

Publications and source records attributed to Wakatsuki, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Large-scale dimensional behavioral profiling dissociates fear memory from locomotor confounds in mice: The necessity of baseline-normalized metrics

Fear conditioning is widely used to assess associative memory in mice, yet percent freezing conflates memory with baseline locomotor and anxiety-related traits. A systematic survey of recent studies (2020-2025) found that fewer than 1% statistically integrate locomotor activity into freezing analyses. Here, we address this gap using a large-scale dataset of >10,000 mice across >160 comparisons, including genetic mutations, pharmacological interventions and aging, tested in 15 standardized behavioral paradigms. Conventional freezing scores covaried strongly with general locomotor activity, obscuring memory-related phenotypes. Multiple factor analysis identified two principal behavioral dimensions, locomotor activity and learning/memory: conventional freezing aligned with the locomotor dimension, whereas freezing subtraction and the activity suppression ratio mapped onto the memory dimension and improved detection of synaptic plasticity phenotypes. These analyses show that baseline locomotor normalization is essential for interpreting fear conditioning as a memory assay and provide an open framework for selecting and reporting locomotor-normalized metrics.

neuroscience↗

bioRxiv2025

A Novel Unorthodox Dimeric Primary Enoyl-CoA Reductase Structure

Enoyl-CoA carboxylases/reductases (ECRs) are enzymes with the fastest carbon dioxide (CO2) fixation capabilities, yet the precise mechanisms behind their assembly and catalytic activity are structurally not yet fully understood. Here, we employed cryo X-ray crystallography to reveal the dimeric structural organization of a novel ECR, isolated from mesophilic Mesorhizobium metallidurans (M. metallidurans). We examined the interactions in silico and compared oligomerization of our dimeric ECR from M. metallidurans (ECRMm_Dim) with tetrameric ECR from Burkholderia ambifaria (ECRBa_Tet)by using size exclusion chromatography in solution. Our in silico analysis revealed that specific residues in the M. metallidurans ECR that preclude tetramer formation, which could affect the enzymes catalytic activity. Additionally, we compared primary ECR sequences and structural variations between K. setae and M. metallidurans to explore their evolutionary relationships, along with their functional diversity. Our study presents the first example of a dimeric ECR structure which may provide new insights into how dimerization versus tetramerization may have an impact on catalytic function. By detailing how different oligomeric states influence enzyme activity and exploring active site conformational changes, we may offer a further understanding of ECR assembly. This work paves the way for future research into the precise molecular mechanisms that drive ECRs exceptional overall catalytic activity, efficiency and efficacy.

biochemistry↗

bioRxiv2025

Crystal Structure of 3-Hydroxypropionyl-CoA Synthetase (ADP-forming) from Nitrosopumilus maritimus

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle in thaumarchaeota contributes significantly to global organic carbon fixation as the most energetically efficient aerobic carbon fixation pathway. The thaumarchaeal 3-Hydroxypropionyl-CoA Synthetase (ADP-forming; Nmar_1309) is crucial to this efficiency, utilizing ATP to ADP catalysis. This first reported structure of Nmar_1309 reveals a homodimer with a unique subdomain organization ([3-4-1-2-5]) and a distinct linker between subdomains 4 and 1. The presence of bound substrates including 3-hydroxypropionate, non-hydrolyzable ATP (ADPNP), and a phosphate suggests an intermediate state mimicking a reaction step immediately preceding the formation of a 3-hydroxypropionyl-phosphohistidine. Conformational differences were observed between the two chains of the homodimer, likely influenced by the binding of a single ADPNP molecule in one chain. Phylogenetic analysis suggests that while 4HB synthetases may have evolved earlier in the evolutionary timeline, 3HP synthetases in Thaumarchaeota may have occurred after the Great Oxygenation Event. These structural data provide further characterization of the 3HP/4HB cycle and, in conjunction with the structure of 4-hydroxybutyryl-CoA synthetase, Nmar_0206, provide baseline structures of the key ADP-forming Acyl-CoA synthetases within this pathway.

