Helix Packing Research Articles

Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases.

The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt Λ-shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signal-inducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having a physiologically representative lipid composition has not been observed. We address this limitation by employing coarse-grained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations.

Atomistic simulations of polysaccharide materials for insights into their crystal structure, nanostructure, and dissolution mechanism

AbstractCrystalline polysaccharides are abundant in nature and can be transformed into highly functional materials. However, the molecular basis for the formation of higher-order structures remains unclear. Computer simulation is an advanced tool for modeling macromolecular structures, and the atomistic simulations provide valuable information on the crystalline polysaccharides. Fiber deformation, crystalline transition, and novel nanostructures of cellulose were characterized through molecular dynamics simulations and density functional theory calculations of models of molecular chain sheets extracted from the crystal structure of the cellulose polymorphs. Extended ensemble molecular dynamics simulations were applied to analyze the artificial crystal structure of non-natural amylose analog polysaccharides, revealing the hexagonal packing of double helices through the self-assembly of molecular chains dispersed in aqueous solution. Dissolution simulations of the cellulose and chitin crystalline fibers revealed that the anions of ionic liquids, with their solvation power, played a key role in the cleavage of intermolecular hydrogen bonds in the crystal structure, whereas the cations contributed to irreversible molecular chain dispersion. The good correlation between the actual solubility of polysaccharides and the predicted number of intermolecular hydrogen bonds prompted the development of a platform that combined simulations and machine learning for high-throughput screening of solvents for cellulose and chitin.

A Collagen Triple Helix without the Super Helical Twist.

Collagens are ubiquitous in biology functioning as the backbone of the extracellular matrix, forming the primary structural components of key immune system complexes, and fulfilling numerous other structural roles in a variety of systems. Despite this, there is limited understanding of how triple helices, the basic collagen structural units, pack into collagenous assemblies. Here we use a peptide self-assembly system to design collagenous assemblies based on the C1q collagen-like region. Using cryo-EM we solve a structure of one assembly to 3.5 Å resolution and build an atomic model. From this, we identify a triple helix conformation with no superhelical twist, starkly in contrast to the canonical right-handed triple helix. This non-twisting region allows for unique hydroxyproline stacking between adjacent triple helices and also results in the formation of an exposed cavity with rings of hydrophobic amino acids packed symmetrically. We find no precedent for such an arrangement of collagen triple helices and have designed mutant assemblies to probe key stabilizing amino acid interactions in the complex. The mutations behave as predicted by our atomic model. Our findings, combined with the extremely limited experimental structural data on triple helix packing in the literature, suggest that collagen and collagen-like assemblies may adopt a far more varied conformational landscape than previously appreciated. We hypothesize that this is particularly likely adjacent to the termini of these helices and at discontinuities to the required Xaa-Yaa-Gly repeating primary sequence; a discontinuity found in the majority of this class of proteins and in many collagen-associated diseases.

Optimized periphery-core interface increases fitness of the Bacillus subtilisglmS ribozyme.

Like other functional RNAs, ribozymes encode a conserved catalytic center supported by peripheral domains that vary among ribozyme sub-families. To understand how core-periphery interactions contribute to ribozyme fitness, we compared the cleavage kinetics of all single base substitutions at 152 sites across the Bacillus subtilis glmS ribozyme by high-throughput sequencing (k-seq). The in vitro activity map mirrored phylogenetic sequence conservation in glmS ribozymes, indicating that biological fitness reports all biochemically important positions. The k-seq results and folding assays showed that most deleterious mutations lower activity by impairing ribozyme self-assembly. All-atom molecular dynamics simulations of the complete ribozyme revealed how individual mutations in the core or the IL4 peripheral loop introduce a non-native tertiary interface that rewires the catalytic center, eliminating activity. We conclude that the need to avoid non-native helix packing powerfully constrains the evolution of tertiary structure motifs in RNA.

ARTEMIS: a method for topology-independent superposition of RNA 3D structures and structure-based sequence alignment.

