Structure

Proteins perform essential roles across nearly all cellular processes, and accurate three-dimensional structures remain critical for elucidating structure-function relationships and studies on drug discovery. Cryo-electron microscopy (cryo-EM), X-ray crystallography, and nuclear magnetic resonance can provide detailed structural information. However, for many proteins, structural information is available only as lower-resolution experimental data or sparse data. Such information is more difficult to translate into accurate atomic coordinates; a common example is low-resolution cryo-EM density maps. In parallel, mass spectrometry-based methods, including ion mobility (IM-MS), offer rapid, broadly applicable structural descriptors such as collisional cross section (CCS), a global measure of molecular shape and size, but CCS values also do not provide atomistic detail. Here we present CRIM (cryo-EM + IM-MS), an integrative Rosetta scoring function that combines low-resolution cryo-EM density information with IM-MS derived CCS as restraints to improve monomeric protein structure prediction. CRIM incorporates the Rosetta REF2015 (RS) energy with a CCS agreement penalty (computed via PARCS) and an electron-density agreement term (elec_dens_fast). We tested CRIM on an ideal dataset of 60 monomeric proteins using simulated CCS values and density maps. Across the ideal dataset, the CRIM score function improved or maintained prediction quality for many targets, reducing the mean RMSD from 3.65 [A] (RS) to 2.90 [A] and increasing the mean TM-score from 0.88 to 0.90. Furthermore, an experimental benchmark dataset of 54 proteins was curated to include either experimental cryo-EM maps or published CCS values. On the experimental dataset, CRIM similarly improved model selection, lowering the mean RMSD from 6.65 [A] to 4.38 [A] and raising the mean TM-score from 0.73 to 0.79. In comparison to AlphaFold3 predictions, CRIM frequently yielded competitive predictions and was able to substantially outperform AlphaFold3 for select difficult targets where sparse experimental restraints provide strong discriminatory power. The CRIM score function is freely available within the Rosetta software suite and provides a practical framework for leveraging complementary IM-MS and cryo-EM data to improve monomeric protein structure prediction.

Microtubules are cytoskeletal filaments typically characterized by a discontinuous helical lattice of /{beta}-tubulin heterodimers. Microtubules can also adopt variable lattice architectures both in vitro and in cellular contexts. Pseudo-helical averaging processing strategies have been developed to generate cryo-EM reconstructions of microtubules with and without decorating protein-binding partners, but these pipelines can be difficult to implement for the average user, especially for undecorated filaments. Here, we describe MiCSPARC, a cryo-EM processing pipeline developed around CryoSPARC (Punjani et al., 2017), which leverages automated particle picking and fast 3D refinement times in CryoSPARC to determine structures of both decorated and undecorated microtubules. We generated reconstructions of undecorated GDP microtubules, as well as kinesin-1 motor domain-decorated GMPCPP filaments at resolutions of up to 2.8 [A], demonstrating the robustness of the pipeline. Based on its convenient implementation and ability to routinely generate high-resolution, seam-corrected microtubule reconstructions, MiCSPARC should provide a valuable tool for understanding microtubule dynamics, microtubule-associated proteins, and microtubule-targeting agents.

Cellular transport processes along microtubules are often facilitated by multi-motor complexes, which are connected by adapter proteins and cargoes. The nuclear pore protein Nup358, for example, interacts with the dynein adapter Bicaudal D2 (BicD2), which in turn recruits minus-end directed dynein motors and plus-end directed kinesin-1 motors for a nuclear positioning pathway that is essential for brain development. How motor recruitment is regulated by interactions of BicD2 with Nup358 is not well understood. Here, we characterize the structure of a minimal complex of kinesin-1 light chain 2 (KLC2), Nup358 and BicD2 by cryo-electron microscopy and small angle X-ray scattering. KLC2/Nup358 assumes a rod-like structure that increases in thickness, when BicD2 is bound. The addition of BicD2 also shifts the KLC2/Nup358/BicD2 complex towards a 2:2:2 stoichiometry, promoting dimerization at lower protein concentrations than without BicD2. Similarly, the presence of the Nup358/KLC2 interaction results in a shift towards a 2:2:2 stoichiometry. Based on these results, we hypothesize that KLC2 and BicD2 are recruited to Nup358 in a cooperative manner, and cooperativity may be promoted by modulation of the oligomeric state.

