Small Helix Research Articles

The Crystal Structure of the Domain of Unknown Function1480 (DUF1480) From Klebsiella pneumoniae.

Domains of unknown function (DUFs) continue to comprise a significant portion of bacterial proteomes, with more than 20% of bacterial proteins remaining annotated as DUFs. The characterization of their molecular structure can provide valuable insight that is not captured by the primary sequence analysis, thus providing a segue into the identification of the molecular function of DUF representatives. Here, we present the crystal structure of KPN_02352 from Klebsiella pneumoniae subsp. pneumoniae, a DUF1480 domain-containing protein, which was determined to be 1.75 Å resolution. Representatives of the DUF1480 family are found broadly across Enterobacterales and have been previously shown to contribute to the antibiotic response. Our structural analysis suggests that DUF1480 is comprised of a six-stranded split barrel fold featuring a small alpha helix that is positioned to cap one end of the split barrel. DUF1480 was found to be monomeric in solution, and harbors structural similarity to response regulators. The crystal structure of DUF1480 is the first experimental insight into the molecular structure of this conserved protein family, revealing several conserved features that may be functionally relevant.

Quantification of flows in a rectangular channel of a single-screw extruder with a small helix angle based on the energy dissipation rate

Quantification of flows in a rectangular channel of a single-screw extruder with a small helix angle based on the energy dissipation rate

Crystal structure of Kunitz-type trypsin inhibitor: Entomotoxic effect of native and encapsulated protein targeting gut trypsin of Tribolium castaneum Herbst

Trypsin inhibitors are known to act against insect pests by inhibiting proteases of the digestive tract. In this study, we report structural and functional characterization of ∼ 19 kDa Albizia procera Kunitz-type trypsin inhibitor (ApKTI) protein with potential bio-insecticidal applications. Crystal structure of ApKTI protein has been refined to 1.42 Å and molecular structure (8HNR) showed highly beta sheeted conformation including 12 beta sheets, 15 loops and two small alpha helices. Docking between predicted model of Tribolium castaneum trypsin (TcPT) and 8HNR produced a stable complex (−11.3 kcal/mol) which reflects the inhibitory potential of ApKTI against insect gut trypsin. Significant mortality was observed in all life stages of T. castaneum including egg, larvae, pupae and adults with a 3.0 mg native ApKTI treatment in comparison to negative control. Although standard trypsin inhibitor (Glycine max trypsin inhibitors; GmKTI; 3.0 mg) produced maximum reduction against all above life stages; however, a non-significant mortality difference was observed in comparison to 3.0 mg native ApKTI. The study further explores the synthesis and characterization of Graphene (GNPs) and Zinc oxide (ZnONPs) nanoparticles, followed by the optimization of ApKTI and GmKTI loading on both nanoparticles to evaluate their enhanced insecticidal effectiveness. Encapsulated proteins showed significant mortality against T. castaneum across all concentrations, with GNPs proving more effective than ZnONPs. Additionally, encapsulated GmKTI produced significant mortality of eggs compared to loaded ApKTI treatments while other life stages were non-significantly affected by two proteins. This research highlights the importance of encapsulated ApKTI protein for eco-friendly pest management strategies.

HIV-1 Intasomes Assembled with Excess Integrase C-Terminal Domain Protein Facilitate Structural Studies by Cryo-EM and Reveal the Role of the Integrase C-Terminal Tail in HIV-1 Integration.

Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.

Investigation of the Solid-State Interactions in Lyophilized Human G-CSF Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at -20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and -20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.

Synergetic enrichment of aggrecan in nucleus pulposus cells by scAAV6-shRNA-mediated knockdown of aggrecanase-1 and aggrecanase-2.