Non-coding RNAs play a major role in diverse processes in living cells with their sequence and spatial structure serving as the principal determinants of their function. Superposition of RNA 3D structures is the most accurate method for comparative analysis of RNA molecules and for inferring structure-based sequence alignments. Topology-independent superposition is particularly relevant, as evidenced by structurally similar RNAs with sequence permutations such as tRNA and Y RNA. To date, state-of-the-art methods for RNA 3D structure superposition rely on intricate heuristics, and the potential for topology-independent superposition has not been exhausted. Recently, we introduced the ARTEM method for unrestrained pairwise superposition of RNA 3D modules and now we developed it further to solve the global RNA 3D structure alignment problem. Our new tool ARTEMIS significantly outperforms state-of-the-art tools in both sequentially-ordered and topology-independent RNA 3D structure superposition. Using ARTEMIS we discovered a helical packing motif to be preserved within different backbone topology contexts across various non-coding RNAs, including multiple ribozymes and riboswitches. We anticipate that ARTEMIS will be essential for elucidating the landscape of RNA 3D folds and motifs featuring sequence permutations that thus far remained unexplored due to limitations in previous computational approaches.

Spontaneous Dimerization and Distinct Packing Modes of Transmembrane Domains in Receptor Tyrosine Kinases.

The insulin receptor (IR) and the insulin-like growth factor-1 receptor (IGF1R) are homodimeric transmembrane glycoproteins that transduce signals across the membrane on binding of extracellular peptide ligands. The structures of IR/IGF1R fragments in apo and liganded states have revealed that the extracellular subunits of these receptors adopt Λ-shaped configurations to which are connected the intracellular tyrosine kinase (TK) domains. The binding of peptide ligands induces structural transitions in the extracellular subunits leading to potential dimerization of transmembrane domains (TMDs) and autophosphorylation in TKs. However, the activation mechanisms of IR/IGF1R, especially the role of TMDs in coordinating signal-inducing structural transitions, remain poorly understood, in part due to the lack of structures of full-length receptors in apo or liganded states. While atomistic simulations of IR/IGF1R TMDs showed that these domains can dimerize in single component membranes, spontaneous unbiased dimerization in a plasma membrane having physiologically representative lipid composition has not been observed. We address this limitation by employing coarse-grained (CG) molecular dynamics simulations to probe the dimerization propensity of IR/IGF1R TMDs. We observed that TMDs in both receptors spontaneously dimerized independent of their initial orientations in their dissociated states, signifying their natural propensity for dimerization. In the dimeric state, IR TMDs predominantly adopted X-shaped configurations with asymmetric helical packing and significant tilt relative to the membrane normal, while IGF1R TMDs adopted symmetric V-shaped or parallel configurations with either no tilt or a small tilt relative to the membrane normal. Our results suggest that IR/IGF1R TMDs spontaneously dimerize and adopt distinct dimerized configurations.

Plasmid DNA Complexes in Powder Form Studied by Spectroscopic and Diffraction Methods.

Currently, new functional materials are being created with a strong emphasis on their ecological aspect. Materials and devices based on DNA biopolymers, being environmentally friendly, are therefore very interesting from the point of view of applications. In this paper, we present the results of research on complexes in the powder form based on plasmid DNA (pDNA) and three surfactants with aliphatic chains containing 16 carbon atoms (cetyltrimethylammonium chloride, benzyldimethylhexadecylammonium chloride and hexadecylpyridinium chloride). The X-ray diffraction results indicate a local hexagonal packing of DNA helices in plasmid DNA complexes, resembling the packing for corresponding complexes based on linear DNA. Based on the Fourier-transform infrared spectroscopy results, the DNA conformation in all three complexes was determined as predominantly of A-type. The two relaxation processes revealed by dielectric spectroscopy for all the studied complexes are connected with two different contributions to total conductivity (crystallite part and grain boundaries). The crystallite part (grain interior) was interpreted as an oscillation of the polar surfactant head groups and is dependent on the conformation of the surfactant chain. The influence of the DNA type on the properties of the complexes is discussed, taking into account our previous results for complexes based on linear DNA. We showed that the type of DNA has an impact on the properties of the complexes, which has not been demonstrated so far. It was also found that the layer of pDNA-surfactant complexes can be used as a layer with variable specific electric conductivity by selecting the frequency, which is interesting from an application point of view.