biophysics↗

bioRxiv2025

Deciphering the altered conformational states of bifunctional thaumarchaeal crotonyl-CoA hydratase and 3-hydroxypropionyl-CoA dehydratase from Nitrosopumilus maritimus

The thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle represents one of the most efficient mechanisms for CO2 fixation discovered to date. Within this cycle, the enzyme encoded by Nmar_1308 from Nitrosopumilus maritimus SCM1 plays a crucial role due to its dual functionality as both a crotonyl-CoA hydratase (CCAH) and a 3-hydroxypropionyl-CoA dehydratase (3HPD). Although the importance of a bifunctional enzyme for lowering the cost of biosynthesis, the details of structural dynamics are still missing. Here, in addition to our cryogenic temperature structures, we determined the first ambient temperature structures of the Nmar_1308 protein by Serial Femtosecond X-ray Crystallography (SFX). The determined structures capture previously unobserved conformational dynamics of the Nmar_1308 protein, providing invaluable information for future synthetic biology applications.

biochemistry↗

bioRxiv2025

Magnetic resonance control of reaction yields through genetically-encoded protein:flavin spin-correlated radicals in a live animal

Radio-frequency (RF) magnetic fields can influence reactions involving spin-correlated radical pairs. This provides a mechanism by which RF fields can influence living systems at the biomolecular level. Here we report the modification of the emission of various red fluorescent proteins (RFPs), in the presence of a flavin cofactor, induced by a combination of static and RF magnetic fields. Resonance features in the protein fluorescence intensity were observed near the electron spin resonance frequency at the corresponding static magnetic field strength. This effect was measured at room temperature both in vitro and in the nematode C. elegans, genetically modified to express the RFP mScarlet. These observations suggest that the magnetic field effects measured in RFP-flavin systems are due to quantum-correlated radical pairs. Our experiments demonstrate that RF magnetic fields can influence dynamics of reactions involving RFPs in biologically relevant conditions, and even within a living animal. These results have implications for the development of a new class of genetic tools based on RF manipulation of genetically-encoded quantum systems.

biophysics↗

bioRxiv2025

Combining MicroED and native mass spectrometry for structural discovery of enzyme-biosynthetic inhibitor complexes

With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach resolves structures of the epoxide-based cysteine protease inhibitor, and natural product, E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. The combined structural power of MicroED and the analytical capabilities of native mass spectrometry (ED-MS) allows assignment of papain structures bound to E-64-like ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors, and crude biosynthetic reactions. ED-MS further discriminates the highest-affinity ligand soaked into microcrystals from a broad inhibitor cocktail, and identifies multiple similarly high-affinity ligands soaked into microcrystals simultaneously. This extends to libraries of printed ligands dispensed directly onto TEM grids and later soaked with papain microcrystal slurries. ED-MS identifies papain binding to its preferred natural products, by showing that two analogues of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analogue from crude biosynthetic reactions, without purification. This illustrates the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets.

biophysics↗

bioRxiv2023

4D Crystallography Captures Transient IF1-Ribosome Dynamics in Translation Initiation

Initiation factor 1 (IF1) is one of multiple key ligands involved in the initiation of mRNA translation, a highly dynamic and carefully-orchestrated process. However, details surrounding IF1 transient interactions with the small 30S ribosomal subunit remain incompletely understood despite characterization of unbound and fully-bound 30S states. Improvements in X-ray light sources and crystallographic techniques are now enabling time-resolved structural studies at near-physiological temperature and near-atomic resolution and thus the structural investigation of such dynamic processes. Here, we employed time-resolved serial femtosecond X-ray crystallography (TR-SFX) to probe the binding of IF1 to the small 30S ribosomal subunit in real time. Our time-resolved structural data demonstrates transient cryptic short-, mid-, and long-range allostery among different regions of the small 30 ribosomal subunit during IF1 binding, revealing small- and large-scale protein-target interactions and dynamics within intermediate macromolecular states at unprecedented temporal and spatial resolution. These data represent one of the first such 4D crystallographic studies assessing protein-protein and protein-RNA interactions and could serve as the basis for subsequent studies of the ribosome and of the multitudinous dynamic processes which underpin biology, and therefore, of life.