Connexin gap junction channels enable the direct exchange of molecules and ions between cells. Channel opening is regulated by various physiological stimuli. Human connexin26 gap junction channels close in response to elevated levels of CO2 in a process that is independent of pH, with structures demonstrating a [CO2]-dependent conformational change. Cx26 from the lungfish, Lepidosiren paradoxa is also CO2 sensitive. Here we solve its structure at high [CO2]. We observe an open conformation of the protein where the N-terminal helix that influences the aperture of the pore is pulled away from the centre. This conformation is stabilised by the presence of a detergent binding between TM3 and TM4 that would prevent the conformational changes necessary to close the protein upon exchange to high [CO2]. The structure supports a mechanism in which the conformation of a motif shown to be important for CO2 sensitivity is correlated with the opening of the pore.

Mutations in MAPT, the tau gene, give rise to forms of frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17T), with abundant filamentous tau inclusions in brain cells. Some mutations that encode missense and deletion variants can give rise to a clinical picture of Picks disease and filaments made of three-repeat tau. Here we report the electron cryo-microscopy (cryo-EM) structures of tau filaments from individuals with MAPT mutations D252V, G272V, S320F and {Delta}G389-I392. The two-layered Pick fold was present in the individuals with mutations D252V and {Delta}G389-I392. By contrast, mutations G272V and S320F gave rise to a more open variant of the Pick fold, with residues 272-341 rotated by 20-25{degrees} with respect to the rest of the structure. These findings show that missense mutations within the filament core can modify the Pick fold, generating closely related structural variants. In addition, we were able to reconstitute the Pick fold and some of its variants using seeded assembly with recombinant 0N3R tau carrying 12 serine or threonine to aspartate substitutions (PAD12) and missense mutations D252V, G272V or S320F. This work provides a foundation for the development of structure-based diagnostic and therapeutic approaches.

In vertebrate phototransduction, the G protein-coupled receptor rhodopsin activates the -subunit of transducin (GT), which, upon binding the {gamma} subunits of phosphodiesterase-6 (PDE6), stimulates the hydrolysis of cGMP. We reported a cryoEM structure for a complex containing two constitutively active GT (GT*) subunits coupled by a bivalent antibody bound to PDE6 that demonstrated a striking displacement of both PDE{gamma} subunits from the PDE/PDE{beta} catalytic sites and suggested an alternating-site mechanism for PDE6 activation. Here, we use site-directed spin labeling (SDSL) and double electron-electron resonance spectroscopy (DEER) to probe PDE6 conformational changes upon GT* binding. Both spin-labelled Cys68 on wild-type PDE{gamma} and a spin-labelled cysteine residue substituted for Ile64 on PDE{gamma} demonstrate that PDE{gamma} has highly flexible C-termini that transiently bind to the PDE/PDE{beta} heterodimer. Binding of GT* to PDE6 with the competitive inhibitor udenafil occupying its catalytic sites alters the positions of the PDE{gamma} subunits in agreement with the striking changes shown in the cryoEM structure for this complex, whereas coupling the GT* subunits to the bivalent antibody does not affect the DEER distributions observed for PDE6 bound to GT*. However, binding of the slow hydrolyzing 8-Br-cGMP substrate in the presence of GT* causes a dramatic increase in the separation and spread of the spin-labelled PDE{gamma} subunits, thereby revealing a previously unobserved conformation of PDE6 associated with catalysis. These studies indicate that whereas inhibitors trap GT*-PDE6 complexes in an inactive state as represented by the cryoEM structure, the binding of both substrate and GT* produces a dynamic active state consistent with an alternating site mechanism.

G protein-coupled receptors (GPCRs) are critical regulators of human physiology and major drug targets. Although structural studies have provided valuable insights, determining GPCR structures remains challenging, especially for inactive state receptors. Recent advances in cryo-electron microscopy (cryo-EM) have enabled structural determination of small GPCRs by using fusion partner proteins and binders to increase molecular weight. However, current methods require extensive experimental screening of fusion constructs. Widely adopted strategies, such as BRIL-Fab complexes, also face limitations due to inherent flexibility. Here, we introduce a streamlined and universal pipeline that integrates an in silico fusion construct screening program, NOAH (NOAH: NOn-experimental, AI-assisted High-throughput construct screening), with a de novo designed fusion protein called ARK1 (ARtificially-designed fiducial marKer). We validate the efficacy of NOAH by determining the structures of the vasopressin V2 receptor (V2R) bound to the clinical antagonist tolvaptan and the partial agonist OPC51803, as well as the bradykinin B2 receptor (B2R) bound to the clinical antagonist icatibant, thereby elucidating their activation and deactivation mechanisms. Furthermore, we demonstrate the capability of NOAH-ARK1 by solving the tolvaptan-bound V2R structure at higher resolution and showcase the methods versatility by determining the structure of lysophosphatidic acid receptor 2 (LPA2) bound to the antagonist Ki16425. This approach eliminates the need for time-consuming and labor-intensive construct optimization, providing a rapid and widely applicable solution for high-resolution GPCR structure determination and drug discovery.

