Cryo-electron Microscopy Research Articles

Cryo-EM structures of the zinc transporters ZnT3 and ZnT4 provide insights into their transport mechanisms.

Zinc transporters (ZnTs) act as H+/Zn2+ antiporters, crucial for zinc homeostasis. Brain-specific ZnT3 expressed in synaptic vesicles transports Zn2+ from the cytosol into vesicles and is essential for neurotransmission, with ZnT3 dysfunction associated with neurological disorders. Ubiquitously expressed ZnT4 localized to lysosomes facilitates the Zn2+ efflux from the cytosol to lysosomes, mitigating the cell injury risk. Despite their importance, the structures and Zn2+ transport mechanisms remain unclear. We characterized the three-dimensional structures of human ZnT3 (inward-facing) and ZnT4 (outward-facing) using cryo-electron microscopy. By combining these structures, we assessed the conformational changes that could occur within the transmembrane domain during Zn2+ transport. Our results provide a structural basis for a more comprehensive understanding of the H+/Zn2+ exchange mechanisms exhibited by ZnTs.

Vesicle Picker: A tool for efficient identification of membrane protein complexes in vesicles

Electron cryomicroscopy (cryo-EM) has recently allowed determination of near-atomic resolution structures of membrane proteins and protein complexes embedded in lipid vesicles. However, particle selection from electron micrographs of these vesicles can be challenging due to the strong signal contributed from the lipid bilayer. This challenge often requires iterative and laborious particle selection workflows to generate a dataset of high-quality particle images for subsequent analysis. Here we present Vesicle Picker, an open-source program built on the Segment Anything model. Vesicle Picker enables automatic identification of vesicles in cryo-EM micrographs with high recall and precision. It then exhaustively selects all potential particle locations, either at the perimeter or uniformly over the surface of the projection of the vesicle. The program is designed to interface with cryoSPARC, which performs both upstream micrograph processing and downstream single particle image analysis. We demonstrate Vesicle Picker’s utility by determining a high-resolution map of the vacuolar-type ATPase from micrographs of native synaptic vesicles (SVs) and identifying an additional protein or protein complex in the SV membrane.

Cryo-EM structure of the heteromeric TRPC1/TRPC4 channel.

Transient receptor potential (TRP) ion channels have a crucial role as cellular sensors, mediating diverse physical and chemical stimuli. The formation of heteromeric structures expands the functionality of TRP channels; however, their molecular architecture remains largely unknown. Here we present the cryo-electron microscopy structures of the human TRPC1/TRPC4 heteromer in the apo and antagonist-bound states, both consisting of one TRPC1 subunit and three TRPC4 subunits. The heteromer structure reveals a distinct ion-conduction pathway, including an asymmetrically constricted selectivity filter and an asymmetric lower gate, primarily attributed to the incorporation of TRPC1. Through a structure-guided electrophysiological assay, we show that both the selectivity filter and the lower part of the S6 helix participate in deciding overall preference for permeating monovalent cations. Moreover, we reveal that the introduction of one lysine residue of TRPC1 into the tetrameric central cavity is enough to render one of the most important functional consequences of TRPC heteromerization: reduced calcium permeability. Our results establish a framework for addressing the structure-function relationship of the heteromeric TRP channels.

VMXm - A sub-micron focus macromolecular crystallography beamline at Diamond Light Source.

VMXm joins the suite of operational macromolecular crystallography beamlines at Diamond Light Source. It has been designed to optimize rotation data collections from protein crystals less than 10 µm and down to below 1 µm in size. The beamline has a fully focused beam of 0.3 × 2.3 µm (vertical × horizontal) with a tuneable energy range (6-28 keV) and high flux (1.6 × 1012 photons s-1 at 12.5 keV). The crystals are housed within a vacuum chamber to minimize background scatter from air. Crystals are plunge-cooled on cryo-electron microscopy grids, allowing much of the liquid surrounding the crystals to be removed. These factors improve the signal-to-noise during data collection and the lifetime of the microcrystals can be prolonged by exploiting photoelectron escape. A novel in vacuo sample environment has been designed which also houses a scanning electron microscope to aid with sample visualization. This combination of features at VMXm allows measurements at the physical limits of X-ray crystallography on biomacromolecules to be explored and exploited.

Interaction of Arginine-Rich Surfactant-like Peptide Nanotubes with Liposomes.