biophysics↗

bioRxiv2023

Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-EM structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 [A] resolution (extending to 2.2 [A] at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 [A] resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the elevator-with-an-operator mechanism of TRAP transporters.

biochemistry↗

bioRxiv2023

Crystal Structure of 4-Hydroxybutyryl-CoA Synthetase (ADP-forming): A Key Enzyme in the Thaumarchaeal Hydroxypropionate/Hydroxybutyrate cycle

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle from ammonia-oxidizing Thaumarchaeota is currently considered the most energy-efficient aerobic carbon fixation pathway. The Nitrosopumilus maritimus 4-hydroxybutyryl-CoA synthetase (ADP-forming; Nmar_0206) represents one of several enzymes from this cycle that exhibit increased efficiency over crenarchaeal counterparts. This enzyme reduces energy requirements on the cell, reflecting thaumarchaeal success in adapting to low-nutrient environments. Here we show the structure of Nmar_0206 from Nitrosopumilus maritimus SCM1, which reveals a highly conserved interdomain linker loop between the CoA-binding and ATP-grasp domains. Phylogenetic analysis suggests the widespread prevalence of this loop and highlights both its underrepresentation within the PDB and structural importance within the (ATP-forming) acyl- CoA synthetase (ACD) superfamily. This linker is shown to have a possible influence on conserved interface interactions between domains, thereby influencing homodimer stability. These results provide a structural basis for the energy efficiency of this key enzyme in the modified 3HP/4HB cycle of Thaumarchaeota.

biophysics↗

bioRxiv2022

Structure and mechanism of the tripartite ATP-independent periplasmic (TRAP) transporter

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential carboxylate- and sulfonate-containing nutrients into the cytoplasm. Unlike other secondary active transporters, TRAP transporters cannot receive their substrates directly, but do so indirectly via a secreted soluble substrate-binding protein. How a sodium-driven secondary active transporter is strictly coupled to a passenger-carrying substrate-binding domain is poorly understood. Here, we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 [A] resolution. SiaM has 12-TMs that come together to form a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium-coupled elevator transporter VcINDY. Interestingly, the SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, indicating TRAP transporters may operate as monomers, rather than the typically observed oligomers. We have identified the Na+ and sialic acid binding sites in SiaM and confirmed a strict dependence on the substrate-binding protein SiaP for uptake. We have determined the SiaP crystal structure that, together with co-evolution driven docking studies, provides a molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We conclude that TRAP proteins are conceptually a marriage between an ABC importer and a secondary active transporter, which we describe herein as an elevator-with-an-operator mechanism.

molecular biology↗

bioRxiv2021

Structural and functional characterization of NEMO cleavage by SARS-CoV-2 3CLpro

In addition to its essential role in viral polyprotein processing, the SARS-CoV-2 3C-like (3CLpro) protease can cleave human immune signaling proteins, like NF-{kappa}B Essential Modulator (NEMO) and deregulate the host immune response. Here, in vitro assays show that SARS-CoV-2 3CLpro cleaves NEMO with fine-tuned efficiency. Analysis of the 2.14 [A] resolution crystal structure of 3CLpro C145S bound to NEMO226-235 reveals subsites that tolerate a range of viral and host substrates through main chain hydrogen bonds while also enforcing specificity using side chain hydrogen bonds and hydrophobic contacts. Machine learning- and physics-based computational methods predict that variation in key binding residues of 3CLpro- NEMO helps explain the high fitness of SARS-CoV-2 in humans. We posit that cleavage of NEMO is an important piece of information to be accounted for in the pathology of COVID-19.