We report the discovery of a new class of local minima that has severely limited the accuracy of macromolecular models. Termed density misfit barrier traps, these minima explain much of the poor fit between macromolecular models and experimental data relative to that of smaller molecules: not just high R factors, but distorted chemical geometry. We postulated that proteins exist as an ensemble of conformations that each have good geometry, but refinement algorithms have been unable to converge to them due to a tangling phenomenon arising from these traps. To demonstrate, a synthetic ground truth data set was generated, consisting of a 2-member ensemble with excellent geometry. A series of starting models, each trapped in increasingly difficult local minima, were prepared, a unified validation score defined, and an open Challenge issued. This Challenge inspired algorithms for escaping such traps, and new programs have been released that are expected to substantially improve the accuracy of macromolecular ensemble models. SynopsisA synthetic 2-member conformational ensemble of a small protein and corresponding electron density data was generated to demonstrate how topological local minima hinder simultaneous agreement with density data and chemical geometry restraints in conventional structure refinement.

Phosphatidylinositol 4-kinase alpha (PI4KA) is an essential lipid kinase that generates phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol (PI) at the plasma membrane (PM). PI4P is a precursor for PIP2 and PIP3 lipid signalling, with PI4P serving a critical role in maintaining PM identity and asymmetry. Given the important roles of PI4KA in myriad processes, understanding how it is regulated is of immense importance. Here, we have identified that PI4KA can be phosphorylated in its dimerization domain (pY1154) and kinase domain (pY2090) by a cohort of tyrosine kinases. Phosphorylation of pY2090 significantly impairs lipid kinase activity of PI4KA but does not alter its recruitment to membranes by its regulatory protein EFR3. Cryo-EM and HDX-MS analysis reveals that phosphorylation does not result in dramatic conformational changes but instead causes localised changes in the k12 C-terminal helix of PI4KA. Phosphorylation of the C-terminal helix is found in multiple PI3Ks and PI4Ks, suggesting this may be a evolutionarily conserved regulatory mechanism. Overall, our work reveals a novel regulatory mechanism for PI4KA that directly alters its lipid kinase activity.

Several members of the adhesion subfamily of G protein-coupled receptors (aGPCRs) are capable of self-activation by an internal agonist sequence (aka the Stachel) thats exposed upon removal or conformational changes of the N-terminal fragment of the receptor. Synthetic peptides derived from the Stachel sequence can be used as exogenous agonists. In the inactive form, the Stachel is sequestered as the {beta}13-strand within the GPCR Autoproteolysis-INducing (GAIN) domain, but it engages the seven transmembrane region as a helix when it is either an intramolecular sequence or a synthetic peptide. Little is known about the molecular details underlying this transition, but we hypothesize that a disordered conformation is central to this intermediate state in receptor activation. Despite the primarily helical Stachel AlphaFold3 and Pepfold4 models predicted with high confidence for the entire aGPCR subfamily, disorder predictions and biophysical experiments reveal a predominantly disordered conformation in solution. Investigating the ADGRG6/GPR126 Stachel peptide, circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments reveal a predominantly random coil conformation in aqueous buffer, polar detergent micelles, and zwitterionic lipids. Titration of trifluoroethanol uncovered a two-state equilibrium between an unfolded and helix-containing conformation with NMR localizing a single-turn helix to residues L846-L849. Taken together, these data indicate the ADGRG6/GPR126 Stachel peptide is primarily disordered, but small populations may adopt a helix-containing conformer that seems to support a conformational-selection activation mechanism.