The interaction of the surfactant-like peptide (SLP) R3L12 bearing three cationic arginine residues with model liposomes is investigated in aqueous solution at various pH values, under conditions for which the SLP self-assembles into nanotubes. The structure of liposomes of model anionic lipid DPPG [1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)], or zwitterionic lipid DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine] is probed using small-angle X-ray scattering and cryogenic-transmission electron microscopy. The unilamellar vesicles of DPPG are significantly restructured in the presence of R3L12, especially at low pH, and multilamellar vesicles of DPPE are also restructured under these conditions. The SLP promotes the release of cargo encapsulated in the vesicles as probed by calcein fluorescence, with notably higher release for anionic DPPG vesicles. Laurdan fluorescence experiments to probe membrane fluidity (lipid chain ordering) show that R3L12 destabilizes the lipid gel phase, especially for anionic DPPG. This model nanotube-forming SLP has promise as a pH-sensitive release system for vesicle-encapsulated cargo.

A structural snapshot of the multiple working states of the Mpox virus helicase-primase D5.

The Mpox virus (or Monkeypox virus, MPXV) uses its own encoded proteins to form a replication machine that replicates the viral genome in the host cell cytoplasm, making this machinery a key target for antiviral drug design. The D5 (also known as the OPG117 or E5) protein, a bi-functional helicase-primase enzyme, is crucial in the MPXV replication machinery and genome uncoating process. Recently, cryo-electron microscopy (cryo-EM) structures of D5 in multiple states have been determined. These structures have elucidated the full trajectory of the MPXV D5 helicase-primase as it moves along single-stranded DNA, providing unprecedented advancements in the molecular dynamics and unwinding mechanism. This structural snapshot describes the structural features of the D5 protein and dissects the broader implications of its pivotal role in MPXV replication.

Cryo-EM reveals transition states of the Acinetobacter baumannii F1-ATPase rotary subunits γ and ε, unveiling novel compound targets.

Priority 1: critical WHO pathogen Acinetobacter baumannii depends on ATP synthesis and ATP:ADP homeostasis and its bifunctional F1FO-ATP synthase. While synthesizing ATP, it regulates ATP cleavage by its inhibitory ε subunit to prevent wasteful ATP consumption. We determined cryo-electron microscopy structures of the ATPase active A. baumannii F1-αßγεΔ134-139 mutant in four distinct conformational states, revealing four transition states and structural transformation of the ε's C-terminal domain, forming the switch of an ATP hydrolysis off- and an ATP synthesis on-state based. These alterations go in concert with altered motions and interactions in the catalytic- and rotary subunits of this engine. These A. baumannii interacting sites provide novel pathogen-specific targets for inhibitors, with the aim of ATP depletion and/or ATP synthesis and growth inhibition. Furthermore, the presented diversity to other bacterial F-ATP synthases extends the view of structural elements regulating such a catalyst.

Asymmetric fluctuation of overlapping dinucleosome studied by cryoelectron microscopy and small-angle X-ray scattering.

Nucleosome remodelers modify the local structure of chromatin to release the region from nucleosome-mediated transcriptional suppression. Overlapping dinucleosomes (OLDNs) are nucleoprotein complexes formed around transcription start sites as a result of remodeling, and they consist of two nucleosome moieties: a histone octamer wrapped by DNA (octasome) and a histone hexamer wrapped by DNA (hexasome). While OLDN formation alters chromatin accessibility to proteins, the structural mechanism behind this process is poorly understood. Thus, this study investigated the characteristics of structural fluctuations in OLDNs. First, multiple structures of the OLDN were visualized through cryoelectron microscopy (cryoEM), providing an overview of the tilting motion of the hexasome relative to the octasome at the near-atomistic resolution. Second, small-angle X-ray scattering (SAXS) revealed the presence of OLDN conformations with a larger radius of gyration than cryoEM structures. A more complete description of OLDN fluctuation was proposed by SAXS-based ensemble modeling, which included possible transient structures. The ensemble model supported the tilting motion of the OLDN outlined by the cryoEM models, further suggesting the presence of more diverse conformations. The amplitude of the relative tilting motion of the hexasome was larger, and the nanoscale fluctuation in distance between the octasome and hexasome was also proposed. The cryoEM models were found to be mapped in the energetically stable region of the conformational distribution of the ensemble. Exhaustive complex modeling using all conformations that appeared in the structural ensemble suggested that conformational and motional asymmetries of the OLDN result in asymmetries in the accessibility of OLDN-binding proteins.

