Cryo-EM Structure of Phospholipase Cε Defines N-terminal Domains and their Roles in Activity.

Phospholipase Cε (PLCε) increases intracellular Ca 2+ and protein kinase C (PKC) activity in the cardiovascular system in response to stimulation of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). The ability of PLCε to respond to these diverse inputs is due, in part, to multiple, conformationally dynamic regulatory domains. However, this heterogeneity has also limited structural studies of the lipase to either individual domains or its catalytic core. Here, we report the 3.9 Šreconstruction of the largest fragment of PLCε to date in complex with an antigen binding fragment (Fab). The structure reveals that PLCε contains a pleckstrin homology (PH) domain and four tandem EF hands, including subfamily-specific insertions and intramolecular interactions with the catalytic core. The structure, together with a model of the holoenzyme, suggest that part of the N-terminus and PH domain form a continuous surface that could engage cytoplasmic leaflets of the plasma and perinuclear membranes, contributing to activity. Functional characterization of this surface confirm it is critical for maximum basal and G protein-stimulated activities. This study provides new insights into the autoinhibited, basal conformation of PLCε and the first mechanistic insights into how it engages cellular membranes for activity.

Engineered Protein-G variants for plug-and-play applications.

We have developed a portfolio of antibody-based modules that can be prefabricated as standalone units and snapped together in plug-and-play fashion to create uniquely powerful multifunctional assemblies. The basic building blocks are derived from multiple pairs of native and modified Fab scaffolds and protein G (PG) variants engineered by phage display to introduce high pair-wise specificity. The variety of possible Fab-PG pairings provides a highly orthogonal system that can be exploited to perform challenging cell biology operations in a straightforward manner. The simplest manifestation allows multiplexed antigen detection using PG variants fused to fluorescently labeled SNAP-tags. Moreover, Fabs can be readily attached to a PG-Fc dimer module which acts as the core unit to produce plug-and-play IgG-like assemblies, and the utility can be further expanded to produce bispecific analogs using the "knobs into holes" strategy. These core PG-Fc dimer modules can be made and stored in bulk to produce off-the-shelf customized IgG entities in minutes, not days or weeks by just adding a Fab with the desired antigen specificity. In another application, the bispecific modalities form the building block for fabricating potent Bispecific T-cell Engagers (BiTEs), demonstrating their efficacy in cancer cell-killing assays. Additionally, the system can be adapted to include commercial antibodies as building blocks, greatly increasing the target space. Crystal structure analysis reveals that a few strategically positioned interactions engender the specificity between the Fab-PG variant pairs, requiring minimal changes to match the scaffolds for different possible combinations. This plug-and-play platform offers a user-friendly and versatile approach to enhance the functionality of antibody-based reagents in cell biology research.

Cryo-EM Structures of Clostridium perfringens Enterotoxin Bound to its Human Receptor, Claudin-4.

Pathogenic strains of Clostridium perfringens secrete an enterotoxin (CpE) that causes prevalent, severe, and sometimes deadly gastrointestinal disorders in humans and domesticated animals. CpE binds selectively to membrane protein receptors called claudins on the apical surfaces of small intestinal epithelium. Claudins normally construct tight junctions that regulate epithelial paracellular transport but are hijacked from doing so by CpE and are instead led to form claudin/CpE small complexes. Small complexes are building blocks for assembling oligomeric ß-barrel pores that penetrate the plasma membrane and induce gut cytotoxicity. Here we present structures of CpE in complexes with its native claudin receptor in humans, claudin-4, at 4.0 and 2.8 Å using cryogenic electron microscopy. The structures reveal the overall architecture of the small complex, that the small complex can be kinetically trapped, and resolve its key features; like the residues used in claudin/CpE complex binding, the orientation of CpE relative to the membrane, and CpE-induced structural changes to claudin-4. Further, the structures allude to the biophysical procession from small complex to cytotoxic ß-barrel pore used by CpE during pathogenesis and the role of trypsin in this process. In full, this work elucidates the structure and mechanism of claudin-bound CpE pore assembly and provides strategies to obstruct its formation to treat CpE-induced gastrointestinal diseases.

Structural Insights into the Roles of PARP4 and NAD + in the Human Vault Cage.

