Structure

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.

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 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.

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.

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.

Affinity chromatography is a powerful and therefore popular method for the purification of proteins for structural studies. The success of the technique relies on the specificity of the interaction between the target protein and the affinity resin. Here, we present the identification of two protein contaminants isolated from HEK293 cell lysate following affinity purification of twin Strep-tagged or FLAG-tagged proteins. The contaminants were identified as human propionyl-coenzyme A carboxylase (hPCC) and protein arginine methyltransferase 5 in complex with methylosome protein 50 (PRMT5:MEP50) via a combination of cryo-EM data processing and proteomic analyses. This report serves to illustrate how these contaminants may appear in cryo-EM datasets and to highlight the paramount importance of affinity chromatography resin specificity for efficient protein purification.

Ligand-gated ion channels are critical mediators of electrochemical signal transduction across evolution. The bacterial channel DeCLIC constitutes a provocative model system for structure, function, and dynamics in this family, including a modulatory N-terminal domain (NTD). Previous closed structures of DeCLIC support a rationale for its inhibition by calcium; however, properties of its open state remain unclear. Here we used cryogenic electron microscopy under acidic conditions to determine a previously unreported conformation of DeCLIC with an expanded pore. This structure was relatively stable and permeable in simulations, and agreed with the average low-pH solution structure by small-angle neutron scattering. In the absence of calcium, an alternative closed class exhibited dynamic rearrangements in the NTD. We propose that our expanded-pore structure corresponds to a functional open state of DeCLIC, while calcium-site and NTD dynamics drive channel closure, providing a detailed template for modulatory mechanisms in the larger channel family.

Recent advances in cryo-electron microscopy and cryo-electron tomography have dramatically increased the number of class A G protein-coupled receptor (GPCR) structures, especially in previously inaccessible G protein-bound, active-like conformations. The increased structural diversity provides a unique opportunity to explore the conformational landscape underlying GPCR activation. To this end, we developed a machine learning (ML) framework that utilizes experimental structural data to elucidate the activation dynamics of class A GPCRs. We find that receptors can populate both inactive and active-like conformations even in the absence of ligand or G protein, providing a structural basis for agonist-free basal activity. Agonist binding shifts this conformational ensemble towards the active state but does not fully stabilize it. Instead, a stable active state is only established upon G protein binding, which locks the receptor in its active conformation. These results support a hybrid activation mechanism in which ligand binding follows conformational selection, while transducer engagement is governed by induced fit. Beyond clarifying class A GPCR activation, the openly available and modifiable ML framework provides a practical tool for analyzing newly determined structures, investigating the mechanisms of action of other GPCR classes and protein families, and guiding structure-based drug discovery in important pharmacological superfamilies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/714415v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19d1327org.highwire.dtl.DTLVardef@1549782org.highwire.dtl.DTLVardef@a6dfaaorg.highwire.dtl.DTLVardef@1a650ce_HPS_FORMAT_FIGEXP M_FIG C_FIG

The suppression of type I interferon (IFN) signalling by the ISG15-USP18-STAT2 inhibitory complex (ISG15 IC) is an established regulatory mechanism of the antiviral response. However, a molecular understanding of how the ISG15 IC forms to suppress IFN signalling is still emerging. Here, we use AlphaFold modelling in conjunction with biochemical and biophysical approaches to elucidate the interactions of this multiprotein assembly. Our analysis identified a unique STAT2 binding loop (SBL) in USP18, which is critical for the USP18-STAT2 association. Further biochemical characterisation through site-directed mutagenesis confirmed the importance of residues within and surrounding the SBL, enabling the design of mutants with both increased and decreased binding affinities. Moreover, several USP18 and STAT2 patient mutations severely disrupted this interaction. Lastly, using influenza B virus (IBV) and Zika virus (ZIKV) proteins, we investigated the influence of these viral effector proteins on these interactions. Taken together, these results provide much-needed insights into a key aspect of IFN signalling control.

The delta-type ionotropic glutamate receptors (iGluRs) GluD1 and GluD2 are ligand-gated ion channels that are fundamental for regulating both excitatory and inhibitory synapses. Rising evidence points to the role of GluD1 in the development of neurological diseases. However, the ultrastructure of human GluD1 (hGluD1) and the molecular basis for its ligand-gating remain unclear. Here, we define the structure of hGluD1 and resolve its ligand-gating mechanism using cryo-electron microscopy (cryoEM) and single channel bilayer recording. While hGluD1 exhibits a non-swapped architecture, it contains conserved iGluR moieties that enable ligand-gating, such as a ligand-binding domain (LBD) tethered to a transmembrane ion channel. Binding of the neurotransmitter {gamma}-aminobutyric acid (GABA) or D-serine to the LBD enables cation influx through the hGluD1 ion channel. Our findings delineate the molecular architecture and function of hGluD1, provide foundations for understanding patient mutations in hGluD1, and will invigorate therapeutic development against hGluD1.