The air-water interface (AWI) remains the primary barrier to routine high-resolution cryo-EM structure determination, driving protein adsorption, structural denaturation, and restricted particle orientations during vitrification. Here, we describe a simple and broadly applicable strategy to mitigate these effects using the mild non-ionic detergent n-decyl-{beta}-D-maltopyranoside (DM). Addition of DM at low millimolar concentrations immediately prior to vitrification consistently suppresses AWI-driven artifacts, resulting in improved angular sampling, reduced structural damage, and enhanced reconstruction quality across diverse macromolecular systems. Using this approach, we obtained a high-resolution reconstruction of the 65 kDa Nucleophosmin 1 pentamer, a target previously limited by severe preferred orientation issues. We further show that DM promotes isotropic particle distributions for high-resolution reconstruction of hemagglutinin, transthyretin, as well as suppressing denaturation of aldolase while stabilizing its C-terminus. Our results indicate that DM effectively passivates deleterious air-water interface interactions without compromising particle integrity. These results establish DM as an effective additive for improving the robustness of single-particle cryo-EM sample preparation. O_FIG O_LINKSMALLFIG WIDTH=174 HEIGHT=200 SRC="FIGDIR/small/716008v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@108a6edorg.highwire.dtl.DTLVardef@10728b4org.highwire.dtl.DTLVardef@1014b2eorg.highwire.dtl.DTLVardef@1eed745_HPS_FORMAT_FIGEXP M_FIG C_FIG

The protein kinase C-related kinase (PKN) family of serine/threonine kinases consists of PKN1, PKN2 and PKN3, all of which are Rho family GTPase effectors. PKNs have three N-terminal Homology Region 1 (HR1) domains (HR1a, HR1b and HR1c), which form antiparallel coiled coils, which in two cases interact with Rho family GTPases, activating the kinase. The PKNs are implicated in several important cellular processes, including cytoskeletal regulation, cell adhesion, gene expression and cell cycle progression, and are also implicated in cancer. Here we have investigated the roles of the HR1 domains in PKN oligomerisation. We show that PKN1 HR1a is a dimer and that the HR1c domain drives further oligomerization. We have mapped the interactions between the HR1 domains and used an integrative approach to model HR1-containing PKN1 dimers. Biophysical analysis shows that RhoA forms a 1:2 complex with HR1a, resulting in a rearrangement of the HR1a dimer, an outcome supported by SAXS models. In contrast, Rac1 binds to monomeric HR1a, suggesting that this GTPase activates PKN1 via a different mechanism. These data provide structural insight into interactions between HR1 domains and the Rho family proteins and their potential consequences for PKN1 activation.

Hydrogen bond (H-bond) restraints are critical for NMR structure determination, yet their experimental identification can be challenging for marginally stable structures that afford insufficient protection from (H/D) exchange in D2O. As an alternative, we explored the use of NMR between pH 10 and 11 conditions that promote rapid exchange, for identifying backbone amide protons involved in H-bonds. We analyzed [~]750 amide sites distributed across ten proteins with known structures. Persistence of amide protons at high pH in standard 2D 1H-15N HSQC spectra for 15N-labeled proteins in H2O, or TOCSY for unlabeled proteins, identifies H-bonds with [~]91% accuracy that exceeds the [~]80% accuracy of traditional H/D exchange experiments in D2O. For two -helical coiled coils and three globular proteins, we performed alkaline unfolding experiments taking advantage of amide NMR signal attenuation from unstructured polypeptides. Increasing the sample pH led to a progressive loss of native amide proton NMR signals, revealing an unfolding hierarchy where "foldons" remaining at the highest pH values had the most persistent H-bonds under EX1 exchange conditions. The foldons observed at high pH are consistent with partially folded structures previously characterized near neutral pH by native state hydrogen exchange, equilibrium unfolding, and protein fragment studies. For {beta}-sheet proteins, foldons correspond to regions with high inter-residue contact density, whereas in coiled coils they demarcate regions with high -helical propensity. High-pH NMR experiments provide a sensitive, fast, inexpensive, and broadly applicable approach to map H-bonding in marginally stable or partially folded proteins. Additionally, they offer the opportunity to explore uncharted protein dynamics and unfolding pathways under basic pH conditions.

The coronavirus envelope (E) protein is a viroporin that plays a key role in viral assembly, release, budding and pathogenesis. E protein forms oligomeric ion channels that can activate immune responses. However, high-resolution structural data for its extramembrane domains is limited. The C-terminal domain of SARS-CoV has been shown previously to form amyloid fibers, and here we show that these fibers can modulate the shape of liposomes. The propensity to form fibrils, and their effect on liposomes, was examined for sequences belonging to the four clades of coronaviruses. Electron microscopy data shows that the C-terminal domain in E protein adopts a filamentous structure. These findings demonstrate the potential of these peptides to modulate membranes, providing a possible mechanism by which E protein interacts with membranes in the host cell.