Vault is a massive ribonucleoprotein complex found across Eukaryota. The major vault protein (MVP) oligomerizes into an ovular cage, which contains several minor vault components (MVCs) and is thought to transport transiently bound "cargo" molecules. Vertebrate vaults house a poly (ADP-ribose) polymerase (known as PARP4 in humans), which is the only MVC with known enzymatic activity. Despite being discovered decades ago, the molecular basis for PARP4's interaction with MVP remains unclear. In this study, we determined the structure of the human vault cage in complex with PARP4 and its enzymatic substrate NAD + . The structures reveal atomic-level details of the protein-binding interface, as well as unexpected NAD + -binding pockets within the interior of the vault cage. In addition, proteomics data show that human vaults purified from wild-type and PARP4-depleted cells interact with distinct subsets of proteins. Our results thereby support a model in which PARP4's specific incorporation into the vault cage helps to regulate vault's selection of cargo and its subcellular localization. Further, PARP4's proximity to MVP's NAD + -binding sites could support its enzymatic function within the vault.

Development of Luminescent Biosensors for Calprotectin.

Calprotectin, a metal ion-binding protein complex, plays a crucial role in the innate immune system and has gained prominence as a biomarker for various intestinal and systemic inflammatory and infectious diseases, including inflammatory bowel disease (IBD) and tuberculosis (TB). Current clinical testing methods rely on enzyme-linked immunosorbent assays (ELISAs), limiting accessibility and convenience. In this study, we introduce the Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement. FESCA utilizes two new fragment antigen binding proteins (Fabs), CP16 and CP17, that bind to different epitopes of the calprotectin complex. These Fabs are fused with split NanoLuc luciferase fragments, enabling the reconstitution of active luciferase upon binding to calprotectin either in solution or in varied immobilized assay formats. FESCA's output luminescence can be measured with standard laboratory equipment as well as consumer-grade cell phone cameras. FESCA can detect physiologically relevant calprotectin levels across various sample types, including serum, plasma, and whole blood. Notably, FESCA can detect abnormally elevated native calprotectin from TB patients. In summary, FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples, with the potential to improve the diagnosis and monitoring of inflammatory diseases, especially in at-home or point-of-care settings.

Structural and biophysical insights into targeting of claudin-4 by a synthetic antibody fragment.

Claudins are a 27-member family of ~25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. As the backbone of tight junction structure and function, claudins are attractive targets for modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. Here we report the development of a synthetic antibody fragment (sFab) that binds human claudin-4 and the determination of a high-resolution structure of it bound to claudin-4/enterotoxin complexes using cryogenic electron microscopy. Structural and biophysical results reveal this sFabs mechanism of select binding to human claudin-4 over other homologous claudins and establish the ability of sFabs to bind hard-to-target claudins to probe tight junction structure and function. The findings provide a framework for tight junction modulation by sFabs for tissue-selective therapies.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.

Structural analysis and conformational dynamics of a holo-adhesion GPCR reveal interplay between extracellular and transmembrane domains.

Adhesion G Protein-Coupled Receptors (aGPCRs) are key cell-adhesion molecules involved in numerous physiological functions. aGPCRs have large multi-domain extracellular regions (ECR) containing a conserved GAIN domain that precedes their seven-pass transmembrane domain (7TM). Ligand binding and mechanical force applied on the ECR regulate receptor function. However, how the ECR communicates with the 7TM remains elusive, because the relative orientation and dynamics of the ECR and 7TM within a holoreceptor is unclear. Here, we describe the cryo-EM reconstruction of an aGPCR, Latrophilin3/ADGRL3, and reveal that the GAIN domain adopts a parallel orientation to the membrane and has constrained movement. Single-molecule FRET experiments unveil three slow-exchanging FRET states of the ECR relative to the 7TM within the holoreceptor. GAIN-targeted antibodies, and cancer-associated mutations at the GAIN-7TM interface, alter FRET states, cryo-EM conformations, and receptor signaling. Altogether, this data demonstrates conformational and functional coupling between the ECR and 7TM, suggesting an ECR-mediated mechanism of aGPCR activation.

Structure of antiviral drug bulevirtide bound to hepatitis B and D virus receptor protein NTCP.

Cellular entry of the hepatitis B and D viruses (HBV/HDV) requires binding of the viral surface polypeptide preS1 to the hepatobiliary transporter Na+-taurocholate co-transporting polypeptide (NTCP). This interaction can be blocked by bulevirtide (BLV, formerly Myrcludex B), a preS1 derivative and approved drug for treating HDV infection. Here, to elucidate the basis of this inhibitory function, we determined a cryo-EM structure of BLV-bound human NTCP. BLV forms two domains, a plug lodged in the bile salt transport tunnel of NTCP and a string that covers the receptor's extracellular surface. The N-terminally attached myristoyl group of BLV interacts with the lipid-exposed surface of NTCP. Our structure reveals how BLV inhibits bile salt transport, rationalizes NTCP mutations that decrease the risk of HBV/HDV infection, and provides a basis for understanding the host specificity of HBV/HDV. Our results provide opportunities for structure-guided development of inhibitors that target HBV/HDV docking to NTCP.

Chaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme binding.