Degenerative disk disease (DDD) that aggravates structural deterioration of intervertebral disks (IVDs) can be accompanied by painful inflammation and immunopathological progressions. Current surgical or pharmacological therapies cannot repair the structure and function of IVDs. Nucleus pulposus (NP) cells are crucial for the preservation or restoration of IVDs by balancing the anabolic and catabolic factors affecting the extracellular matrix. Imbalanced anabolic and catabolic factors cause increased degradation of aggrecan. Aggrecanases A Disintegrin And Metalloproteinase with ThromboSpondin motifs (ADAMTS)4 and ADAMTS5 are the main degrading enzymes of aggrecan. Previously, we characterized adeno-associated virus (AAV6) as the most suitable serotype with marked NP cellular tropism and demonstrated that ADAMTS4 could be silenced by self-complementary adeno-associated virus grade 6 small helix ribonucleic acid (scAAV6-shRNA) in NP cells of degeneration grade III, which resulted in enrichment of aggrecan. Nonetheless, neither scAAV6-shRNA-mediated inhibition of ADAMTS5 nor joint inhibitions of ADAMTS4 and ADAMTS5 have been investigated, although both enzymes are regulated by analogous proinflammatory cytokines and have the same cleavage sites in aggrecan. Therefore, we attempted scAAV6-shRNA-mediated inhibitions of both enzymes in NP cells of degeneration grade IV to increase efficacies in treatments of DDD. The degeneration grade of IVDs in patients was determined by magnetic resonance imaging (MRI) before surgical operations. After isolation and culturing of NP cells, cells were transduced with scAAV6-shRNAs targeting ADAMTS4 or ADAMTS5. Transduced cells were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR), fluorescence microscopy, flow cytometry-assisted cell sorting (FACS), MTT assay (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay), immunoblotting, and enzyme-linked immunosorbent assay (ELISA). Joint transduction of NP cells exhibited high transduction efficacies (98.1%), high transduction units (TU) (1381 TU/Cell), and no effect on cell viability or proliferation. Above all joint treatments resulted in effective knockdown of ADAMTS4 (92.8%) and ADAMTS5 (93.4%) along with additive enrichment of aggrecan (113.9%). Treatment effects were significant for more than 56 days after transduction (P < 0.001). In conclusion, scAAV6-shRNA-mediated combined molecular therapy could be very valuable for more effective, durable, and less immunogenic treatment approaches in DDD.

Closed-Form Expression of Radiation Characteristics for Electrically Small Spherical Helix Antennas

To understand the radiation mechanism of an electrically small spherical helix antenna, we develop a theory on the radiation characteristics of the antenna. An analytical model of the antenna presuming a current on the wire to be sinusoidally distributed is proposed and analyzed with the spherical wave expansion. The radiation efficiency, radiation resistance, and radiation patterns are obtained in closed-form expression. The radiation efficiency evidently varies with the surface area of the wire and the radiation resistance depends on the square of the length of the wire. The obtained result for the radiation pattern illustrates the tilt of the pattern caused by the modes asymmetric to the z-axis. The radiation efficiency formula indicates a good agreement between the simulation and measurement result. In addition, the radiation resistance of the theoretical and simulation results exhibits good agreement. Considering the effect of the feeding structure of the fabricated antenna, the radiation resistance of the analytical model can be treated as a reasonable result. The result of radiation pattern also shows good agreement between the simulation and measurement results excluding a small contribution from the feeding cable acting as a scatterer.

Low-velocity impact resistance behaviors of bionic double-helicoidal composite laminates

To improve the low-velocity impact resistance of composite laminates, a bionic double-helicoidal composite laminate (BDHCL) emulated from the micro-structure of coelacanth scales is proposed. The micro-structure is a double helicoidal Bouligand structure in which two adjacent collagen layers are arranged into an orthogonal bilayer and then rotating at a small helix angle. Three types of helicoidal configurations with different derivatives are designed, including sine rotation angle (SRA), linear rotation angle (LRA), and exponential rotation angle (ERA). The progressive intra-laminar and inter-laminar damage model is adopted to capture the failure behaviors of material. The impact behaviors of BDHCLs with several helix angles and included angles of the bilayer are studied. The results show that the double-helicoidal Bouligand structure has a higher resistance load and shallower penetration depth. The impact resistance of ERA laminate has the maximum improvement among the above three types of helix angle. A certain included angle of bilayer for the BDHCL will exhibit excellent impact resistance.

Investigating the role of SERBP1 in translation.

Idle ribosomes are inactive 80S monosomes that lack mRNA and are common throughout eukaryotes. In yeast, they promote starvation recovery and translational reactivation. In metazoan, their physiological functions and the mechanisms governing their abundance are unclear. Current hypotheses posit that they play a role in fine tuning protein synthesis under conditions requiring tight regulation such as cell development and plasticity. Previously, we determined cryo-EM structures of idle ribosomes containing serpine 1 mRNA binding protein (SERBP1) and phosphorylated eukaryotic elongation factor 2 (p-eEF2). SERBP1 binds to the 40S head via a small helix and threads through the mRNA channel, where it interacts with eEF2. Here, we term these regions ‘anchor’ and ‘thread’, respectively. Stimulation of eEF2 kinase (eEF2K) increases the abundance of SERBP1/p-eEF2 idle ribosomes and prevents their recycling in vitro. SERBP1 also regulates the stability of specific mRNAs and contains motifs that drive phase separation. We propose that different regions of SERBP1 mediate its functions in translation and localization in liquid phase separation. We designed structure-informed truncations and mutations and examined their effect on protein synthesis in an in vitro translation system. We found that recombinant full-length SERBP1 strongly inhibits translation. A C-terminal truncation including the thread and anchor regions shows a similar inhibitory effect, attenuated upon mutating the binding sites with eEF2. The SERBP1 anchor alone does not affect protein synthesis. eEF2K strongly inhibits translation and this effect is not enhanced in the presence of the anchor. Collectively, our data suggest that the SERBP1 thread and specifically the residues interacting with eEF2 are critical for attenuation of protein synthesis, likely by promoting the formation of idle ribosomes. Further studies will illuminate if other SERBP1 regions are implicated in this process and in phase separation, and if eEF2K acts synergistically to inhibit translation.

