Single Conformational State Research Articles

Allosteric activation of VCP, an AAA unfoldase, by small molecule mimicry

The loss of function of AAA (ATPases associated with diverse cellular activities) mechanoenzymes has been linked to diseases, and small molecules that activate these proteins can be powerful tools to probe mechanisms and test therapeutic hypotheses. Unlike chemical inhibitors that can bind a single conformational state to block enzyme function, activator binding must be permissive to different conformational states needed for mechanochemistry. However, we do not know how AAA proteins can be activated by small molecules. Here, we focus on valosin-containing protein (VCP)/p97, an AAA unfoldase whose loss of function has been linked to protein aggregation-based disorders, to identify druggable sites for chemical activators. We identified VCP ATPase Activator 1 (VAA1), a compound that dose-dependently stimulates VCP ATPase activity up to ~threefold. Our cryo-EM studies resulted in structures (ranging from ~2.9 to 3.7 Å-resolution) of VCP in apo and ADP-bound states and revealed that VAA1 binds an allosteric pocket near the C-terminus in both states. Engineered mutations in the VAA1-binding site confer resistance to VAA1, and furthermore, modulate VCP activity. Mutation of a phenylalanine residue in the VCP C-terminal tail that can occupy the VAA1 binding site also stimulates ATPase activity, suggesting that VAA1 acts by mimicking this interaction. Together, our findings uncover a druggable allosteric site and a mechanism of enzyme regulation that can be tuned through small molecule mimicry.

Multiple conformational states assembly of multidomain proteins using evolutionary algorithm based on structural analogues and sequential homologues

With the breakthrough of AlphaFold2, nearly all single-domain protein structures can be built at experimental resolution. However, accurate modelling of full-chain structures of multidomain proteins, particularly all relevant conformations for those with multiple states remain challenging. In this study, we develop a multidomain protein assembly method, M-SADA, for assembling multiple conformational states. In M-SADA, a multiple population-based evolutionary algorithm is proposed to sample multiple conformational states under the guidance of multiple energy functions constructed by combining homologous and analogous templates with inter-domain distances predicted by deep learning. On a developed benchmark dataset containing 72 multidomain proteins with multiple conformational states, the performance of M-SADA is significantly better than that of AlphaFold2 on multiple conformational states modelling, where 29/72 (40.3%) of proteins can be assembled with a TM-score > 0.90 for two highly distinct conformational states with M-SADA. Furthermore, M-SADA is tested on a developed benchmark dataset containing 296 multidomain proteins with single conformational state, and results show that the average TM-score of M-SADA on the best models is 0.913, which is 5.2% higher than that of AlphaFold2 models (0.868). Results on CASP15 multidomain targets also show that M-SADA can predict new domain arrangements when individual domain structures are correct.

PBPK Modeling of PAXLOVIDTM: Incorporating Rotamer Conversion Kinetics to Advanced Dissolution and Absorption Model

PAXLOVID™ is a combination medicine of nirmatrelvir tablets co-packaged with ritonavir tablets. Nirmatrelvir is a peptidomimetic inhibitor of SARS-CoV2 main protease (Mpro), developed for the treatment of COVID-19. Ritonavir is co-administered as a pharmacokinetics (PK) enhancer to inhibit CYP3A mediated metabolism increasing exposures of nirmatrelvir. In the solid form, nirmatrelvir exists in a stable single conformational state (ANTI form). However, nirmatrelvir exhibits atropisomerism in solution whereby upon dissolution the ANTI rotational isomer reversibly converts to another conformation state (SYN form). Nirmatrelvir rotamer conversion follows pseudo first order kinetics with a conversion half-life of approximately 15 min in aqueous solutions, which is on a similar time scale of diffusion mediated dissolution from the solid form. In vitro dissolution studies further indicated that rotamer conversion is one of the processes controlling nirmatrelvir dissolution. It was hypothesized that rotamer conversion kinetics would affect oral absorption of nirmatrelvir in vivo. Consequently, a physiologically based pharmacokinetic (PBPK) model for Paxlovid was developed in Simcyp™ using the advanced dissolution, absorption, and metabolism model (ADAM) by incorporating rotamer conversion kinetics to achieve a more mechanistic description of nirmatrelvir oral absorption. The results demonstrate that the established absorption model with rotamer kinetics adequately described observed clinical data from various nirmatrelvir doses, dosage forms, and dosing regimens. The predicted vs. observed AUCinf and Cmax ratios were within 2-fold. The model has been internally used to inform clinical studies and dose recommendations for pediatrics.