Pathogenic bacteria, such as Pseudomonas aeruginosa, depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in P. aeruginosa clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5 Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR's heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations.

Engineered antigen-binding fragments for enhanced crystallization of antibody:antigen complexes.

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.

The Molecular Basis of Human ALKBH3 Mediated RNA N1 -methyladenosine (m1 A) Demethylation.

N1 -methyladenosine (m1 A) is a prevalent post-transcriptional RNA modification, and the distribution and dynamics of the modification play key epitranscriptomic roles in cell development. At present, the human AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family member ALKBH3 is the only known mRNA m1 A demethylase, but its catalytic mechanism remains unclear. Here, we present the structures of ALKBH3-oligo crosslinked complexes obtained with the assistance of a synthetic antibody crystallization chaperone. Structural and biochemical results showed that ALKBH3 utilized two ß-hairpins (ß4-loop-ß5 and ß'-loop-ß'') and the α2 helix to facilitate single-stranded substrate binding. Moreover, a bubble-like region around Asp194 and a key residue inside the active pocket (Thr133) enabled specific recognition and demethylation of m1 A- and 3-methylcytidine (m3 C)-modified substrates. Mutation of Thr133 to the corresponding residue in the AlkB Fe(II)/α-ketoglutarate-dependent dioxygenase family members FTO or ALKBH5 converted ALKBH3 substrate selectivity from m1 A to N6 -methyladenosine (m6 A), as did Asp194 deletion. Our findings provide a molecular basis for understanding the mechanisms of substrate recognition and m1 A demethylation by ALKBH3. This study is expected to aid structure-guided design of chemical probes for further functional studies and therapeutic applications.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification, and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain has eluded the field for over fifty years. The MFS transporter FLVCR1 was recently determined to be a choline transporter, and while this protein is not highly expressed at the blood-brain barrier (BBB), its relative FLVCR2 is. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus, and embryonic lethality, but the physiological role of FLVCR2 is unknown. Here, we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in the inward- and outward-facing states using cryo-electron microscopy to 2.49 and 2.77 Å resolution, respectively. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of neurotherapeutics into the brain.

Structure of human drug transporters OATP1B1 and OATP1B3.

The organic anion transporting polypeptides OATP1B1 and OATP1B3 are membrane proteins that mediate uptake of drugs into the liver for subsequent conjugation and biliary excretion, a key step in drug elimination from the human body. Polymorphic variants of these transporters can cause reduced drug clearance and adverse drug effects such as statin-induced rhabdomyolysis, and co-administration of OATP substrates can lead to damaging drug-drug interaction. Despite their clinical relevance in drug disposition and pharmacokinetics, the structure and mechanism of OATPs are unknown. Here we present cryo-EM structures of human OATP1B1 and OATP1B3 bound to synthetic Fab fragments and in functionally distinct states. A single estrone-3-sulfate molecule is bound in a pocket located in the C-terminal half of OATP1B1. The shape and chemical nature of the pocket rationalize the preference for diverse organic anions and allow in silico docking of statins. The structure of OATP1B3 is determined in a drug-free state but reveals a bicarbonate molecule bound to the conserved signature motif and a histidine residue that is prevalent in OATPs exhibiting pH-dependent activity.

Chaperone-assisted cryo-EM structure of P. aeruginosa PhuR reveals molecular basis for heme uptake.

Pathogenic bacteria, such as Pseudomonas aeruginosa, depend on scavenging heme for the acquisition of iron, an essential nutrient. The TonB-dependent transporter (TBDT) PhuR is the major heme uptake protein in P. aeruginosa clinical isolates. However, a comprehensive understanding of heme recognition and TBDT transport mechanisms, especially PhuR, remains limited. In this study, we employed single-particle cryogenic electron microscopy (cryo-EM) and a phage display-generated synthetic antibody (sAB) as a fiducial marker to enable the determination of a high-resolution (2.5 Å) structure of PhuR with a bound heme. Notably, the structure reveals iron coordination by Y529 on a conserved extracellular loop, shedding light on the role of tyrosine in heme binding. Biochemical assays and negative-stain EM demonstrated that the sAB specifically targets the heme-bound state of PhuR. These findings provide insights into PhuR's heme binding and offer a template for developing conformation-specific sABs against outer membrane proteins (OMPs) for structure-function investigations.

ACKR3-arrestin2/3 complexes reveal molecular consequences of GRK-dependent barcoding.