The emergence of SARS-CoV-2 has caused millions of deaths and excess morbidity in the worldwide population. In addition to its respiratory symptoms, SARS-CoV-2 has become known for its neurotropism and long-term neurological sequelae, with a post-acute infection syndrome commonly referred to as long-COVID. Next to the host receptor angiotensin-converting enzyme 2 (ACE2) additional interactions of the SARS-CoV-2 spike (S) protein have been described for neuronal co-receptors specific to the nervous system including cell adhesion protein contactin 1 (CNTN1). Details of the spike-CNTN1 interaction have remained elusive. Here, we quantified the spike-CNTN1 interaction by surface plasmon resonance and resolved the structure of the complex by single particle cryo-electron microscopy (cryo-EM). Spike and CNTN1 interact with nanomolar affinity, driven by an avidity effect and mediated by the horseshoe moiety of CNTN1. The cryo-EM structure reveals that the CNTN1 Ig1-4 horseshoe is wedged in between two receptor binding domains (RBDs) and interacts, through Ig3, with a unique receptor interface at the base of the RBD in the up-conformation. This receptor interface is not previously described for other spike receptors but overlaps with the epitopes of several neutralizing monoclonal antibodies. Comparison of our data with available spike structures suggests one spike trimer can bind three CNTN1 molecules, or alternatively, different co-receptors such as ACE2 and CNTN1, simultaneously. These findings shed new light on the molecular determinants of SARS-CoV-2 neurotropism.

AbstractThe development of machine learning models for protein-ligand interactions is fundamentally constrained by the quality and diversity of available structural data. Existing databases of protein-ligand complexes present researchers with an unsatisfying trade-off: carefully curated collections such as PDBBind and HiQBind offer high structural reliability but cover only a narrow slice of the Protein Data Bank (PDB), while large-scale resources like PLInder provide broad coverage at the expense of rigorous quality control. Here, we introduce CROWN (Curated Repository Of Well-resolved Non-covalent interactions), a machine learning-ready dataset that reconciles this tension by applying a comprehensive, fully automated preprocessing pipeline to the PLInder database. Starting from 649,915 protein-ligand interaction systems, CROWN applies a series of interleaved quality filters and processing stages addressing crystallographic resolution, ligand identity, pocket completeness, structural repair, interaction quality, and protonation at physiological pH. A distinguishing feature of the pipeline is a final constrained energy minimisation step using custom flat-bottomed restraints, which balances crystallographic evidence with relaxation of intramolecular strain. This step -- absent from existing protein-ligand datasets -- produces structurally uniform complexes by reconciling the heterogeneous refinement practices of different crystallographers and structure determination protocols, without distorting the experimentally observed binding geometry. The resulting dataset of 153,005 complexes represents a roughly four-fold increase in protein and species diversity over PDBBind and HiQBind, while maintaining rigorous structural standards. Importantly, CROWN adopts a geometry-centric design philosophy that treats the 3D arrangement of atoms at the binding interface as a self-consistent source of information, rather than relying on externally measured binding affinities that cover only a fraction of known structures and introduce well-documented biases. We anticipate that CROWN will serve as a broadly useful resource for training generative models of protein-ligand binding poses, developing scoring functions, and benchmarking interaction prediction methods.

Calculation of density maps from atomic models is essential for structural studies using crystallography and electron cryo-microscopy (cryoEM). These maps serve various purposes, including atomic model building, refinement, visualization, and validation. However, accurately comparing model-calculated maps to experimental data poses challenges, particularly because the resolution of cryoEM experimental maps varies across the map. Traditional crystallography methods generate finite-resolution maps with uniform resolution throughout the unit cell volume, while most modern software in cryoEM employ Gaussian-like functions to generate these maps, which does not adequately account for atomic model parameters and resolution. Recent work by Urzhumtsev & Lunin (2022, IUCr Journal, 9, 728-734) introduces a novel method for computing atomic model maps that incorporate local resolution and can be expressed as analytically differentiable functions of all atomic parameters. This approach enhances the accuracy of matching atomic models to experimental maps. In this paper, we detail the implementation of this method in CCTBX and Phenix. SynopsisNew tools implemented in CCTBX and Phenix allow the calculation of variable-resolution maps through a sum of atomic images expressed as analytic functions of all atomic parameters, along with their associated local resolution.