De novo design of highly selective miniprotein inhibitors of integrins αvβ6 and αvβ8

The RGD (Arg-Gly-Asp)-binding integrins αvβ6 and αvβ8 are clinically validated cancer and fibrosis targets of considerable therapeutic importance. Compounds that can discriminate between homologous αvβ6 and αvβ8 and other RGD integrins, stabilize specific conformational states, and have high thermal stability could have considerable therapeutic utility. Existing small molecule and antibody inhibitors do not have all these properties, and hence new approaches are needed. Here we describe a generalized method for computationally designing RGD-containing miniproteins selective for a single RGD integrin heterodimer and conformational state. We design hyperstable, selective αvβ6 and αvβ8 inhibitors that bind with picomolar affinity. CryoEM structures of the designed inhibitor-integrin complexes are very close to the computational design models, and show that the inhibitors stabilize specific conformational states of the αvβ6 and the αvβ8 integrins. In a lung fibrosis mouse model, the αvβ6 inhibitor potently reduced fibrotic burden and improved overall lung mechanics, demonstrating the therapeutic potential of de novo designed integrin binding proteins with high selectivity.

Conformational trapping of an ABC transporter in polymer lipid nanoparticles.

ATP-binding cassette (ABC) proteins play important roles in cells as importers and exporters but as membrane proteins they are subject to well-known challenges of isolating pure and stable samples for study. One solution to this problem is to use styrene-maleic acid lipid particles (SMALPs). Styrene-maleic acid (SMA) can be added directly to membranes, forming stable nanoparticles incorporating membrane proteins and lipids. Here we use Sav1866, a well-characterised bacterial protein, as a proxy for ABC proteins in general. We show that stable and monodispersed Sav1866 can be purified at high yield using SMA. This protein can be used for biophysical characterisations showing that its overall structure is consistent with existing evidence. However, like other ABC proteins in SMALPs it does not hydrolyse ATP. The lack of ATPase activity in ABC–SMALPs may result from conformational trapping of the proteins in SMALPs. Undertaken in a controlled manner, conformational trapping is a useful tool to stabilise protein samples into a single conformation for structural studies. Due to their inability to hydrolyse ATP, the conformation of Sav1866–SMALPs cannot be altered using ATP and vanadate after purification. To achieve controlled trapping of Sav1866–SMALPs we show that Sav1866 in crude membranes can be incubated with ATP, magnesium and sodium orthovanadate. Subsequent solubilisation and purification with SMA produces a sample of Sav1866–SMALPs with enhanced stability, and in a single conformational state. This method may be generally applicable to vanadate-sensitive ABC proteins and overcomes a limitation of the SMALP system for the study of this protein family.

Multiscale molecular kinetics by coupling Markov state models and reaction-diffusion dynamics.

A novel approach to simulate simple protein-ligand systems at large time and length scales is to couple Markov state models (MSMs) of molecular kinetics with particle-based reaction-diffusion (RD) simulations, MSM/RD. Currently, MSM/RD lacks a mathematical framework to derive coupling schemes, is limited to isotropic ligands in a single conformational state, and lacks multiparticle extensions. In this work, we address these needs by developing a general MSM/RD framework by coarse-graining molecular dynamics into hybrid switching diffusion processes. Given enough data to parameterize the model, it is capable of modeling protein-protein interactions over large time and length scales, and it can be extended to handle multiple molecules. We derive the MSM/RD framework, and we implement and verify it for two protein-protein benchmark systems and one multiparticle implementation to model the formation of pentameric ring molecules. To enable reproducibility, we have published our code in the MSM/RD software package.

Conformational ensemble of a multidomain protein explored by Gd3+ electron paramagnetic resonance

Despite their importance in function, the conformational state of proteins and its changes are often poorly understood, mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution nuclear magnetic resonance (NMR) study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work, we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially intergadolinium distance measurement using double electron-electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic NMR. The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.