Mechano-responsive chiral gold(I) complexes with aggregation- and crystallization-induced circularly polarized luminescence

Chiral materials with circularly polarized luminescence (CPL) have attracted extensive attention due to their unique optical properties. However, it is still a challenge to obtain efficient CPL materials. In this work, noticeable CPL signals were obtained through simple crystallization in the classical aggregation-caused quenching (ACQ)-typed chloro-gold(I) complexes (R)-BINAP-AuCl and (S)-BINAP-AuCl. They exhibited typical crystallization-induced emission (CIE) and crystallization-induced CPL behaviors. Importantly, after rational modification by introducing carbazole units, the resulting carbazole-gold(I) complexes (R)-BINAP-AuCz and (S)-BINAP-AuCz showed remarkable aggregation-induced phosphorescent emission (AIPE) and unique aggregation-induced CPL characteristics due to the formation of highly ordered helical packing modes in the nano-aggregate state. More interestingly, the solids of chiral carbazole-gold(I) complexes (R)-BINAP-AuCz and (S)-BINAP-AuCz could sensitively respond to external stimuli and exhibited mechano-responsive CPL performance, which was tuned by the intermolecular C–H···π interactions. Thus, this work provided a new way for constructing high-performance and smart CPL materials.

Probing the energy barriers and stages of membrane protein unfolding using solid-state NMR spectroscopy.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.

Ligand Chirality Transfer from Solution State to the Crystalline Self-Assemblies in Circularly Polarized Luminescence (CPL) Active Lanthanide Systems.

The synthesis of a family of chiral and enantiomerically pure pyridyl-diamide (pda) ligands that upon complexation with europium [Eu(CF3SO3)3] result in chiral complexes with metal centered luminescence is reported; the sets of enantiomers giving rise to both circular dichroism (CD) and circularly polarized luminescence (CPL) signatures. The solid-state structures of these chiral metallosupramolecular systems are determined using X-ray diffraction showing that the ligand chirality is transferred from solution to the solid state. This optically favorable helical packing arrangement is confirmed by recording the CPL spectra from the crystalline assembly by using steady state and enantioselective differential chiral contrast (EDCC) CPL Laser Scanning Confocal Microscopy (CPL-LSCM) where the two enantiomers can be clearly distinguished.

The irreplaceable glycine: glycine homologs destabilize the collagen triple helix

Collagen, the most widespread protein in animals, contains glycine as every third amino acid. This regular repetition of glycine residues is required for the tight packing of the collagen triple helix. In this work, we substitute glycine with its homolog β-alanine in collagen model peptides (CMPs) and study the effect of β-alanine on the formation of triple helices. While β-alanine in the middle of CMP sequences is incompatible with triple helix formation, terminal β-alanine residues are tolerated. The work highlights the critical role of glycine in collagen.

Cycloplatinated(II) metallomesogens and their binary-mediated chirality transfer and amplificated deep-red circularly polarized luminescence with ultrahigh dissymmetry factor over 0.13

