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Beamlines/Technical Area	Publication Details	Published
I02-Macromolecular Crystallography	Crystallization and X-ray analysis of monodisperse human properdin Dennis Vestergaard Pedersen , Margot Revel , Trine Amalie Fogh Gadeberg , Gregers Rom Andersen Acta Crystallographica Section F Structural Biology Communications 75 DOI: 10.1107/S2053230X18018150 Diamond Proposal Number(s): [13062] Show Abstract ▼ Abstract: The 54 kDa protein properdin, also known as factor P (FP), plays a major role in the complement system through the stabilization of the alternative pathway convertases. FP circulates in the blood as cyclic dimers, trimers and tetramers, and this heterogeneity challenges detailed structural insight into the mechanism of convertase stabilization by FP. Here, the generation of an intact FP monomer and a variant monomer with the third thrombospondin repeat liberated is described. Both FP monomers were excised from recombinant full-length FP containing internal cleavage sites for TEV protease. These FP monomers could be crystallized, and complete data sets extending to 2.8 Å resolution for the intact FP monomer and to 3.5 Å resolution for the truncated variant were collected. The principle of specific monomer excision and domain removal by the insertion of a protease cleavage site may be broadly applicable to structural studies of oligomeric, flexible and modular proteins.	Feb 2019
I02-Macromolecular Crystallography I03-Macromolecular Crystallography I24-Microfocus Macromolecular Crystallography	Trapping IgE in a closed conformation by mimicking CD23 binding prevents and disrupts FcεRI interaction Frederic Jabs , Melanie Plum , Nick S. Laursen , Rasmus K. Jensen , Brian Mølgaard , Michaela Miehe , Marco Mandolesi , Michèle M. Rauber , Wolfgang Pfützner , Thilo Jakob , Christian Möbs , Gregers R. Andersen , Edzard Spillner Nature Communications 9 DOI: 10.1038/s41467-017-02312-7 Diamond Proposal Number(s): [13062] Open Access Show Abstract ▼ Abstract: Anti-IgE therapeutics interfere with the ability of IgE to bind to its receptors on effector cells. Here we report the crystal structure of an anti-IgE single-domain antibody in complex with an IgE Fc fragment, revealing how the antibody inhibits interactions between IgE and the two receptors FcεRI and CD23. The epitope overlaps only slightly with the FcεRI-binding site but significantly with the CD23-binding site. Solution scattering studies of the IgE Fc reveal that antibody binding induces a half-bent conformation in between the well-known bent and extended IgE Fc conformations. The antibody acts as functional homolog of CD23 and induces a closed conformation of IgE Fc incompatible with FcεRI binding. Notably the antibody displaces IgE from both CD23 and FcεRI, and abrogates allergen-mediated basophil activation and facilitated allergen binding. The inhibitory mechanism might facilitate strategies for the future development of anti-IgE therapeutics for treatment of allergic diseases.	Jan 2018
I04-Macromolecular Crystallography	Introducing site-specific cysteines into nanobodies for mercury labelling allows de novo phasing of their crystal structures Simon Boje Hansen , Nick Stub Laursen , Gregers Rom Andersen , Kasper R. Andersen Acta Crystallographica Section D Structural Biology 73, 804 - 813 DOI: 10.1107/S2059798317013171 Diamond Proposal Number(s): [13062] Open Access Show Abstract ▼ Abstract: The generation of high-quality protein crystals and the loss of phase information during an X-ray crystallography diffraction experiment represent the major bottlenecks in the determination of novel protein structures. A generic method for introducing Hg atoms into any crystal independent of the presence of free cysteines in the target protein could considerably facilitate the process of obtaining unbiased experimental phases. Nanobodies (single-domain antibodies) have recently been shown to promote the crystallization and structure determination of flexible proteins and complexes. To extend the usability of nanobodies for crystallographic work, variants of the Nb36 nanobody with a single free cysteine at one of four framework-residue positions were developed. These cysteines could be labelled with fluorophores or Hg. For one cysteine variant (Nb36-C85) two nanobody structures were experimentally phased using single-wavelength anomalous dispersion (SAD) and single isomorphous replacement with anomalous signal (SIRAS), taking advantage of radiation-induced changes in Cys–Hg bonding. Importantly, Hg labelling influenced neither the interaction of Nb36 with its antigen complement C5 nor its structure. The results suggest that Cys–Hg-labelled nanobodies may become efficient tools for obtaining de novo phase information during the structure determination of nanobody–protein complexes.	Oct 2017

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