Atypical chemokine receptor 3 (ACKR3, also known as CXCR7) is a scavenger receptor that regulates extracellular levels of the chemokine CXCL12 to maintain responsiveness of its partner, the G protein-coupled receptor (GPCR), CXCR4. ACKR3 is notable because it does not couple to G proteins and instead is completely biased towards arrestins. Our previous studies revealed that GRK2 and GRK5 install distinct distributions of phosphates (or "barcodes") on the ACKR3 carboxy terminal tail, but how these unique barcodes drive different cellular outcomes is not understood. It is also not known if arrestin2 (Arr2) and 3 (Arr3) bind to these barcodes in distinct ways. Here we report cryo-electron microscopy structures of Arr2 and Arr3 in complex with ACKR3 phosphorylated by either GRK2 or GRK5. Unexpectedly, the finger loops of Arr2 and 3 directly insert into the detergent/membrane instead of the transmembrane core of ACKR3, in contrast to previously reported "core" GPCR-arrestin complexes. The distance between the phosphorylation barcode and the receptor transmembrane core regulates the interaction mode of arrestin, alternating between a tighter complex for GRK5 sites and heterogenous primarily "tail only" complexes for GRK2 sites. Arr2 and 3 bind at different angles relative to the core of ACKR3, likely due to differences in membrane/micelle anchoring at their C-edge loops. Our structural investigations were facilitated by Fab7, a novel Fab that binds both Arr2 and 3 in their activated states irrespective of receptor or phosphorylation status, rendering it a potentially useful tool to aid structure determination of any native GPCR-arrestin complex. The structures provide unprecedented insight into how different phosphorylation barcodes and arrestin isoforms can globally affect the configuration of receptor-arrestin complexes. These differences may promote unique downstream intracellular interactions and cellular responses. Our structures also suggest that the 100% bias of ACKR3 for arrestins is driven by the ability of arrestins, but not G proteins, to bind GRK-phosphorylated ACKR3 even when excluded from the receptor cytoplasmic binding pocket.

Antiviral HIV-1 SERINC restriction factors disrupt virus membrane asymmetry.

The host proteins SERINC3 and SERINC5 are HIV-1 restriction factors that reduce infectivity when incorporated into the viral envelope. The HIV-1 accessory protein Nef abrogates incorporation of SERINCs via binding to intracellular loop 4 (ICL4). Here, we determine cryoEM maps of full-length human SERINC3 and an ICL4 deletion construct, which reveal that hSERINC3 is comprised of two α-helical bundles connected by a ~ 40-residue, highly tilted, "crossmember" helix. The design resembles non-ATP-dependent lipid transporters. Consistently, purified hSERINCs reconstituted into proteoliposomes induce flipping of phosphatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine. Furthermore, SERINC3, SERINC5 and the scramblase TMEM16F expose PS on the surface of HIV-1 and reduce infectivity, with similar results in MLV. SERINC effects in HIV-1 and MLV are counteracted by Nef and GlycoGag, respectively. Our results demonstrate that SERINCs are membrane transporters that flip lipids, resulting in a loss of membrane asymmetry that is strongly correlated with changes in Env conformation and loss of infectivity.

Conformation-specific Synthetic Antibodies Discriminate Multiple Functional States of the Ion Channel CorA.

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg2+, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg2+. Two sABs from these selections, C12 and C18, showed different degrees of Mg2+-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg2+-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg2+-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg2+] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.

Cryo-EM structures of a synthetic antibody against 22 kDa claudin-4 reveal its complex with Clostridium perfringens enterotoxin.

Claudins are a family of ∼25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. Humans have 27 subtypes, which homo- and hetero-oligomerize to impart distinct properties and physiological functions to tissues and organs. As the structural and functional backbone of tight junctions, claudins are attractive targets for therapeutics capable of modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties-these traits also make therapy development a challenge. We have developed a synthetic antibody fragment (sFab) that binds human claudin-4 and used it to resolve structures of its complex with Clostridium perfringens enterotoxin (CpE) using cryogenic electron microscopy (cryo-EM). The resolution of the structures reveals the architectures of 22 kDa claudin-4, the 14 kDa C-terminal domain of CpE, and the mechanism by which this sFab binds claudins. Further, we elucidate the biochemical and biophysical bases of sFab binding and demonstrate that this molecule exhibits subtype-selectivity by assaying homologous claudins. Our results provide a framework for developing sFabs against hard-to-target claudins and establishes the utility of sFabs as fiducial markers for determining cryo-EM structures of this small membrane protein family at resolutions that surpass X-ray crystallography. Taken together, this work highlights the ability of sFabs to elucidate claudin structure and function and posits their potential as therapeutics for modulating tight junctions by targeting specific claudin subtypes.

Conformation-specific synthetic antibodies discriminate multiple functional states of the ion channel CorA.

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg 2+ , and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg 2+ . Two sABs from these selections, C12 and C18, showed different degrees of Mg 2+ -sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg 2+ -depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg 2+ -depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg 2+ ] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.