Incorporating supramolecular chirality and high emission into metallomesogens has great significance in the field of flexible electronic devices because of their emergent capacities for enlarged chiroptical activities and either thermotropic or solution-processability. In this work, an enantiomeric pair of chiral Pt(II) metallomesogens (S)/(R)-Pt1 were prepared through laboratory-synthesized chiral picolinic acid ligands and phenylpyridine derivatives to study their mesophase behaviors, chiroptical properties, and chirality transfer and amplification. The solutions of (S)/(R)-Pt1 emitted green phosphorescence at 508 nm, but the amorphous form displayed orange-red phosphorescence at 616 nm. Upon gentle grinding or heating, the emission color of Pt1 was dramatically changed with a bathochromic shift of 47 and 51 nm, respectively, because of emerged intermolecular π···π and Pt⋯Pt interactions in all aggregation states. Furthermore, the chiroptical properties and LC self-assembly model were proposed by Density Functional Theory (DFT) calculations and chiroptical spectrum measurements. The enantiotopic (S)/(R)-Pt1 complexes exhibited electronic circular dichroism (CD) and circularly polarized luminescence (CPL) activities in solution with a smaller asymmetry factor (±5 × 10−4) by chiral disturbance. This pair of exclusively enantiomeric complexes exhibited smectic mesophase behavior in the range of 31.4–76.5 °C but induced unsuccessfulness of helical packing in LC states due to weak induction ability. Contrastively, binary-component mediated supramolecular helical formation and amplificated deep-red CPL switch with ultrahigh dissymmetry factor over ±0.13 in helical twist grain boundary phases (TGBA*) mesophase has been realized.

Impact of Ion-Mixing Entropy on Orientational Preferences of DNA Helices: FRET Measurements and Computer Simulations.

Biological processes require DNA and RNA helices to pack together in specific interhelical orientations. While electrostatic repulsion between backbone charges is expected to be maximized when helices are in parallel alignment, such orientations are commonplace in nature. To better understand how the repulsion is overcome, we used experimental and computational approaches to investigate how the orientational preferences of DNA helices depend on the concentration and valence of mobile cations. We used Förster resonance energy transfer (FRET) to probe the relative orientations of two 24-bp helices held together via a freely rotating PEG linker. At low cation concentrations, the helices preferred more "cross"-like orientations over those closer to parallel, and this preference was reduced with increasing salt concentrations. The results were in good quantitative agreement with Poisson-Boltzmann (PB) calculations for monovalent salt (Na+). However, PB underestimated the ability of mixtures of monovalent and divalent ions (Mg2+) to reduce the conformational preference. As a complementary approach, we performed all-atom molecular dynamics (MD) simulations and found better agreement with the experimental results. While MD and PB predict similar electrostatic forces, MD predicts a greater accumulation of Mg2+ in the ion atmosphere surrounding the DNA. Mg2+ occupancy is predicted to be greater in conformations close to the parallel orientation than in conformations close to the crossed orientation, enabling a greater release of Na+ ions and providing an entropic gain (one bound ion for two released). MD predicts an entropy gain larger than that of PB because of the increased Mg2+ occupancy. The entropy changes have a negligible effect at low Mg2+ concentrations because the free energies are dominated by electrostatic repulsion. However, as the Mg2+ concentration increases, charge screening is more effective and the mixing entropy produces readily detectable changes in packing preferences. Our results underline the importance of mixing entropy of counterions in nucleic acid interactions and provide a new understanding on the impact of a mixed ion atmosphere on the packing of DNA helices.

Exploring computational tools for improved structural design and stability of helical AApeptides

Peptidomimetics have garnered increased attention due to their unique structural properties and self-assembly, as well as their promising applications in material sciences and peptidomimetic drug design. N-acetylated-N-aminoethyl amino acid oligomers (AApeptides), a novel class of helical foldamer, have been effectively utilized to imitate protein helices and regulate protein-protein interactions. While the structures of a series of AApeptides have been determined experimentally, the chemical diversity of AApeptides is impeding the advancement of peptide inhibitor development and high-level architecture design through purely experimental methods. Consequently, there's an urgent need for effective computational tools to facilitate the structural design of more complex systems using AApeptides. While a general AMBER force field (GAFF) can be applied to simulate AApeptides, it is crucial to evaluate the accuracy of such a force field and consider alternatives to enhance accuracy. In this study, we employed molecular dynamics simulations (MD) to assess the stability of a helical AApeptide. Our findings indicate that GAFF alone is insufficient to stabilize the helical structure of our AApeptide. We suggest the use of restraints derived from experimentally determined structures to guide the simulation and maintain this helical structure. Although any set of restraint definitions used in this study can simulate helical packing, a minimum of three points per AApeptide building block restraint is necessary to accurately reproduce the hydrogen bonding pattern.