Characterization of rapeseed protein supramolecular structures obtained by aqueous extractions

Aqueous extraction of rapeseed has the potential to produce mildly refined yet functional protein ingredients. However, the colloidal structures of these mildly refined ingredients are still not fully understood. Therefore, the aim of this work was to examine the structures present in rapeseed extracts by analyzing their scattering characteristics across different length scales. Rapeseed protein concentrates were obtained after aqueous extractions at acidic (pH 5.7) or alkaline (pH 8.5) conditions. The structures were investigated using complementary techniques, cryo-transmission electron microscopy, small angle X-ray scattering and small angle neutron scattering. To understand the scattering contribution of various components at characteristic length scales, D2O contrast variation was performed, and SAXS analysis for extracts at pH 5.7 treated with pectinase were compared to those untreated. All suspensions contained globular proteins within a network of soluble polysaccharides as observed by Cryo-TEM. SANS analysis with different D2O/H2O ratios demonstrated that at low q, both proteins and polysaccharide contributed to scattering, suggesting the presence of large complexes containing both. The signal for pH 8.5 suspensions was mainly attributed to oleosomes. SAXS measurements agreed with the SANS data, and pH 5.7 suspensions measured after pectin hydrolysis revealed that the complexes were formed by rapeseed globulins (∼4 nm) and polysaccharide chains. This research highlights the importance of understanding the protein-polysaccharide interactions occurring in rapeseed aqueous extracts, to fully utilize these less refined fractions as value-added ingredients in future sustainable foods.

Scalable protein design using optimization in a relaxed sequence space.

Machine learning (ML)-based design approaches have advanced the field of de novo protein design, with diffusion-based generative methods increasingly dominating protein design pipelines. Here, we report a "hallucination"-based protein design approach that functions in relaxed sequence space, enabling the efficient design of high-quality protein backbones over multiple scales and with broad scope of application without the need for any form of retraining. We experimentally produced and characterized more than 100 proteins. Three high-resolution crystal structures and two cryo-electron microscopy density maps of designed single-chain proteins comprising up to 1000 amino acids validate the accuracy of the method. Our pipeline can also be used to design synthetic protein-protein interactions, as validated experimentally by a set of protein heterodimers. Relaxed sequence optimization offers attractive performance with respect to designability, scope of applicability for different design problems, and scalability across protein sizes.

Structural basis for regulation of a CBASS-CRISPR-Cas defense island by a transmembrane anti-σ factor and its ECF σ partner.

How CRISPR-Cas and cyclic oligonucleotide-based antiphage signaling systems (CBASS) are coordinately deployed against invaders remains unclear. We show that a locus containing two CBASS and one type III-B CRISPR-Cas system, regulated by the transmembrane anti-σ DdvA and its cognate extracytoplasmic function (ECF) σ DdvS, can defend Myxococcus xanthus against a phage. Cryo-electron microscopy reveals DdvA-DdvS pairs assemble as arrow-shaped transmembrane dimers. Each DdvA periplasmic domain adopts a separase/craspase-type tetratricopeptide repeat (TPR)-caspase HetF-associated with TPR (TPR-CHAT) architecture with an incomplete His-Cys active site, lacking three α-helices conserved among CHAT domains. Each active site faces the dimer interface, raising the possibility that signal-induced caspase-like DdvA autoproteolysis in trans precedes RseP-mediated intramembrane proteolysis and DdvS release. Nuclear magnetic resonance reveals a DdvA cytoplasmic CHCC-type zinc-bound three-helix bundle that binds to DdvS σ2 and σ4 domains, undergoing σ4-induced helix extension to trap DdvS. Altogether, we provide structural-mechanistic insights into membrane anti-σ-ECF σ regulation of an antiviral CBASS-CRISPR-Cas defense island.

Current Technologies in Snake Venom Analysis and Applications

This comprehensive review explores the cutting-edge advancements in snake venom research, focusing on the integration of proteomics, genomics, transcriptomics, and bioinformatics. Highlighting the transformative impact of these technologies, the review delves into the genetic and ecological factors driving venom evolution, the complex molecular composition of venoms, and the regulatory mechanisms underlying toxin production. The application of synthetic biology and multi-omics approaches, collectively known as venomics, has revolutionized the field, providing deeper insights into venom function and its therapeutic potential. Despite significant progress, challenges such as the functional characterization of toxins and the development of cost-effective antivenoms remain. This review also discusses the future directions of venom research, emphasizing the need for interdisciplinary collaborations and new technologies (mRNAs, cryo-electron microscopy for structural determinations of toxin complexes, synthetic biology, and other technologies) to fully harness the biomedical potential of venoms and toxins from snakes and other animals.