Presynaptic inhibitory GPCR (Gi/o GPCR) signaling is an essential regulatory mechanism in vertebrate physiology. Near the presynaptic active zone, Gi/o GPCR activation releases G-protein {beta}{gamma} heterodimers (G{beta}{gamma}) which act to inhibit synaptic vesicle fusion through either modulation of Ca2+ entry via voltage-gated Ca2+ channels, or by direct interactions with the core exocytotic machinery comprised of the ternary SNARE complex downstream of Ca2+ influx. The precise molecular mechanism underlying G{beta}{gamma}-SNARE mediated inhibition has remained unclear due to lack of structural data for the G{beta}{gamma}-SNARE complex. We address this long-standing question here by stabilizing the interaction between G{beta}1{gamma}2 and a pre-fusion ternary SNARE mimetic and determining the structure using single-particle cryo-EM. We used our cryo-EM envelope to build an atomic level prediction of the interaction interface. We validated key interaction sites predicted by our model at the C-terminus of SNAP-25 using site directed mutagenesis and biochemical affinity measurements. Additionally, we found that G{beta}1{gamma}2 and a fragment of the regulatory protein complexin can engage the pre-fusion SNARE complex simultaneously. On the basis of these results, we propose a model in which G{beta}1{gamma}2 acts on the partially zipped SNARE complex at a late stage in the vesicle docking and priming cycle. In the model, the amino-terminal coiled-coil of G{beta}1{gamma}2 forms an interface with the C-terminus of the target membrane SNARE (t-SNARE) complex to prevent complete incorporation of the vesicle SNARE (v-SNARE) into the core SNARE helical bundle, thus blocking vesicle approach to the plasma membrane. The {beta}-propeller domain of G{beta}1 may also sterically hinder vesicle approach. Together these results provide crucial structural insights into the mechanism of binding of G{beta}{gamma} to the SNARE complex, and lends essential insights into the critical role of GPCR signaling to the SNARE complex in modulating synaptic vesicle fusion.

TRPC3 is a calcium-permeable, non-selective cation channel that is activated by DAG. It is expressed in several tissues, especially in the cerebellum, and has been implicated in various human diseases. Despite recent progress in understanding the structural mechanism of TRPC3, how the channel opens remains elusive. Here, we present structures of hTRPC3 in an agonist-free resting state, determined using a DAG-binding site mutant. We also present the structure of hTRPC3 in a DAG-bound open state, determined using a constitutively active "moonwalker" (T561A) mutant. These structures, together with electrophysiological results, reveal that the T561A mutation activates hTRPC3 by disrupting a polar interaction with N652. A newly formed {pi}-bulge in S6 leads to rotation and outward tilting of the lower half of S6, resulting in dilation of the pore and thus channel opening. Agonist DAG stabilizes hTRPC3 in the open conformation. BTDM exerts its inhibitory effect by pushing S5 and S6 back to the center to close the pore, while preserving the {pi}-bulge. These results shed light on the opening mechanism of hTRPC3.

Although machine learning has transformed protein structure prediction of folded protein ground states with remarkable accuracy, intrinsically disordered proteins and regions (IDPs/IDRs) are defined by diverse and dynamical structural ensembles that are predicted with low confidence by algorithms such as AlphaFold and RoseTTAFold. We present a new machine learning method, IDPForge (Intrinsically Disordered Protein, FOlded and disordered Region GEnerator), that exploits a transformer protein language diffusion model to create all-atom IDP ensembles and IDR disordered ensembles that maintains the folded domains. IDPForge does not require sequence-specific training, back transformations from coarse-grained representations, nor ensemble reweighting, as in general the created IDP/IDR conformational ensembles show good agreement with solution experimental data, and options for biasing with experimental restraints are provided if desired. We envision that IDPForge with these diverse capabilities will facilitate integrative and structural studies for proteins that contain intrinsic disorder, and is available as an open source resource for general use.