Molecular dynamics simulations of the calmodulin-induced α-helix in the SK2 calcium-gated potassium ion channel

The family of small-conductance Ca2+-activated potassium ion channels (SK channels) is composed of four members (SK1, SK2, SK3, and SK4) involved in neuron-firing regulation. The gating of these channels depends on the intracellular Ca2+ concentration, and their sensitivity to this ion is provided by calmodulin (CaM). This protein binds to a specific region in SK channels known as the calmodulin-binding domain (CaMBD), an event which is essential for their gating. While CaMBDs are typically disordered in the absence of CaM, the SK2 channel subtype displays a small prefolded α-helical region in its CaMBD even if CaM is not present. This small helix is known to turn into a full α-helix upon CaM binding, although the molecular-level details for this conversion are not fully understood yet. In this work, we offer new insights on this physiologically relevant process by means of enhanced sampling, atomistic Hamiltonian replica exchange molecular dynamics simulations, providing a more detailed understanding of CaM binding to this target. Our results show that CaM is necessary for inducing a full α-helix along the SK2 CaMBD through hydrophobic interactions with V426 and L427. However, it is also necessary that W431 does not compete for these interactions; the role of the small prefolded α-helix in the SK2 CaMBD would be to stabilize W431 so that this is the case. In conclusion, our findings provide further insight into a key interaction between CaM and SK channels that is important for channel sensitivity to Ca2+.

Probing small ribosomal subunit RNA helix 45 acetylation across eukaryotic evolution.

NAT10 is an essential enzyme that catalyzes N4-acetylcytidine (ac4C) in eukaryotic transfer RNA and 18S ribosomal RNA. Recent studies suggested that rRNA acetylation is dependent on SNORD13, a box C/D small nucleolar RNA predicted to base-pair with 18S rRNA via two antisense elements. However, the selectivity of SNORD13-dependent cytidine acetylation and its relationship to NAT10’s essential function remain to be defined. Here, we demonstrate that SNORD13 is required for acetylation of a single cytidine of human and zebrafish 18S rRNA. In-depth characterization revealed that SNORD13-dependent ac4C is dispensable for human cell growth, ribosome biogenesis, translation and development. This loss of function analysis inspired a cross-evolutionary survey of the eukaryotic rRNA acetylation ‘machinery’ that led to the characterization of many novel metazoan SNORD13 genes. This includes an atypical SNORD13-like RNA in Drosophila melanogaster which guides ac4C to 18S rRNA helix 45 despite lacking one of the two rRNA antisense elements. Finally, we discover that Caenorhabditis elegans 18S rRNA is not acetylated despite the presence of an essential NAT10 homolog. Our findings shed light on the molecular mechanisms underlying SNORD13-mediated rRNA acetylation across eukaryotic evolution and raise new questions regarding the biological and evolutionary relevance of this highly conserved rRNA modification.

Back Cover: Accelerated Mechanochemistry in Helical Polymers (Angew. Chem. Int. Ed. 14/2022)

Solution polymer mechanochemistry is well understood in random coil morphology. In their Research Article (e202115325), Charles E. Diesendruck and Hang Zhang assess covalent mechanochemistry when α-helices are present in covalent macromolecules. Poly(γ-benzyl glutamate)s (PBG) with different helicities are prepared by tuning the enantiomeric excess of the feed monomer. Ultrasonication is used to induce solvodynamic shear, leading to the scission of the polymer into smaller helices.