Fusion Proteins as Tools to Discriminate the Biased Signaling Profile of β 2 Adrenergic Receptor Ligands

Introduction The β2 Adrenoceptor (β2AR) is a seven-transmembrane domain receptor that induces a cellular response by activating a unique signaling pathway dependent on the conformational state of the receptor. At least two signaling pathways have been characterized for the β2AR: the canonical Gs pathway and the β-arrestin2 (βarr2) pathway. So far, the determination of the signaling preference of multiple β2AR ligands (biased signaling) is compared to the reference β2AR endogenous ligand, epinephrine (EPI), which by convention is defined as unbiased. However, the absolute biased signaling of β2AR ligands on the basis for the distinct active conformational states of the β2AR is unknown. Thus, we used novel fusion proteins (fp) made of the β2AR receptor fused to Gαs (β2AR-Gs) or to βarr2 (β2AR-βarr2) to stabilize the β2AR conformation and characterize the pharmacological properties (affinity, efficacy, and potency) of the β2AR ligands. Our central hypothesis is that by restricting signaling to a single conformational state of the receptor, we will be able to characterize the biased signaling of β2AR ligands (independent of epinephrine). Methods For pharmacological characterization, we used HEK293 cells, a common cell line widely used to study the β2AR biased signaling. We stably transfected each fp (β2AR-Gs or β2AR-βarr2) independently into HEK293 cells. Stably transfected HEK293 cells with wild-type (WT) β2AR and untransfected HEK293 cells served as experimental controls. Using homologous time-resolving fluorescence, we measured cAMP production and ERK phosphorylation as the surrogates for β2AR signaling via Gs and βarr2, respectively. Results For the Gs protein signaling pathway, our data suggest ICI has a higher affinity for the inhibition of cAMP production by isoproterenol (ISO) in both the β2AR-βarr2 and β2AR-Gs fp (apparent pA2=10.38 and 10.16, respectively), compared to both control groups (WT β2AR pA2=9.42; untransfected HEK293 pA2=9.21). Furthermore, the ligands ISO and EPI showed a higher potency for the β2AR-Gs fp compared to the β2AR-βarr2 fp (Table-1). Regarding the βarr2 signaling pathway, EPI showed higher potency at ERK phosphorylation in cells transfected with β2AR-βarr2 vs β2AR-Gs fp (pEC50=7.0 vs 6.365). Conclusion Our preliminary results suggest that the affinity of ICI as well as the potency of EPI might change based on the conformational state of the β2AR. Ongoing binding affinity studies will further develop the absolute biased signaling profile of each β2AR ligand and reveal if the reason for the differences in potency are due to their differing affinities for the conformational states.