Bacteria microcompartments (BMCs) are pseudo-organelles comprised of a self-assembling, semi-permeable protein shell, most commonly enclosing components of enzymatic pathways. -Carboxysomes (-CBs) are anabolic BMCs known for their role in sequestering Rubisco, the enzyme responsible for carbon fixation in plants, algae and bacteria, along with an upstream enzyme and an assembly protein. Rubisco has low selectivity for its substrate, CO2, and has a slow enzymatic turnover rate, resulting in an inefficient metabolic pathway. Within the -CB, Rubisco has been observed at a range of concentrations and with either a liquid-like assembly or a pseudo-lattice of polymerized fibrils. The biophysical origins of the fibril ultrastructure organization are unclear; however, it is only observed inside -CBs. Quantitative knowledge of the binding constants and energies for assembly and maintenance of these fibrils is critical for understanding this organization and Rubisco regulation, but quantitative methods for in situ analysis of Rubisco polymerization have been lacking. Here, we present an approach to convert tomography-derived -CB volumes and Rubisco particle positions into polymerization binding curves. We used this procedure to determine the Rubisco polymerization constants, including the nucleus size (n) and equilibrium polymerization constant (Kpol). The adopted modeling approach is consistent with in situ constraints, such as concentration-dependent binding interactions and confinement. This approach offers a powerful tool to evaluate both in vitro and potentially in vivo biomolecular interactions, both of Rubisco and of other proteins and polymers suitable for analysis by cryo-electron tomography. Significance StatementCryogenic electron tomography (cryoET) is a powerful method to resolve structures of proteins in their native environment at subnanometer-level resolution. Because tomography data retains spatial relationships of all particles, it intrinsically contains information about component (e.g., protein) binding interactions. Here, we use Rubisco polymerization in -carboxysomes as a model system to demonstrate that quantitative, biochemical binding analysis is possible with cryoET.

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are tetrameric ER Ca2+ channels that shape intracellular Ca2+ signaling in response to IP3, regulating many physiological processes. The structural basis for subtype-specific regulation of three subtypes (IP3R-1-3) remains incompletely understood, due to the lack of IP3R-2 structures. Here, we determined cryo-electron microscopy (cryo-EM) structures of human IP3R-2 in distinct conformations with and without IP3, Ca2+, and ATP. These structures define the conformational ensembles adopted by IP3R-2 and delineate ligand-binding interactions. Comparison with rat IP3R-1 and human IP3R-3 highlights shared architectural features and isoform-specific differences that underlie subtype-specific functional properties. We also resolved structures of IP3R-2 clusters, providing insight into mechanisms of ligand-dependent clustering. Together, these findings establish a structural framework for human IP3R-2, linking ligand recognition to conformational transitions and interchannel interfaces, and illuminate how subtype-specific features and clustering may shape cellular Ca2+ signaling.

Eukaryotes have distinct nuclear genes for tryptophanyl-tRNA synthetase (TrpRS). Human mitochondrial (Hmt) TrpRS (also WARS2) shares only 14% sequence identity with human cytoplasmic (Hc)TrpRS, but 41% with Bacillus stearothermophilus (Bs)TrpRS. Tryptophan binding to BsTrpRS is largely promoted by hydrophobic interactions and recognition of the indole nitrogen by side chains of Met129 and Asp132. The non-reactive analog indolmycin can recruit unique polar interactions to form an active-site metal coordination that lies off the normal mechanistic path, enhancing affinity to BsTrpRS and other prokaryotic TrpRS enzymes by 1500-fold over its tryptophan substrate. By contrast, human WARS2, complements nonpolar interactions for tryptophan binding with additional electrostatic and hydrogen bonding interactions that are inconsistent with indolmycin binding. We report here a 1.82 [A] crystal structure of an HmtTrpRS* indolmycin*Mn2+*ATP complex, showing that mitochondrial and bacterial enzymes use similar determinants to bind both ATP and indolmycin. ATP forms tight electrostatic interactions between the catalytic metal ion and a non-bridging oxygen atom from each phosphate group. Hydrogen bonds between the oxazolinone group and active-site residues create an off-path ground-state configuration. This arrangement closely mimics that in the corresponding BsTrpRS complex but varies greatly from ATP binding to HcTrpRS, Moreover, isothermal titration calorimetry demonstrates that, as for BsTrpRS, Mg2+*ATP, but not ATP alone, enhances indolmycin binding affinity [~]100-fold with a supplemental {Delta}({Delta}G) of [~] -3 kcal/mol. Structural, thermodynamic, and kinetic similarities confirm our previous conclusion that a reinforced ground-state Mg2+ ion configuration contributes to the high indolmycin affinity in the bacterial system.