Quinones are an integral component of electron transfer processes in photosynthetic and mitochondrial respiratory proteins. One such photosynthetic protein, Photosystem I, is an essential photooxidoreductase found in all oxygenic phototrophs. To better understand quinone chemistry and to form a basis for protein engineering, the menB gene in the model cyanobacterium Synechocystis sp. PCC 6803 was interrupted, blocking the biosynthesis of phylloquinone and causing it to be replaced by exchangeable plastoquinone-9 in the A1A and A1B quinone-binding sites of Photosystem I. This genetic variant has been instrumental in bioenergy research, enabling incorporation of a range of substituted and isotopically labeled quinones. Despite numerous valuable studies, the interpretation of biophysical data has been limited by a lack of structural data. To address this, we present the high-resolution cryo-EM structures of Photosystem I from the {Delta}menB variant containing (a) exchangeable plastoquinone-9 and (b) exogenously added 2-ethyl-1,4-napthoquinone at 1.90- and 2.05-[A] resolution, respectively. Unexpectedly, the quinones in the A1A and A1B sites of Photosystem I, previously believed to have similar binding affinities, are found to be asymmetric in their ability to bind and exchange plastoquinone-9. This work reveals new and important insight into the molecular basis for Photosystem I activity in the {Delta}menB variant, the power of metabolic plasticity to maintain protein stability, and the requirement for protein instability to facilitate ligand exchange.

Purified proteins are routinely flash frozen for use in functional and structural studies, providing a convenient way to reproduce results across complex experiments. Rho guanine-nucleotide exchange factors (RhoGEFs) are no exception to this practice, yet the effects of freezing on their activity and stability remain largely uncharacterized. This gap potentially affects the characterization of these important enzymes and how results are interpreted with respect to their prospective use as therapeutic targets. Here, we tested the isolated DH/PH tandems of P-Rex1, P-Rex2, and PRG under different cryoprotectant conditions and monitored activity and thermostability over time after flash freezing. Our results show a clear divergence between the activity of fresh and frozen purified RhoGEF protein samples in as little as one week for some conditions. Specifically, the variability in data collected on frozen samples was greatly increased. Despite these differences, thermostability seems to be preserved for much longer timepoints across RhoGEFs. Moreover, despite eventual changes in both activity and thermostability with respect to freezing, there are no obvious changes in global conformation between fresh and frozen samples of the isolated P-Rex2 DH/PH tandem. From our data, there are few generalizable trends between the different RhoGEFs and no single cryoprotective agent tested was a silver bullet to preserve both activity and thermostability across RhoGEFs. Overall, our findings emphasize the unpredictable effects of freezing RhoGEFs. As such, RhoGEF freezing should be carefully characterized for each protein and critically viewed when comparing analyses between different studies.

Glutamate racemase (MurI) catalyzes the stereochemical interconversion of L-glutamate to D-glutamate, a key element of bacterial peptidoglycan biosynthesis. In this study, we present the crystal structure of Helicobacter pylori glutamate racemase at 1.43 [A] and in monoclinic symmetry, as previously reported models, but different unit-cell parameters. The present model contains a single dimer and retains the previously described head-to-head dimer arrangement. The differences between the models arise from variations in unit-cell parameters, which lead to altered crystal packing interactions rather than changes in the quaternary assembly. The monomeric fold and active-site architecture remain conserved and are consistent with the catalytic features described for bacterial glutamate racemases. This structure provides an updated, high-resolution structural model for H. pylori glutamate racemase and highlights the variability in crystal packing within the same space group.

KdpFABC is a hetero-tetrameric potassium pump that uses ATP to import potassium and thereby maintain homeostasis in bacteria under stress conditions. KdpA is a channel-like subunit with a selectivity filter that binds potassium from the periplasm. K+ then moves through a [~]40[A]-long intramembrane tunnel to reach a canonical binding site in KdpB. KdpB is a P-type ATPase that orchestrates conformational changes associated with the Post-Albers reaction cycle, involving E1 and E2 conformations and formation of an aspartyl phosphate intermediate as a way of coupling ATP hydrolysis to K+ transport. To elucidate the associated structural changes in a lipid environment, we reconstituted wild-type KdpFABC into lipid nanodiscs and used cryo-EM to image the complex under active turnover. The resulting six high resolution (2.1-2.7 [A]) structures provide new insight into the sequence of allosteric changes that produce (1) occlusion of K+ at the canonical binding site and (2) expulsion of K+ from this site and into a low-affinity release site. The structures also reveal two types of lipids bound to the complex. Specifically, two structural lipids bind at subunit interfaces and [~]20 annular lipids are seen at the periphery of the complex. In addition, we tested functional effects of mutations to residues at the KdpA/KdpB interface. ATPase and transport assays were used to document functional defects that reflect delipidation of structurally compromised complexes. We conclude that lipids play an integral role in structure and function of the KdpFABC complex. SignificanceKdpFABC uses ATP to transport potassium across the plasma membrane of E. coli. To further our understanding of its mechanism, we put purified KdpFABC molecules into membrane bilayers and used cryo-EM to capture structures during active transport. We have thereby produced structures representing all major states of the transport cycle with a high degree of precision. Analysis of these structures reveals new details about two key steps in this cycle and shows lipid molecules bound to the protein. We then introduced mutations at the interface between the two main subunits, which controls passage of potassium across the membrane. Activity measurements reveal how the protein depends on lipid to stabilize the structure and facilitate transport.