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Summary The lectin pathway of complement is activated by complexes comprising a recognition component (mannose-binding lectin, serum ficolins, collectin-LK or collectin-K1) and a serine protease (MASP-1 or MASP-2). MASP-1 activates MASP-2, and MASP-2 cleaves C4 and C4b-bound C2. To clarify activation, new crystal structures of Ca2+-bound MASP dimers were determined, together with their solution structures from X-ray scattering, analytical ultracentrifugation, and atomistic modeling. Solution structures of the CUB1-EGF-CUB2 dimer of each MASP indicate that the two CUB2 domains were tilted by as much as 90° compared with the crystal structures, indicating considerable flexibility at the EGF-CUB2 junction. Solution structures of the full-length MASP dimers in their zymogen and activated forms revealed similar structures that were much more bent than anticipated from crystal structures. We conclude that MASP-1 and MASP-2 are flexible at multiple sites and that this flexibility may permit both intra- and inter-complex activation., Highlights • Ca2+-bound MASP crystal structures reveal binding sites for MBL and other ligands • The MASP crystal structures predict linear domain structures • Solution structures of MASP-1 and MASP-2 are bent by as much as 90° from linearity • Flexibility in MASP dimers may permit both intra- and inter-complex activation, Nan et al. show that MASPs, the serine proteases responsible for initiating the lectin pathway of complement activation, are much more flexible in solution than previously thought. This flexibility probably facilitates both intra- and inter-complex activation when mannan-binding lectin-MASP complexes bind to pathogen surfaces.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies.