IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure

Abstract

The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.

The low-resolution structure of nHDL reconstituted with DMPC with and without cholesterol reveals a mechanism for particle expansion

“…Before studies employing contrast variation small-angle neutron scattering (SANS) (see below), the overall conformation of apoA1 within a lipid containing HDL particles was inferred from crystal structure data from lipid free apoA1 mutant forms ( 19,20 ), as well as measured distance constraints between inter-and intrachain amino acids in mutated or derivatized apoA1 using FRET or ESR ( 12 ), and in native apoA1 by cross-linking MS studies ( 11,15,21,22 ). Further structural information has been obtained from amide bond hydrogen/deuterium exchange mass spectrometry ( 14,23,24 )}, and cryo-electron tomography ( 25 ).…”

Single Particle Tomography in EMAN2

“…Of note, it has been suggested that direct Fourier inversion can produce more accurate 3-D reconstructions (Heymann et al 2008) than the more traditional WBP and SIRT (Radermacher 2006) methods used in the field. Furthermore, since gold fiducials are absent from tilt series in cases where they might interact with the specimen and, when present, they undergo beam-induced motion resulting in unavoidable errors in whole-frame alignment (Comolli, & Downing 2005), subtiltseries can allow for more accurate individual per particle alignment, refinement and reconstruction, provided that the data have enough contrast, as demonstrated for negatively stained data (Iwasaki et al 2005) and cryoET data (Zhang, & Ren 2012).…”

107 Genomes to hit moleculesIn Silico: a country path today, a highway tomorrow: a case study of Chikungunya

“…FETR can tolerate certain levels of image distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. Since this approach can obtain the structure of a single-instance macromolecule, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single ''snapshot'' of molecule structure determination (Zhang & Ren, 2012). FETR/IPET provides a completely new opportunity for a single-macromolecule structure determination, and could be used to study the dynamic character, equilibrium fluctuation, to reveal macromolecular mechanism, and even to track the intermediate state of the reaction of macromolecules Zhang & Ren, 2010).…”