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Nipah virus (NiV) and Hendra virus (HeV) are zoonotic henipaviruses (HNVs) responsible for outbreaks of encephalitis and respiratory illness. The entry of HNVs into host cells requires the attachment (G) and fusion (F) glycoproteins, which are the main targets of antibody responses. To understand viral infection and host immunity, we determined a cryo–electron microscopy structure of the NiV G homotetrameric ectodomain in complex with the nAH1.3 broadly neutralizing antibody Fab fragment. We show that a cocktail of two nonoverlapping G-specific antibodies neutralizes NiV and HeV synergistically and limits the emergence of escape mutants. Analysis of polyclonal serum antibody responses elicited by vaccination of macaques with NiV G indicates that the receptor binding head domain is immunodominant. These results pave the way for implementing multipronged therapeutic strategies against these deadly pathogens.

Building accurate protein models into moderate resolution (3-5Å) cryo-electron microscopy (cryo-EM) maps is challenging and error-prone. While the majority of solved cryo-EM structures are at these resolutions, there are few model validation metrics that can precisely evaluate the local quality of atomic models built into these maps. We have developed MEDIC (Model Error Detection in Cryo-EM), a robust statistical model to identify residue-level errors in protein structures built into cryo-EM maps. Trained on a set of errors from obsoleted protein structures, our model draws off two major sources of information to predict errors: the local agreement of model and map compared to expected, and how “native-like” the neighborhood around a residue looks, as predicted by a deep learning model. MEDIC is validated on a set of 28 structures that were subsequently solved to higher-resolutions, where our model identifies the differences between low- and high-resolution structures with 68% precision and 60% recall. We additionally use this model to rebuild 12 deposited structures, fixing 2 sequence registration errors, 51 areas with improper secondary structure, 51 incorrect loops, and 16 incorrect carbonyls, showing the value of this approach to guide model building.

While native scaffolds offer a large diversity of shapes and topologies for enzyme engineering, their often unpredictable behavior in response to sequence modification makes de novo generated scaffolds an exciting alternative. Here we explore the customization of the backbone and sequence of a de novo designed eight stranded β-barrel protein to create catalysts for a retro-aldolase model reaction. We show that active and specific catalysts can be designed in this fold and use directed evolution to further optimize activity and stereoselectivity. Our results support previous suggestions that different folds have different inherent amenability to evolution and this property could account, in part, for the distribution of natural enzymes among different folds.

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.

SUMMARYThe actin cytoskeleton is critical for cell migration, morphogenesis, endocytosis, organelle dynamics, and cytokinesis. To support diverse cellular processes, actin filaments form a variety of structures with specific architectures and dynamic properties. Key proteins specifying actin filaments are tropomyosins. Non-muscle cells express several functionally non-redundant tropomyosin isoforms, which differentially control the interactions of other proteins, including myosins and ADF/cofilin, with actin filaments. However, the underlying molecular mechanisms have remained elusive. By determining the cryogenic electron microscopy structures of actin filaments decorated by two functionally distinct non-muscle tropomyosin isoforms, Tpm1.6 and Tpm3.2, we reveal that actin filament conformation remains unaffected upon binding. However, Tpm1.6 and Tpm3.2 follow different paths along the major groove of the actin filament, providing an explanation for their incapability to co-polymerize on actin filaments. The structures and biochemical work also elucidate the molecular basis underlying specific roles of Tpm1.6 and Tpm3.2 in myosin II activation and protecting actin filaments from ADF/cofilin-catalysed severing.

Potassium-coupled chloride transporters (KCCs) play crucial roles in regulating cell volume and intracellular chloride concentration. They are characteristically inhibited under isotonic conditions via phospho-regulatory sites located within the cytoplasmic termini. Decreased inhibitory phosphorylation in response to hypotonic cell swelling stimulates transport activity, and dysfunction of this regulatory process has been associated with various human diseases. Here, we present cryo-EM structures of human KCC3b and KCC1, revealing structural determinants for phospho-regulation in both N- and C-termini. We show that phospho-mimetic KCC3b is arrested in an inward-facing state in which intracellular ion access is blocked by extensive contacts with the N-terminus. In another mutant with increased isotonic transport activity, KCC1 Delta 19, this interdomain interaction is absent, likely due to a unique phospho-regulatory site in the KCC1 N-terminus. Furthermore, we map additional phosphorylation sites as well as a previously unknown ATP/ADP-binding pocket in the large C-terminal domain and show enhanced thermal stabilization of other CCCs by adenine nucleotides. These findings provide fundamentally new insights into the complex regulation of KCCs and may unlock innovative strategies for drug development.