A comprehensive survey of long-range tertiary interactions and motifs in non-coding RNA structures.

Understanding the 3D structure of RNA is key to understanding RNA function. RNA 3D structure is modular and can be seen as a composition of building blocks of various sizes called tertiary motifs. Currently, long-range motifs formed between distant loops and helical regions are largely less studied than the local motifs determined by the RNA secondary structure. We surveyed long-range tertiary interactions and motifs in a non-redundant set of non-coding RNA 3D structures. A new dataset of annotated LOng-RAnge RNA 3D modules (LORA) was built using an approach that does not rely on the automatic annotations of non-canonical interactions. An original algorithm, ARTEM, was developed for annotation-, sequence- and topology-independent superposition of two arbitrary RNA 3D modules. The proposed methods allowed us to identify and describe the most common long-range RNA tertiary motifs. Along with the prevalent canonical A-minor interactions, a large numberof previously undescribed staple interactions were observed. The most frequent long-range motifs were found to belong to three main motif families: planar staples, tilted staples, and helical packing motifs.

Temperature-Induced Nanostructure Transition for Supramolecular Gelation in Water.

Thermogel is an injectable biomaterial that functions at body temperatures due to the ease of the sol-to-gel transition. However, most conventional physically cross-linked thermogels generally have relatively low stiffness, which limits various biomedical applications, particularly for stem-cell-based studies. While chemical cross-linking through double-network (DN) structures can increase the stiffness of the hydrogel, they generally lack injectable and thermoresponsive properties due to strong covalent bonds between molecules. To address this challenge, we have developed a temperature-induced nanostructure transition (TINT) system for preparing physical DN supramolecular hydrogels. These hydrogels possess injectable, thermoreversible characteristics and relatively high storage modulus (G'), which increases ∼14-fold from 20 to 37 °C (body temperature). Our bottom-up strategy is based on the co-assembly of aromatic peptide (Ben-FF) and poly(ethylene glycol) (PEG) to form a thermogel at 37 °C through a nanofiber dissociation pathway that differs from the well-known micelle aggregation or polymer shrinkage mechanisms. Peptide molecules form helical packing and weak, noncovalent interactions with PEG, resulting in co-assembled metastable nanofibers. Thermal perturbation initiates lateral dissociation of nanofibers into extensively cross-linked DN nanostructures and subsequent hydrogelation (ΔG = -13.32 kJ/mol). The TINT hydrogel is nontoxic to human mesenchymal stem cells and supports enhanced cell adhesion, suggesting the potential of this strategy in the applications of tissue engineering and regenerative medicine.

The intrinsically disordered region of coronins fine-tunes oligomerization and actin polymerization

Coronins play critical roles in actin network formation. The diverse functions of coronins are regulated by the structured N-terminal β propeller and the C-terminal coiled coil (CC). However, less is known about a middle "unique region" (UR), which is an intrinsically disordered region (IDR). The UR/IDR is an evolutionarily conserved signature in the coronin family. By integrating biochemical and cell biology experiments, coarse-grained simulations, and protein engineering, we find that the IDR optimizes the biochemical activities of coronins invivo and invitro. The budding yeast coronin IDR plays essential roles in regulating Crn1 activity by fine-tuning CC oligomerization and maintaining Crn1 as a tetramer. The IDR-guided optimization of Crn1 oligomerization is critical for F-actin cross-linking and regulation of Arp2/3-mediated actin polymerization. The final oligomerization status and homogeneity of Crn1 are contributed by three examined factors: helix packing, the energy landscape of the CC, and the length and molecular grammar of the IDR.