molecular biology↗

bioRxiv2021

Structural Insights into Bifunctional Thaumarchaeal Crotonyl-CoA Hydratase and 3-Hydroxypropionyl-CoA Dehydratase from Nitrosopumilus maritimus

The ammonia-oxidizing thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle is one of the most energy-efficient CO2 fixation cycles discovered thus far. The protein encoded by Nmar_1308 (from Nitrosopumilus maritimus SCM1) is a promiscuous enzyme that catalyzes two essential reactions within the thaumarchaeal 3HP/4HB cycle, functioning as both a crotonyl-CoA hydratase (CCAH) and 3- hydroxypropionyl-CoA dehydratase (3HPD). In performing both hydratase and dehydratase activities, Nmar_1308 reduces the total number of enzymes necessary for CO2 fixation in Thaumarchaeota, reducing the overall cost for biosynthesis. Here, we present the first high-resolution crystal structure of this bifunctional enzyme with key catalytic residues in the thaumarchaeal 3HP/4HB pathway.

biochemistry↗

bioRxiv2020

Structural adaptation of oxygen tolerance in 4-hydroxybutyrl-CoA dehydratase, a key enzyme of archaeal carbon fixation

Autotrophic microorganisms that convert inorganic carbon into organic matter were key players in the evolution of life on Earth. As the early atmosphere became oxygenated, microorganisms needed to develop mechanisms for oxygen protection, especially those relying on enzymes containing oxygen-sensitive metal clusters (e.g., Fe-S). Here we investigated how 4-hydroxybutyryl-CoA dehydratase (4HBD) - the key enzyme of the 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle for CO2-fixation - adapted as conditions shifted from anoxic to oxic. 4HBD is found in both anaerobic bacteria and aerobic ammonia-oxidizing archaea (AOA). The oxygen-sensitive bacterial 4HBD and oxygen-tolerant archaeal 4HBD share 59 % amino acid identity. To examine the structural basis of oxygen tolerance in archaeal 4HBD, we determined the atomic resolution structure of the enzyme. Two tunnels providing access to the canonical [4Fe-4S] cluster in oxygen-sensitive bacterial 4HBD were closed with four conserved mutations found in all aerobic AOA and other archaea. Further biochemical experiments and molecular dynamics simulations support our findings that restricting access to the active site is the key to oxygen tolerance, explaining how active site evolution drove a major evolutionary transition. Significance statementAutotrophy (primary production) was the first life strategy on Earth. Before photosynthesis (using solar energy to fix carbon dioxide), life relied on chemical reactions for energy. These chemosynthetic reactions are present in all domains of life, including archaea possessing the most energy-efficient carbon fixation pathway - the 3-hydroxypropionate/4-hydroxybutyrate cycle. This efficiency results from enzyme modifications, including enhanced enzyme stability and catalysis of multiple reactions. We reveal the first structure of aerobic 4-hydroxybutyryl-CoA dehydratase (4HBD) from ammonia-oxidizing archaea. These archaea are among the most abundant organisms on the planet, and their 4HBD active site evolved oxygen tolerance to support aerobic metabolism. This modification can provide further insight into enzyme evolution on early earth, as photosynthesis developed and began oxygenating the atmosphere.

evolutionary biology↗

bioRxiv2019

Deconstructing the structural conservation of distantly related bacterial nucleoid-associated proteins using functional chimeras