To contribute to the combat of COVID-2019, we applied structure-based computational docking screens using flexible docking protocol of Rosetta GALigandDock against multiple potential SARS-CoV-2 protein targets, including the Nsp5 3-chymotrypsin-like protease (3CLpro), the Nsp3 ADP ribose phosphatase, the Nsp15 Endoribonuclease, the RNA binding domain of nucleocapsid phosphoprotein, the Nsp16 2'-O-MTase, Nsp14, and Nsp12 RNA-dependent RNA polymerase. Screening against a re-purposing library of 8,395 FDA approved drugs at various stages of drug development and various natural products from DrugBank, we found a total of 124 putative inhibitors with predicted binding ∆G less than -8.9 kcal/mol, including HIV-AIDS drugs Nelfinavir and Tipranavir, targeting 3Clpro with ∆G=-18.8 kcal/mol and ∆G=-16.6 kcal/mol respectively. These primarily involve binders to the Nsp5 3CLpro (37 hits) and the Nsp3 ADP ribose phosphatase (36 hits), with smaller numbers of hits to other targets. These small molecule putative inhibitors suggest a possible avenue for drug repurposing, and the identified compounds should serve as a high-priority list for experimental validation via co-crystallization, enzymatic and cell based assays.

This work reports a general wet-chemistry method to produce Au-Cu-X (X = Pt, Pd, and Ag) trimetallic nanorods using galvanic replacement reaction with Au-Cu nanorods as the templates. The mild conditions, such as low temperature and slow injection of metal precursors, contributed to the slow galvanic replacement reaction and helped keep the rod structure intact. The distribution of Au, Cu and the doping metals was even in the rods as confirmed by elemental mapping. The alloyed trimetallic nanorods showed enhanced catalytic activity for p-nitrophenol reduction after incorporating the third metal. Remarkably, the Au-Cu-Pd and Au-Cu-Pt nanorods show more than an order of magnitude improvement in the mass activities compared to the Au-Cu nanorods. This facile and general synthetic method can be applied to fabricate other multimetallic nanoparticles with varying shapes and compositions.

Hybrid metal–semiconductor nanostructures are promising candidates for photocatalytic applications because of the efficient charge separation in the nanostructure. Silver nanocrystals can be used as seeds to synthesize hybrid nanostructures, providing great optical and electronic properties. But they are not much explored because they tend to get oxidized or sulfurized. Here, we report a general protocol for synthesizing Ag based hybrid metal chalcogenide nanorods using the complex of oleylamine and chalcogenide as a precursor. This method allows for the epitaxial growth of metal chalcogenides without sulfurization of Ag seeds by controlled release of chalcogenides in the reaction process. From the experimental analysis, we propose a growth mechanism of the heteronanorod formation. That is, the metal sulfide firstly grows on one side of the Ag seeds, while the other side of the Ag seed is sulfurized and the Ag2S phase is formed subsequently in the presence of an excess sulfur precursor. The synthetic method is demonstrated to be widely applicable for organic phase metal chalcogenide nanorod growth, including CdS, ZnS, MnS, and CdSe. Moreover, the hybrid nanorods without sulfurization of the Ag seeds exhibit significantly improved photocatalytic activity, due to the efficient charge separation in these materials.

Development of trimetallic nanoparticles can potentially broaden the optical window for plasmonic applications, but can also offer new photocatalysts for organic synthesis. The optical and catalytic properties can be tuned by the ratio between different components and how they are distributed in the nanoparticles, in addition to their size and shape. This work reports a seed-mediated coreduction method for the fabrication of Au-Cu-Ag nanorods in oil phase. The composition and morphology of trimetallic Au-Cu-Ag nanostructures can be controlled by adjusting the injection temperature and the concentration of silver precursor. An additional peak (around 395 nm) is observed in extinction spectrum of trimetallic Au-Cu-Ag nanorods compared to that of bimetallic Au-Cu nanorods, caused by the segregated silver composition in the trimetallic nanorods. The reported synthesis can be used to develop a wide range of trimetallic nanostructures with tunable optical properties and functionality.