Structural studies by cryo-electron tomography of two initiation complexes of the immune system: C1-IgG6 and NAIP5-NLRC4

Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and the killing of target cells, using all four human IgG subclasses. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.
Inflammasomes are high molecular weight protein complexes that play a crucial role in innate immunity by activating caspase-1. Inflammasome formation is initiated when molecules originating from invading microorganisms activate NLRs and induce NLR multimerization. Little is known about the conformational changes involved in NLR activation and the structural organization of NLR multimers. Here we show by cryo-electron tomography that flagellin-induced NAIP5/NLRC4 multimers form right- and left-handed helical polymers with a diameter of 28 nm and a rise of 6.5 nm. Sub tomogram averaging produced an electron density map at 4 nm resolution, which was used for rigid body fitting of NLR subdomains derived from the crystal structure of dormant NLRC4. The resulting structural model of inflammasome-incorporated NLRC4 indicates that a prominent rotation of the LRR domain of NLRC4 is necessary for multimer formation, providing unprecedented insight in the conformational changes that accompany NLR activation.
The resolution of electron tomograms is anisotropic due to geometrical constraints during data collection, such as the limited tilt range and single-axis tilt series acquisition. Acquisition of dual axis-tilt series can decrease these effects. However, in cryo-electron tomography, to limit the electron radiation damage that occurs during imaging, the total dose should not increase and must be fractionated over the two tilt series. Here, we set out to determine whether it is beneficial to fractionate electron dose for recording dual-axis cryo-electron tilt series or whether it is better to perform single-axis acquisition. To assess the quality of tomographic reconstructions in different directions, we introduce conical Fourier shell correlation (cFSCe/o). Employing cFSCe/o, we compared the resolution isotropy of single-axis and dual-axis (cryo-)electron tomograms using even/odd split data sets. We show that the resolution of dual-axis simulated and cryo-electron tomograms in the plane orthogonal to the electron beam becomes more isotropic compared to single-axis tomograms and high-resolution peaks along the tilt axis disappear. cFSCe/o also allowed us to compare different methods for the alignment of dual-axis tomograms. We show that different tomographic reconstruction programs produce varying anisotropic resolution patterns in dual- axis tomograms. We anticipate that cFSCe/o can also be useful for comparisons of acquisition and reconstruction parameters, and different hardware implementations.