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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.