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C-type lectins (CTLs) play key roles in immunity and microbial carbohydrate recognition. In the vector-mosquito Aedes aegypti, the C-type lectin domain-single (CTLD-S) family comprises 34 soluble CTLs whose members are implicated in flavivirus dissemination and microbial homeostasis, yet their organization remains uncharacterized. We combine X-ray crystallography, small-angle X-ray scattering (SAXS), molecular dynamics, and machine learning-based structure prediction to characterize CTLs in Aedes aegypti. We determined the crystal structures of four representative CTLD-S proteins: mosGCTL-1, -3, -6, and -20. All crystals featured an identical homodimer arrangement, positioning both carbohydrate-binding sites on the same molecular face. Dimerization was confirmed in solution and AlphaFold predictions across the entire family indicated that dimer formation may be a unifying feature of CTLD-S proteins. For one mosGCTL structure, paucimannose glycans bound at a Ca2+-dependent site, demonstrating bidentate binding through one dimer. Machine learning-based predictions indicated hundreds of possible CTLD-S heterodimers may be viable, with wide-ranging implications for preferred glycan binding through one dimer. Our findings reveal a conserved dimeric arrangement among mosquito lectins that may underpin ligand recognition relevant to vector–pathogen interactions.

Henipaviruses, such as Nipah virus, can cause deadly illness and constitute WHO blueprint priorities due to their pandemic potential. Their genomes are packaged within a nucleocapsid consisting of viral nucleoproteins (N). Now, it is unclear how the encapsidated genome is released from N to allow the viral polymerase to read its sequence. Here, we present the high-resolution cryo-EM structure of a helical N-RNA filament from Langya henipavirus (LayV), allowing us to identify vertical interactions crucial for assembly. We show that assembly efficiency is sequence-dependent and prefers 5'-genomic sequences. Further, we solve the structure of an RNA-free assembly of LayV-N. Structural comparison of the RNA-bound and RNA-free LayV-N shows a conformational opening and closing, even within the assembled state. Our data suggest that N within nucleocapsids may undergo local conformational changes, switching between closed and open states, to temporarily allow access to the encapsidated RNA without nucleocapsid disruption.

A flurry of recent structural studies have focused on the polymerase complex of the deadly zoonotic pathogen Nipah virus (NiV). These include a report by Sala et al. describing an RNA duplex-bound state. This structure constitutes a snapshot of the complex in an early elongation step of the RNA synthesis cycle.

Despite recent advances in cryo-electron microscopy and artificial intelligence-based model predictions, a significant fraction of structure determinations by macromolecular crystallography still requires experimental phasing, usually by means of single-wavelength anomalous diffraction (SAD) techniques. Most synchrotron beamlines provide highly brilliant beams of X-rays of between 0.7 and 2 Å wavelength. Use of longer wavelengths to access the absorption edges of biologically important lighter atoms such as calcium, potassium, chlorine, sulfur and phosphorus for native-SAD phasing is attractive but technically highly challenging. The long-wavelength beamline I23 at Diamond Light Source overcomes these limitations and extends the accessible wavelength range to λ = 5.9 Å. Here we report 22 macromolecular structures solved in this extended wavelength range, using anomalous scattering from a range of elements which demonstrate the routine feasibility of lighter atom phasing. We suggest that, in light of its advantages, long-wavelength crystallography is a compelling option for experimental phasing.

Human metapneumovirus (HMPV) is a major cause of respiratory illness in young children. The HMPV polymerase (L) binds an obligate cofactor, the phosphoprotein (P). During replication and transcription, the L/P complex traverses the viral RNA genome, which is encapsidated within nucleoproteins (N). An essential interaction between N and a C-terminal region of P tethers the L/P polymerase to the template. This N-P interaction is also involved in the formation of cytoplasmic viral factories in infected cells, called inclusion bodies. To define how the polymerase component P recognizes N-encapsidated RNA (N-RNA) we employed cryogenic electron microscopy (cryo-EM) and molecular dynamics simulations, coupled to activity assays and imaging of inclusion bodies in cells. We report a 2.9 Å resolution structure of a triple-complex between multimeric N, bound to both RNA and the C-terminal region of P. Furthermore, we also present cryo-EM structures of assembled N in different oligomeric states, highlighting the plasticity of N. Combined with our functional assays, these structural data delineate in molecular detail how P attaches to N-RNA whilst retaining substantial conformational dynamics. Moreover, the N-RNA-P triple complex structure provides a molecular blueprint for the design of therapeutics to potentially disrupt the attachment of L/P to its template.