Role of Distinct β2 Adrenergic Receptor Pathways in Mediating Inflammation

Chronic treatment of inflammatory diseases (i.e. asthma) with β2‐adrenergic receptor (β2AR) agonists can paradoxically lead to increased inflammation and poor therapeutic outcomes. The β2AR has at least two signaling pathways: the canonical Gs pathway and the β‐arrestin2 (βarr2) pathway. Both pathways are suggested to be involved in inflammatory disorders that are regulated by immunomodulatory cells, such as the airway epithelium. However, the inflammatory response of the epithelium to β2AR ligands that preferentially (relative to epinephrine) activate one of these pathways (biased signaling) remains unknown. Thus, we will use novel fusion proteins made of the β2AR receptor fused to Gαs (β2AR‐Gs) or to βarr2 (β2AR‐βarr2) to restrict signaling to a single pathway, to characterize the inflammatory profile of β2AR ligands, relative to their pharmacological properties (affinity, efficacy, and potency). Our central hypothesis is that by restricting signaling to a single conformational state of the receptor, we will be able to correlate the signaling of β2AR ligands with their pharmacological properties. Our long‐term goal is to understand how the distinct β2AR pathways mediate the inflammatory response in the epithelium. For pharmacological characterization, we used HEK293 cells. This cell line has been used to generate most of the previous data on β2AR biased signaling and allows for comparisons for experimental rigor. We stably transfected each fusion protein (β2AR‐Gs or β2AR‐βarr2) independently into HEK293 cells. Stably transfected HEK293 cells with wild‐type (WT) β2AR and untransfected HEK293 cells served as experimental controls. Using homologous time‐resolving fluorescence, we measured cAMP production and ERK phosphorylation as the surrogates for β2AR signaling via Gs and βarr2, respectively. For the Gs protein signaling pathway, all transfected cells showed increased constitutive activity compared to untransfected HEK293 cells. β2AR‐βarr2 transfected cells, however, showed the highest sensitivity to ICI 115,881 (ICI), a β2AR inverse agonist/antagonist for both pathways, at decreasing constitutive activity. Conversely, for the βarr2 signaling pathway, only β2AR‐βarr2 transfected cells showed constitutive activity. Furthermore, both signaling pathways (Gs or βarr2) could be modulated by isoproterenol (ISO), an agonist for both pathways. Thus, while the chimeric receptor is constitutively active, the fusion proteins are still able to respond to ligands, allowing us to explore the ‘absolute bias’ of β2AR ligands. Our data suggest ICI has a higher affinity for the inhibition of cAMP production by ISO in both the β2AR‐βarr2 and β2AR‐Gs fusion proteins (apparent pA2=10.38 and 10.16, respectively), compared to both control groups (WT β2AR pA2=9.42; untransfected HEK293 pA2=9.21). Furthermore, ISO showed a higher potency for the β2AR‐Gs fusion protein (pEC50=10.12) compared to cells expressing the β2AR‐βarr2 fusion protein (pEC50=8.98). We conclude that our fusion proteins can be used to generate a gain‐of‐function phenotype for studying our long‐term goals and can be useful tools to explore the signaling bias of β2AR ligands.

Recording and Analyzing Nucleic Acid Distance Distributions with X-Ray Scattering Interferometry (XSI).

Most structural techniques provide averaged information or information about a single predominant conformational state. However, biological macromolecules typically function through series of conformations. Therefore, a complete understanding of macromolecular structures requires knowledge of the ensembles that represent probabilities on a conformational free energy landscape. Here we describe an emerging approach, X-ray scattering interferometry (XSI), a method that provides instantaneous distance distributions for molecules in solution. XSI uses gold nanocrystal labels site-specifically attached to a macromolecule and measures the scattering interference from pairs of heavy metal labels. The recorded signal can directly be transformed into a distance distribution between the two probes. We describe the underlying concepts, present a detailed protocol for preparing samples and recording XSI data, and provide a custom-written graphical user interface to facilitate XSI data analysis. © 2018 by John Wiley & Sons, Inc.

A single NaK channel conformation is not enough for non-selective ion conduction

NaK and other non-selective channels are able to conduct both sodium (Na+) and potassium (K+) with equally high efficiency. In contrast to previous crystallographic results, we show that the selectivity filter (SF) of NaK in native-like lipid membranes adopts two distinct conformations that are stabilized by either Na+ or K+ ions. The atomic differences of these conformations are resolved by solid-state NMR (ssNMR) spectroscopy and molecular dynamics (MD) simulations. Besides the canonical K+ permeation pathway, we identify a side entry ion-conduction pathway for Na+ permeation unique to NaK. Moreover, under otherwise identical conditions ssNMR spectra of the K+ selective NaK mutant (NaK2K) reveal only a single conformational state. Therefore, we propose that structural plasticity within the SF and the selection of these conformations by different ions are key molecular determinants for highly efficient conduction of different ions in non-selective cation channels.

In Vitro Reconstitution of Rab GTPase-dependent Vesicle Clustering by the Yeast Lethal Giant Larvae/Tomosyn Homolog, Sro7