Structural basis for hepatitis B virus restriction by a viral receptor homologue

Macaque restricts hepatitis B virus (HBV) infection because its receptor homologue, NTCP (mNTCP), cannot bind preS1 on viral surface. To reveal how mNTCP loses the viral receptor function, we here solve the cryo-electron microscopy structure of mNTCP. Superposing on the human NTCP (hNTCP)-preS1 complex structure shows that Arg158 of mNTCP causes steric clash to prevent preS1 from embedding onto the bile acid tunnel of NTCP. Cell-based mutation analysis confirms that only Gly158 permitted preS1 binding, in contrast to robust bile acid transport among mutations. As the second determinant, Asn86 on the extracellular surface of mNTCP shows less capacity to restrain preS1 from dynamic fluctuation than Lys86 of hNTCP, resulting in unstable preS1 binding. Additionally, presence of long-chain conjugated-bile acids in the tunnel induces steric hindrance with preS1 through their tailed-chain. This study presents structural basis in which multiple sites in mNTCP constitute a molecular barrier to strictly restrict HBV.

Mefloquine-induced conformational shift in Cx36 N-terminal helix leading to channel closure mediated by lipid bilayer

Connexin 36 (Cx36) forms interneuronal gap junctions, establishing electrical synapses for rapid synaptic transmission. In disease conditions, inhibiting Cx36 gap junction channels (GJCs) is beneficial, as it prevents abnormal synchronous neuronal firing and apoptotic signal propagation, mitigating seizures and progressive cell death. Here, we present cryo-electron microscopy structures of human Cx36 GJC in complex with known channel inhibitors, such as mefloquine, arachidonic acid, and 1-hexanol. Notably, these inhibitors competitively bind to the binding pocket of the N-terminal helices (NTH), inducing a conformational shift from the pore-lining NTH (PLN) state to the flexible NTH (FN) state. This leads to the obstruction of the channel pore by flat double-layer densities of lipids. These studies elucidate the molecular mechanisms of how Cx36 GJC can be modulated by inhibitors, providing valuable insights into potential therapeutic applications.

3D Cryo-Electron Microscopy Reveals the Structure of a 3-Fluorenylmethyloxycarbonyl Zipper Motif Ensuring the Self-Assembly of Tripeptide Nanofibers.

Short peptide-based supramolecular hydrogels appeared as highly interesting materials for applications in many fields. The optimization of their properties relies mainly on the design of a suitable hydrogelator through an empirical trial-and-error strategy based on the synthesis of various types of peptides. This approach is in part due to the lack of prior structural knowledge of the molecular architecture of the various families of nanofibers. The 3D structure of the nanofibers determines their ability to interact with entities present in their surrounding environment. Thus, it is important to resolve the internal structural organization of the material. Herein, using Fmoc-FFY tripeptide as a model amphiphilic hydrogelator and cryo-EM reconstruction approach, we succeeded to obtain a 3.8 Å resolution 3D structure of a self-assembled nanofiber with a diameter of approximately 4.1 nm and with apparently "infinite" length. The elucidation of the spatial organization of such nano-objects addresses fundamental questions about the way short amphiphilic N-Fmoc peptides lacking secondary structure can self-assemble and ensure the cohesion of such a lengthy nanostructure. This nanofiber is organized into a triple-stranded helix with an asymmetric unit composed of two Fmoc-FFY peptides per strand. The three identical amphiphilic strands are maintained together by strong lateral interactions coming from a 3-Fmoc zipper motif. This hydrophobic core of the nanofiber is surrounded by 12 phenyl groups from phenylalanine residues, nonplanar with the six Fmoc groups. Polar tyrosine residues at the C-term position constitute the hydrophilic shell and are exposed all around the external part of the assembly. This fiber has a highly hydrophobic central core with an internal diameter of only 2.4 Å. Molecular dynamics simulations highlight van der Waals and hydrogen bonds between peptides placed on top of each other. We demonstrate that the self-assembly of Fmoc-FFY, whether induced by annealing or by the action of a phosphatase on the phosphorylated precursor Fmoc-FFpY, results in two nanostructures with minor differences that we are unable to distinguish.

Characterization of bi-segmented and tri-segmented recombinant Pichinde virus particles.

