Monday, September 29, 2014
“High Resolution Protein Structural Studies by Single
Particle Helical 3D Reconstruction using Cryo-EM”
Inflammasomes elicit host defense inside cells by activating caspase-1 for cytokine maturation and cell death. AIM2 and NLRP3 are representative sensor proteins in two major families of inflammasomes. The adaptor protein ASC bridges the sensor proteins and caspase-1 to form ternary inflammasome complexes, achieved through pyrin domain (PYD) interactions. PYD monomer structure revealed by NMR studies shows that it is a six-helix bundle. The truncation mutant of only one monomer forms clean fibrils. The monomer consists of 96 amino acid residues, equals to 11kDa mol.wt. and the diameter of the fibril is approximately 2.5 nm. The pyrin domain protein monomer forms nice filaments, which are expected to have helical symmetry. Originally, 3D reconstruction of helical filaments were obtained by using Fourier Bessel (F-B) methods, in which the periodic pattern of layer lines in the 2D power spectra of projected EM images, are indexed to determine helical symmetry parameters (Diaz et al., 2010). A 3D density map is reconstructed in cylindrical coordinates using F-B inversion (Klug et al., 1958).
The overlapping Bessel orders and uncertain indexing of diffraction pattern can prevent the application of F-B approach (Egelman and Stasiak, 1988) to obtain reliable structures. Alternative to this approach is the Iterative Helical Real-Space Refinement (IHRSR) algorithm (Egelman, 2000), it is based on the fruitful single particle approach to determine macromolecular structure, applied to asymmetric or low symmetric structures and also for flexible polymers. It retains the same strategy as single particle approach but modifying it, so that it can refine helical symmetry parameters during the iterative procedures for structure determination. Adding to this, the invention of Direct Electron Detectors help to reach atomic resolution and avoid the ambiguities in indexing the Fourier Bessel Layer lines for initial guess of symmetry parameters to start and refine it with IHRSR. To compliment this, a novel hybrid approach has been introduced where the constraints of 3.7 Å EM map of PYD fibrils is combined with NMR studies of both solid state and solution state to obtain atomic resolution structure for detailed structural analysis.