Intracellular traffic in yeast between the Golgi and the cell surface is mediated by vesicular carriers that tether and fuse in a fashion that depends on the function of the Rab GTPase, Sec4. Overexpression of either of two Sec4 effectors, Sro7 or Sec15, results in the formation of a cluster of post-Golgi vesicles within the cell. Here, we describe a novel assay that recapitulates post-Golgi vesicle clustering in vitro utilizing purified Sro7 and vesicles isolated from late secretory mutants. We show clustering in vitro closely replicates the in vivo clustering process as it is highly dependent on both Sro7 and GTP-Sec4. We also make use of this assay to characterize a novel mutant form of Sro7 that results in a protein that is specifically defective in vesicle clustering both in vivo and in vitro. We show that this mutation acts by effecting a conformational change in Sro7 from the closed to a more open structure. Our analysis demonstrates that the N-terminal propeller needs to be able to engage the C-terminal tail for vesicle clustering to occur. Consistent with this, we show that occupancy of the N terminus of Sro7 by the t-SNARE Sec9, which results in the open conformation of Sro7, also acts to inhibit vesicle cluster formation by Sro7. This suggests a model by which a conformational switch in Sro7 acts to coordinate Rab-mediated vesicle tethering with SNARE assembly by requiring a single conformational state for both of these processes to occur.

The Effects of Thermal Disorder on the Solution-Scattering Profiles of Macromolecules

The solution-scattering profiles of macromolecules are significantly affected by the thermal motions of their atoms, especially at wide scattering angles, even when only a single conformational state is significantly populated in solution. Here it is shown that the impact thermal motions have on the molecular component of the solution-scattering profile of a single-state macromolecule can be predicted accurately if the variances and covariances of the thermal excursions of its atoms from their average positions are known.

Conformation sequence recovery of a non-periodic object from a diffraction-before-destruction experiment

Knowledge of the sequence of different conformational states of a protein molecule is key to better understanding its biological function. A diffraction pattern from a single conformational state can be captured with an ultrafast X-ray Free-Electron Laser (XFEL) before the target is completely annihilated by the radiation. In this paper, we report the first experimental demonstration of conformation sequence recovery using diffraction patterns from randomly ordered conformations of a non-periodic object using the dimensional reduction technique Isomap and coherent diffraction imaging.

Engineering an Ultra-Thermostable β1-Adrenoceptor

Conformational thermostabilisation of G-protein-coupled receptors is a successful strategy for their structure determination. The thermostable mutants tolerate short-chain detergents, such as octylglucoside and nonylglucoside, which are ideal for crystallography, and in addition, the receptors are preferentially in a single conformational state. The first thermostabilised receptor to have its structure determined was the β1-adrenoceptor mutant β1AR-m23 bound to the antagonist cyanopindolol, and recently, additional structures have been determined with agonist bound. Here, we describe further stabilisation of β1AR-m23 by the addition of three thermostabilising mutations (I129V, D322K, and Y343L) to make a mutant receptor that is 31 °C more thermostable than the wild-type receptor in dodecylmaltoside and is 13 °C more thermostable than β1AR-m23 in nonylglucoside. Although a number of thermostabilisation methods were tried, including rational design of disulfide bonds and engineered zinc bridges, the two most successful strategies to improve the thermostability of β1AR-m23 were an engineered salt bridge and leucine scanning mutagenesis. The three additional thermostabilising mutations did not significantly affect the pharmacological properties of β1AR-m23, but the new mutant receptor was significantly more stable in short-chain detergents such as heptylthioglucoside and denaturing detergents such as SDS.

Requirement of open headpiece conformation for activation of leukocyte integrin αXβ2

Negative stain electron microscopy (EM) and adhesion assays show that alpha(X)beta(2) integrin activation requires headpiece opening as well as extension. An extension-inducing Fab to the beta(2) leg, in combination with representative activating and inhibitory Fabs, were examined for effect on the equilibrium between the open and closed headpiece conformations. The two activating Fabs stabilized the open headpiece conformation. Conversely, two different inhibitory Fabs stabilized the closed headpiece conformation. Adhesion assays revealed that alpha(X)beta(2) in the extended-open headpiece conformation had high affinity for ligand, whereas both the bent conformation and the extended-closed headpiece conformation represented the low affinity state. Intermediate integrin affinity appears to result not from a single conformational state, but from a mixture of equilibrating conformational states.