The instability of a helical vortex filament under a free surface

We perform a theoretical investigation of the instability of a helical vortex filament beneath a free surface in a semi-infinite ideal fluid. The focus is on the leading-order free-surface boundary effect upon the equilibrium form and instability of the vortex. This effect is characterised by the Froude number $F_r = U(gh^*)^{-{1}/{2}}$ where $g$ is gravity, and $U = \varGamma /(2{\rm \pi} b^*)$ with $\varGamma$ being the strength, $2{\rm \pi} b^*$ the pitch and $h^*$ the centre submergence of the helical vortex. In the case of $F_r \rightarrow 0$ corresponding to the presence of a rigid boundary, a new approximate equilibrium form is found if the vortex possesses a non-zero rotational velocity. Compared with the infinite fluid case (Widnall, J. Fluid Mech., vol. 54, no. 4, 1972, pp. 641–663), the vortex is destabilised (or stabilised) to relatively short- (or long-)wavelength sub-harmonic perturbations, but remains stable to super-harmonic perturbations. The wall-boundary effect becomes stronger for smaller helix angle and could dominate over the self-induced flow effect depending on the submergence. In the case of $F_r &gt; 0$ , we obtain the surface wave solution induced by the vortex in the context of linearised potential-flow theory. The wave elevation is unbounded when the $m$ th wave mode becomes resonant as $F_r$ approaches the critical Froude numbers ${\mathcal {F}} (m) = (C_0^*/U)^{-1} (mh^*/b^*)^{-{1}/{2}}$ , $m=1, 2, \ldots,$ where $C_0^*$ is the induced wave speed. We find that the new approximate equilibrium of the vortex exists if and only if $F_r &lt; {\mathcal {F}}(1)$ . Compared with the infinite fluid and $F_r \rightarrow 0$ cases, the wave effect causes the vortex to be destabilised to super-harmonic and long-wavelength sub-harmonic perturbations with generally faster growth rate for greater $F_r$ and smaller helix angle.

A short helix regulates conversion of dimeric and 24-meric ferritin architectures

The inter-subunit interaction at the protein interfaces plays a key role in protein self-assembly, through which enabling protein self-assembly controllable is of great importance for preparing the novel nanoscale protein materials with unexplored properties. Different from normal 24-meric ferritin, archaeal ferritin, Thermotoga maritima ferritin (TmFtn) naturally occurs as a dimer, which can assemble into a 24-mer nanocage induced by salts. However, the regulation mechanism of protein self-assembly underlying this phenomenon remains unclear. Here, a combination of the computational energy simulation and key interface reconstruction revealed that a short helix involved interactions at the C4 interface are mainly responsible for the existence of such dimer. Agreeing with this idea, deletion of such short helix of each subunit triggers it to be a stable dimer, which losses the ability to reassemble into 24-meric ferritin in the presence of salts in solution. Further support for this idea comes from the observation that grafting a small helix from human H ferritin onto archaeal subunit resulted in a stable 24-mer protein nanocage even in the absence of salts. Thus, these findings demonstrate that adjusting the interactions at the protein interfaces appears to be a facile, effective approach to control subunit assembly into different protein architectures.

Protein Engineering and HDX Identify Structural Regions of G-CSF Critical to Its Stability and Aggregation.

The protein engineering and formulation of therapeutic proteins for prolonged shelf-life remain a major challenge in the biopharmaceutical industry. Understanding the influence of mutations and formulations on the protein structure and dynamics could lead to more predictive approaches to their improvement. Previous intrinsic fluorescence analysis of the chemically denatured granulocyte colony-stimulating factor (G-CSF) suggested that loop AB could subtly reorganize to form an aggregation-prone intermediate state. Hydrogen deuterium exchange mass spectrometry (HDX-MS) has also revealed that excipient binding increased the thermal unfolding transition midpoint (Tm) by stabilizing loop AB. Here, we have combined protein engineering with biophysical analyses and HDX-MS to reveal that increased exchange in a core region of the G-CSF comprising loop AB (ABI, a small helix, ABII) and loop CD packed onto helix B and the beginning of loop BC leads to a decrease in Tm and higher aggregation rates. Furthermore, some mutations can increase the population of the aggregation-prone conformation within the native ensemble, as measured by the greater local exchange within this core region.

Enriching terpinen-4-ol from tea tree (Melaleuca alternifolia) oil using vacuum fractional distillation: Effect of column and packings on the separation

Two types of columns, three types of packings, and four levels of column height were considered to investigate how column and packings affected the separation in the vacuum fractional distillation process of raw tea tree (Melaleuca alternifolia) oil (TTO). This study discussed those effects on purity, yield, and overall discovery to select the most excellent operating conditions for enriching terpinen-4-ol. After the experiments, the essential oil was successfully separated into two fractions, in which the second one composed mostly the main TTO constituent, terpinen-4-ol. The best result was achieved by conducting the distillation on a 300-mm Hempel column filled with small Fenske helices (10 mm × 2 mm i.d.) at the system pressure of 60 mmHg. GC/MS analysis showed an almost 2.5-fold increase in the content of terpinen-4-ol, from 39.23% to 95.77% after fractionation. Meanwhile, there was 75% of terpinen-4-ol successfully recovered from its parental oil. Hence, the vacuum fraction distillation could be an effective method to enrich the terpinen-4-ol content in TTO.