Mammarenaviruses include several highly virulent pathogens (e.g., Lassa virus) capable of causing severe hemorrhagic fever diseases for which there are no approved vaccines and limited treatment options. Mammarenaviruses are enveloped, bi-segmented ambisense RNA viruses. There is limited knowledge about cellular proteins incorporated into progeny virion particles and their potential biological roles in viral infection. Pichinde virus (PICV) is a prototypic arenavirus used to characterize mammarenavirus replication and pathogenesis. We have developed a recombinant PICV with a tri-segmented RNA genome as a viral vector platform. Whether the tri-segmented virion differs from the wild-type bi-segmented one in viral particle morphology and protein composition has not been addressed. In this study, recombinant PICV (rPICV) virions with a bi-segmented (rP18bi) and a tri-segmented (rP18tri) genome were purified by density-gradient ultracentrifugation and analyzed by cryo-electron microscopy and mass spectrometry. Both virion types are pleomorphic with spherical morphology and have no significant difference in size despite rP18tri having denser particles. Both virion types also contain similar sets of cellular proteins. Among the highly enriched virion-associated cellular proteins are components of the endosomal sorting complex required for transport pathway and vesicle trafficking, such as ALIX, Tsg101, VPS, CHMP, and Ras-associated binding proteins, which have known functions in virus assembly and budding. Other enriched cellular proteins include peripheral and transmembrane proteins, chaperone proteins, and ribosomal proteins; their biological roles in viral infection warrant further analysis. Our study provides important insights into mammarenavirus particle formation and aids in the future development of viral vectors and antiviral discovery.IMPORTANCEMammarenaviruses, such as Lassa virus, are enveloped RNA viruses that can cause severe hemorrhagic fever diseases (Lassa fever) with no approved vaccine and limited therapeutic options. Cellular proteins incorporated into progeny virion particles and their biological roles in mammarenavirus infection have not been well characterized. Pichinde virus (PICV) is a prototypic mammarenavirus used as a surrogate model for Lassa fever. We used cryo-electron microscopy and proteomic analysis to characterize the morphology and protein contents of the purified PICV particles that package either two (bi-segmented) or three (tri-segmented) genomic RNA segments. Our results demonstrate a similar virion morphology but different particle density for the bi- and tri-segmented viral particles and reveal major virion-associated cellular proteins. This study provides important insights into the virus-host interactions that can be used for antiviral development and optimizing arenavirus-based vaccine vectors.

Stimuli-Responsive Vesicles and Hydrogels Formed by a Single-Tailed Dynamic Covalent Surfactant in Aqueous Solutions.

Controlling the hierarchical self-assembly of surfactants in aqueous solutions has drawn much attention due to their broad range of applications, from targeted drug release, preparation of smart material, to biocatalysis. However, the synthetic procedures for surfactants with stimuli-responsive hydrophobic chains are complicated, which restricts the development of surfactants. Herein, a novel single-tailed responsive surfactant, 1-methyl-3-(2-(4-((tetradecylimino) methyl) phenoxy) ethyl)-3-imidazolium bromides (C14PMimBr), was facilely fabricated in situ by simply mixing an aldehyde-functionalized imidazolium cation (3-(2-(4-formylphenoxy) ethyl)-1-methyl imidazolium bromide, BAMimBr) and aliphatic amine (tetradecylamine, TDA) through dynamic imine bonding. With increasing concentration, micelles, vesicles, and hydrogels were spontaneously formed by the hierarchical self-assembly of C14PMimBr in aqueous solutions without any additives. The morphologies of vesicles and hydrogels were characterized by cryogenic transmission electron microscopy and scanning electron microscopy. The mechanical properties and microstructure information of hydrogels were demonstrated by rheological measurement, X-ray diffraction, and density functional theory calculation. In addition, the vesicles could be disassembled and reassembled with the breakage and reformation of imine bonds by adding acid/bubbling CO2 and adding alkali. This work provides a simple method for constructing stimuli-responsive surfactant systems and shows great potential application in targeted drug release, drug delivery, and intelligent materials.