Molecular mechanism of fatty acid activation of FFAR1

FFAR1 is a G-protein-coupled receptor (GPCR) that responds to circulating free fatty acids to enhance glucose-stimulated insulin secretion and release of incretin hormones. Due to the glucose-lowering effect of FFAR1 activation, potent agonists for this receptor have been developed for the treatment of diabetes. Previous structural and biochemical studies of FFAR1 showed multiple sites of ligand binding to the inactive state but left the mechanism of fatty acid interaction and receptor activation unknown. We used cryo-electron microscopy to elucidate structures of activated FFAR1 bound to a Gq mimetic, which were induced either by the endogenous FFA ligand docosahexaenoic acid or γ-linolenic acid and the agonist drug TAK-875. Our data identify the orthosteric pocket for fatty acids and show how both endogenous hormones and synthetic agonists induce changes in helical packing along the outside of the receptor that propagate to exposure of the G-protein-coupling site. These structures show how FFAR1 functions without the highly conserved "DRY" and "NPXXY" motifs of class A GPCRs and also illustrate how the orthosteric site of a receptor can be bypassed by membrane-embedded drugs to confer full activation of G protein signaling.

Nature-inspired synthetic oligourea foldamer channels allow water transport with high salt rejection

•Synthetic, self-assembling, helical oligourea foldamers form artificial water channels •ssNMR, MD simulation, and cryo-EM data show proper insertion of foldamers into liposomes •Water-selective channels are formed by oligomerization of the amphipathic foldamers •4.8 Å hydrophilic pore is present in X-ray crystal structure of foldamer H2OC1 Biomimetic membranes incorporating artificial water channels (AWCs) are being developed for industrial water purification. Designing AWCs to achieve high water permeation with salt rejection is a challenge. We designed and synthesized oligourea foldamers, which form predictable helical structures that can be used to create biomimetic porin-like architectures. Two of these foldamers (H2OC1 and H2OC2) allow superior water permeability and almost total salt rejection across lipid membranes. Solid-state NMR, cryo-EM, and molecular dynamics analyses suggest proper insertion of foldamers into lipid vesicles. The H2OC1 crystal structure shows hydrophilic pores of diameters 4.8 and 6.4 Å. The oligourea helices pack together by hydrophobic and salt bridge interactions to build two channel-like assemblies. Besides their proteolytic stability and microbial resistance, the sequence of foldamers can be tailored to regulate selectivity. The ease of designing, synthesizing, and purifying oligourea foldamers is an added advantage. Our findings can help to develop novel AWCs for water purification applications. Biomimetic membranes incorporating artificial water channels (AWCs) are being developed for industrial water purification. Designing AWCs to achieve high water permeation with salt rejection is a challenge. We designed and synthesized oligourea foldamers, which form predictable helical structures that can be used to create biomimetic porin-like architectures. Two of these foldamers (H2OC1 and H2OC2) allow superior water permeability and almost total salt rejection across lipid membranes. Solid-state NMR, cryo-EM, and molecular dynamics analyses suggest proper insertion of foldamers into lipid vesicles. The H2OC1 crystal structure shows hydrophilic pores of diameters 4.8 and 6.4 Å. The oligourea helices pack together by hydrophobic and salt bridge interactions to build two channel-like assemblies. Besides their proteolytic stability and microbial resistance, the sequence of foldamers can be tailored to regulate selectivity. The ease of designing, synthesizing, and purifying oligourea foldamers is an added advantage. Our findings can help to develop novel AWCs for water purification applications.

Membrane protein isolation and structure determination in cell-derived membrane vesicles

Integral membrane protein structure determination traditionally requires extraction from cell membranes using detergents or polymers. Here, we describe the isolation and structure determination of proteins in membrane vesicles derived directly from cells. Structures of the ion channel Slo1 from total cell membranes and from cell plasma membranes were determined at 3.8Å and 2.7Å resolution, respectively. The plasma membrane environment stabilizes Slo1, revealing an alteration of global helical packing, polar lipid, and cholesterol interactions that stabilize previously unresolved regions of the channel and an additional ion binding site in the Ca2+ regulatory domain. The two methods presented enable structural analysis of both internal and plasma membrane proteins without disrupting weakly interacting proteins, lipids, and cofactors that are essential to biological function.