T-SNARE Protein Conformations Patterned by the Lipid Microenvironment

The spatial distribution of the target (t-)SNARE proteins (syntaxin and SNAP-25) on the plasma membrane has been extensively characterized. However, the protein conformations and interactions of the two t-SNAREs in situ remain poorly defined. By using super-resolution optical techniques and fluorescence lifetime imaging microscopy, we observed that within the t-SNARE clusters syntaxin and SNAP-25 molecules interact, forming two distinct conformations of the t-SNARE binary intermediate. These are spatially segregated on the plasma membrane with each cluster exhibiting predominantly one of the two conformations, representing the two- and three-helical forms previously observed in vitro. We sought to explain why these two t-SNARE intermediate conformations exist in spatially distinct clusters on the plasma membrane. By disrupting plasma membrane lipid order, we found that all of the t-SNARE clusters now adopted a single conformational state corresponding to the three helical t-SNARE intermediates. Together, our results define spatially distinct t-SNARE intermediate states on the plasma membrane and how the conformation adopted can be patterned by the underlying lipid environment.

The effect of β‐methylation on the conformation of α, β‐dehydrophenylalanine: a DFT study

AbstractDehydroamino acids are non‐coded amino acids that offer unique conformational properties. Dehydrophenylalanine (ΔPhe) is most commonly used to modify bioactive peptides to constrain the topography of the phenyl ring in the side chain, which commonly serves as a pharmacophore. The Ramachandran maps (in the gas phase and in CHCl3 mimicking environments) of ΔPhe analogues with methyl groups at the β position of the side chain as well as at the C‐terminal amide were calculated using the B3LYP/6‐31 + G** method. Unexpectedly, β‐methylation alone results in an increase of conformational freedom of the affected ΔPhe residue. However, further modification by introducing an additional methyl group at C‐terminal methyl amide results in a steric crowding that fixes the torsion angle ψ of all conformers to the value 123°, regardless of the Z or E position of the phenyl ring. The number of conformers is reduced and the accessible conformational space of the residues is very limited. In particular, (Z)‐Δ(βMe)Phe with the tertiary C‐terminal amide can be classified as the amino acid derivative that has a single conformational state as it seems to adopt only the β conformation. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.

Molecular dynamics studies of the energetics of translocation in model T7 RNA polymerase elongation complexes

Translocation in the single subunit T7 RNA polymerase elongation complex was studied by molecular dynamics simulations using the posttranslocated crystal structure with the fingers domain open, an intermediate stable in the absence of pyrophosphate, magnesium ions, and nucleotide substrate. Unconstrained and umbrella sampling simulations were performed to examine the energetics of translocations. The extent of translocation was quantified using reaction coordinates representing the average and individual displacements of the RNA-DNA hybrid base pairs with respect to a reference structure. In addition, an unconstrained simulation was also performed for the product complex with the fingers domain closed, but with the pyrophosphate and magnesium removed, in order to examine the local stability of the pretranslocated closed state after the pyrophosphate release. The average spatial movement of the entire hybrid was found to be energetically costly in the post- to pretranslocated direction in the open state, while the pretranslocated state was stable in the closed complex, supporting the notion that the conformational state dictates the global stability of translocation states. However, spatial fluctuations of the RNA 3'-end in the open conformation were extensive, with the typical range reaching 3-4 A. Our results suggest that thermal fluctuations play more important roles in the translocation of individual nucleotides than in the movement of large sections of nucleotide strands: RNA 3'-end can move into and out of the active site within a single conformational state, while a global movement of the hybrid may be thermodynamically unfavorable without the conformational change.

Normal-Mode Refinement of Anisotropic Thermal Parameters for Potassium Channel KcsA at 3.2 Å Crystallographic Resolution

We report a normal-mode method for anisotropic refinement of membrane-protein structures, based on a hypothesis that the global near-native-state disordering of membrane proteins in crystals follows low-frequency normal modes. Thus, a small set of modes is sufficient to represent the anisotropic thermal motions in X-ray crystallographic refinement. By applying the method to potassium channel KcsA at 3.2 A, we obtained a structural model with an improved fit with the diffraction data. Moreover, the improved electron density maps allowed for large structural adjustments for 12 residues in each subunit, including the rebuilding of 3 missing side chains. Overall, the anisotropic KcsA structure at 3.2 A was systematically closer to a 2.0 A KcsA structure, especially in the selectivity filter. Furthermore, the anisotropic thermal ellipsoids from the refinement revealed functionally relevant structural flexibility. We expect this method to be a valuable tool for structural refinement of many membrane proteins with moderate-resolution diffraction data.