Spiral Bevel Gears Nonlinear Vibration Having Radial and Axial Misalignments Effects

In gear transmissions, vibration causes noise and malfunction. In actual applications, misalignments contribute to intensifying the destructive effect of vibrations. In this paper, the nonlinear dynamics of a spiral bevel gear pair, with small helix angle, considering different misalignments, are deeply investigated. Axial misalignment, radial misalignment, and the combination of these two types are considered in this study. The governing equation is numerically solved through an implicit Runge–Kutta scheme. Since the main goal of this study is the analysis of the dynamic scenario, the mesh stiffness of the gear pair is obtained from the literature. The dynamical system is nonlinear and time-varying; it is analyzed through time responses, phase portraits, Poincaré maps, and bifurcation diagrams. Results show that, among the considered three cases with different types of misalignments, the spiral bevel gear with axial misalignment is the worst destructive case; aperiodic, subharmonic, and multiperiod responses are observable for this case. It is interesting that the chaotic responses for the case, having both types of misalignments, are less likely for the case with axial misalignment, only.

Transmission of electromagnetic wave through an in-plane square array of small helices overlying a dielectric slab

Transmission of a linear polarized electromagnetic wave through an infinite square array of small helices overlying a dielectric layer has been investigated by using semi-analytical method. The possibility of an abrupt change of polarization characteristics of the transmitted wave at varying of wavelengths in the vicinity of the waveguide resonance in a dielectric slab has been demonstrated. The phenomenon can prove useful for creation of switching devices, for light polarization control, for fine tuning of laser emission frequency, for precise measurement of the refraction index of dielectric media and for solving some other spectroscopy problems.

Kirkwood-Buff-Derived Force Field for Peptides and Proteins: Applications of KBFF20.

Here, we perform structural, thermodynamic, and kinetics tests of the Kirkwood-Buff-derived force field, KBFF20, for peptides and proteins developed in the previous article. The physical/structural tests measure the ability of KBFF20 to capture the experimental J-couplings for small peptides, to keep globular monomeric and oligomeric proteins folded, and to produce the experimentally relevant expanded conformational ensembles of intrinsically disordered proteins. The thermodynamic-based tests probe KBFF20's ability to quantify the preferential interactions of sodium chloride around native β-lactoglobulin and urea around native lysozyme, to reproduce the melting curves for small helix- and sheet-based peptides, and to fold the small proteins Trp-cage and Villin. The kinetics-based tests quantify how well KBFF20 can match the experimental contact formation rates of small, repeat-sequence peptides of variable lengths and the rotational diffusion coefficients of globular proteins. The results suggest that KBFF20 is naturally able to reproduce properties of both folded and disordered proteins, which we attribute to the use of the Kirkwood-Buff theory as the foundation of the force field's development. However, we show that KBFF20 tends to lose some well-defined secondary structural elements and increases the percentage of coil regions, indicating that the perfect balance of all interactions remains elusive. Nevertheless, we argue that KBFF20 is an improvement over recently modified force fields that require ad hoc interventions to prevent the collapse of intrinsically disordered proteins.

Interferon-Induced Transmembrane Protein 3 Blocks Fusion of Diverse Enveloped Viruses by Altering Mechanical Properties of Cell Membranes.

Interferon-induced transmembrane protein 3 (IFITM3) potently inhibits entry of diverse enveloped viruses by trapping the viral fusion at a hemifusion stage, but the underlying mechanism remains unclear. Here, we show that recombinant IFITM3 reconstituted into lipid vesicles induces negative membrane curvature and that this effect maps to its small amphipathic helix (AH). We demonstrate that AH (i) partitions into lipid-disordered domains where IAV fusion occurs, (ii) induces negative membrane curvature, and (iii) increases lipid order and membrane stiffness. These effects on membrane properties correlate with the fusion-inhibitory activity, as targeting the ectopically expressed AH peptide to the cytoplasmic leaflet of the cell plasma membrane diminishes IAV-cell surface fusion induced by exposure to acidic pH. Our results thus imply that IFITM3 inhibits the transition from hemifusion to full fusion by imposing an unfavorable membrane curvature and increasing the order and stiffness of the cytoplasmic leaflet of endosomal membranes. Our findings reveal a universal mechanism by which cells block entry of diverse enveloped viruses.