Nucleoid-associated proteins (NAPs) are DNA-binding proteins critical for the organization and function of the bacterial chromosome. A subclass of NAPs, including Caulobacter crescentus GapR and Escherichia coli H-NS, preferentially bind AT-rich regions of the nucleoid, but phylogenetic groups that encode GapR rarely encode H-NS. Here, utilizing genetic, biochemical, and biophysical studies of GapR in light of a recent DNA-bound crystal structure of GapR (Guo et al, 2018), we show that although evolutionarily distant, GapR and H-NS possess two regions that are structurally and functionally conserved. These regions are involved in self-association and DNA-binding, even though the two proteins oligomerize and regulate transcription differently. Functional analysis of GapR and H-NS protein chimeras identified structural elements present in H-NS but absent in GapR that rationalize differences in transcriptional regulation. In addition, we identified a sequence element unique to GapR that enables assembly into its tetrameric state. Using fluid-atomic force microscopy, we showed that GapR is capable of bridging DNA molecules in vitro. Together, these results demonstrate that two distantly related NAPs utilize evolutionarily conserved structural elements to serve specialized cellular roles via distinct mechanisms.

molecular biology↗

bioRxiv2019

A Bacterial Surface Layer Protein Exploits Multi-step Crystallization for Rapid Self-assembly

Surface layers (S-layers) are crystalline protein coats surrounding microbial cells. S-layer proteins (SLPs) regulate their extracellular self-assembly by crystallizing when exposed to an environmental trigger. However, molecular mechanisms governing rapid protein crystallization in vivo or in vitro are largely unknown. Here, we demonstrate that the C. crescentus SLP readily crystallizes into sheets in vitro via a calcium-triggered multi-step assembly pathway. This pathway involves two domains serving distinct functions in assembly. The C-terminal crystallization domain forms the physiological 2D crystal lattice, but full-length protein crystallizes multiple orders of magnitude faster due to the N-terminal nucleation domain. Observing crystallization using time-resolved electron cryo-microscopy (Cryo-EM) reveals a crystalline intermediate wherein N-terminal nucleation domains exhibit motional dynamics with respect to rigid lattice-forming crystallization domains. Dynamic flexibility between the two domains rationalizes efficient S-layer crystal nucleation on the curved cellular surface. Rate enhancement of protein crystallization by a discrete nucleation domain may enable engineering of kinetically controllable self-assembling 2D macromolecular nanomaterials.\n\nSignificance StatementMany microbes assemble a crystalline protein layer on their outer surface as an additional barrier and communication platform between the cell and its environment. Surface layer proteins efficiently crystallize to continuously coat the cell and this trait has been utilized to design functional macromolecular nanomaterials. Here, we report that rapid crystallization of a bacterial surface layer protein occurs through a multi-step pathway involving a crystalline intermediate. Upon calcium-binding, sequential changes occur in the structure and arrangement of the protein, which are captured by time-resolved small angle x-ray scattering and transmission electron cryo-microscopy. We demonstrate that a specific domain is responsible for enhancing the rate of self-assembly, unveiling possible evolutionary mechanisms to enhance the kinetics of 2D protein crystallization in vivo.

biophysics↗

bioRxiv2019

Coupled inter-subunit dynamics enable the fastest CO2-fixation by reductive carboxylases

Enoyl-CoA carboxylases/reductases (ECRs) belong to the most efficient CO2-fixing enzymes described to date. However, the molecular mechanisms underlying ECRs extraordinary catalytic activity on the level of the protein assembly remain elusive. Here we used a combination of ambient temperature X-ray Free Electron Laser (XFEL) and cryogenic synchrotron experiments to study the structural organization of the ECR from Kitasatospora setae. K. setae ECR is a homo-tetramer that differentiates into a dimer of dimers of open- and closed-form subunits in the catalytically active state. Using molecular dynamics simulations and structure-based mutagenesis, we show that catalysis is synchronized in K. setae ECR across the pair of two dimers. This conformational coupling of catalytic domains is conferred by individual amino acids to achieve high CO2-fixation rates. Our results provide unprecedented insights into the dynamic organization and synchronized inter- and intra-subunit communications of this remarkably efficient CO2-fixing enzyme during catalysis. Significance StatementFixation of CO2 offers real potential for reaching negative CO2 emissions in bioenergy, and bioproduct utilization. The capture and conversion of atmospheric CO2 remains a challenging task. Existing biological systems can be exploited and optimized for this use. Bacterial enoyl-CoA carboxylases/reductases (ECRs) encompass the fastest CO2-fixing enzymes found in nature to date. However, the mechanisms underlying ECRs extraordinary catalytic activity remain elusive. Our structural, computational, and biochemical results elucidate the dynamic structural organization of the ECR complex and describe how coupled motions of catalytic domains in the ECR tetramer drive carboxylation. This mechanistic understanding is critical for engineering highly efficient CO2-fixing biocatalysts for bioenergy and bioproduct applications.