“Seeing Is Believing”: How Neutron Crystallography Informs Enzyme Mechanisms by Visualizing Unique Water Species

Hydrogen is the lightest atom and composes approximately half of the atomic content in macromolecules, yet their location can only be inferred or predicted in most macromolecular structures. This is because hydrogen can rarely be directly observed by the most common structure determination techniques (such as X-ray crystallography and electron cryomicroscopy). However, knowledge of hydrogen atom positions, especially for enzymes, can reveal protonation states of titratable active site residues, hydrogen bonding patterns, and the orientation of water molecules. Though we know they are present, this vital layer of information, which can inform a myriad of biological processes, is frustratingly invisible to us. The good news is that, even at modest resolution, neutron crystallography (NC) can reveal this layer and has emerged this century as a powerful tool to elucidate enzyme catalytic mechanisms. Due to its strong and coherent scattering of neutrons, incorporation of deuterium into the protein crystal amplifies the power of NC. This is especially true when solvation and the specific participation of key water molecules are crucial for catalysis. Neutron data allow the modeling of all three atoms in water molecules and have even revealed previously unobserved and unique species such as hydronium (D3O+) and deuteroxide (OD−) ions as well as lone deuterons (D+). Herein, we briefly review why neutrons are ideal probes for identifying catalytically important water molecules and these unique water-like species, limitations in interpretation, and four vignettes of enzyme success stories from disparate research groups. One of these groups was that of Dr. Chris G. Dealwis, who died unexpectedly in 2022. As a memorial appreciation of his scientific career, we will also highlight his interest and contributions to the neutron crystallography field. As both the authors were mentored by Chris, we feel we have a unique perspective on his love of molecular structure and admiration for neutrons as a tool to query those structures.

The N-terminal activation function AF-1 domain of ERα interacts directly with the C-terminal AF-2-holding ligand-binding domain to recruit the coactivator proteins.

Cryoelectron microscopy (cryo-EM) clarified the quaternary structure of the DNA complex of coactivator-bound estrogen receptor alpha (ERα), revealing the adjacency of the N-terminal domain (NTD) and C-terminal ligand-binding domain (LBD). ERα-NTD and LBD constitute activation function 1 (AF-1) and activation function 2 (AF-2), respectively. These domains are essential for transcription activation. Their spatial proximity was judged to be essential for ERα to recruit the SRC coactivator proteins. In the present study, we first evaluated untethered free ERα-NTD(AF-1) [residues 1-180] and its-truncated desNTD(AF-1)-ERα [residues 181-595] in a luciferase reporter gene assay. ERα-NTD(AF-1) was completely inactive, whereas desNTD(AF-1)-ERα exhibited 66% activity of wild-type ERα. Surprisingly, ERα-NTD(AF-1) was found to inhibit desNTD(AF-1)-ERα markedly. Therefore, assuming that ERα-NTD(AF-1) must also inhibit wild-type full-length ERα, we co-expressed ERα-NTD(AF-1) and full-length ERα. As expected, ERα-NTD(AF-1) inhibited ERα in a dose-dependent manner, but non-competitively for 17β-estradiol. When their intracellular transport was examined immunocytochemically, ERα-NTD(AF-1) showed a distinct translocation from the cytoplasm to the nucleus, despite being expressed solely in the cytoplasm without full-length ERα. This nuclear translocation was attributable to a direct interaction between ERα-NTD(AF-1) and full-length ERα consisting of the nuclear localization signal. The present results demonstrated that, in full-length ERα, the N-terminally tethered NTD(AF-1) domain collaborates with the C-terminal LBD(AF-2) for coactivator recruitment.

Two Forms of Thick Filament in the Flight Muscle of Drosophila melanogaster.

Invertebrate striated muscle myosin filaments are highly variable in structure. The best characterized myosin filaments are those found in insect indirect flight muscle (IFM) in which the flight-powering muscles are not attached directly to the wings. Four insect orders, Hemiptera, Diptera, Hymenoptera, and Coleoptera, have evolved IFM. IFM thick filaments from the first three orders have highly similar myosin arrangements but differ significantly among their non-myosin proteins. The cryo-electron microscopy of isolated IFM myosin filaments from the Dipteran Drosophila melanogaster described here revealed the coexistence of two distinct filament types, one presenting a tubular backbone like in previous work and the other a solid backbone. Inside an annulus of myosin tails, tubular filaments show no noticeable densities; solid filaments show four paired paramyosin densities. Both myosin heads of the tubular filaments are disordered; solid filaments have one completely and one partially immobilized head. Tubular filaments have the protein stretchin-klp on their surface; solid filaments do not. Two proteins, flightin and myofilin, are identifiable in all the IFM filaments previously determined. In Drosophila, flightin assumes two conformations, being compact in solid filaments and extended in tubular filaments. Nearly identical solid filaments occur in the large water bug Lethocerus indicus, which flies infrequently. The Drosophila tubular filaments occur in younger flies, and the solid filaments appear in older flies, which fly less frequently if at all, suggesting that the solid filament form is correlated with infrequent muscle use. We suggest that the solid form is designed to conserve ATP when the muscle is not in active use.