biophysics↗

bioRxiv2019

Serial femtosecond X-ray diffraction of HIV-1 Gag MA-IP6 microcrystals at ambient temperature

The Human immunodeficiency virus-1 (HIV-1) matrix (MA) domain is involved in the highly regulated assembly process of the virus particles that occur at the host cells plasma membrane. High-resolution structures of the MA domain determined using cryo X- ray crystallography have provided initial insights into the possible steps in the viral assembly process. However, these structural studies have relied on large and frozen crystals in order to reduce radiation damage caused by the intense X-rays. Here, we report the first XFEL study of the HIV-1 MA domains interaction with inositol hexaphosphate (IP6), a phospholipid headgroup mimic. We also describe the purification, characterization and microcrystallization of two MA crystal forms obtained in the presence of IP6. In addition, we describe the capabilities of serial femtosecond X-ray crystallography (SFX) using X-ray free-electron laser (XFEL) to elucidate the diffraction data of MA-IP6 complex microcrystals in liquid suspension at ambient temperature. Two different microcrystal forms of MA-IP6 complex both diffracted to beyond 3.5 [A] resolution, demonstrating the feasibility of using SFX to study the complexes of MA domain of HIV-1 Gag polyprotein with IP6 at near-physiological temperatures. Further optimization of the experimental and data analysis procedures will lead to better understanding of the MA domain of HIV-1 Gag and IP6 interaction at high resolution and provide basis for optimization of the lead compounds for efficient inhibition of the Gag protein recruitment to the plasma membrane prior to virion formation.

biophysics↗

bioRxiv2019

Continuous, Topologically Guided Protein Crystallization Controls Bacterial Surface Layer Self-Assembly

Bacteria assemble the cell envelope using localized enzymes to account for growth and division of a topologically complicated surface1-3. However, a regulatory pathway has not been identified for assembly and maintenance of the surface layer (S-layer), a 2D crystalline protein coat surrounding the curved 3D surface of a variety of bacteria4,5. By specifically labeling, imaging, and tracking native and purified RsaA, the S-layer protein (SLP) from C. crescentus, we show that protein self-assembly alone is sufficient to assemble and maintain the S-layer in vivo. By monitoring the location of newly produced S-layer on the surface of living bacteria, we find that S-layer assembly occurs independently of the site of RsaA secretion and that localized production of new cell wall surface area alone is insufficient to explain S-layer assembly patterns. When the cell surface is devoid of a pre-existing S-layer, the location of S-layer assembly depends on the nucleation characteristics of SLP crystals, which grow by capturing RsaA molecules freely diffusing on the outer bacterial surface. Based on these observations, we propose a model of S-layer assembly whereby RsaA monomers are secreted randomly and diffuse on the lipopolysaccharide (LPS) outer membrane until incorporated into growing 2D S-layer crystals. The complicated topology of the cell surface enables formation of defects, gaps, and grain boundaries within the S-layer lattice, thereby guiding the location of S-layer assembly without enzymatic assistance. This unsupervised mechanism poses unique challenges and advantages for designing treatments targeting cell surface structures or utilizing S-layers as self-assembling macromolecular nanomaterials. As an evolutionary driver, 2D protein self-assembly rationalizes the exceptional S-layer subunit sequence and species diversity6.

biophysics↗