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

Ren G (2012) IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single- Molecule Structure. PLoS ONE 7(1): e30249. doi:10.1371/journal.pone.0030249 IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure Lei Zhang 0 Gang Ren 0 Wenqing Xu, University of Washington, United States of America 0 Molecular Foundry, Lawrence Berkeley National Laboratory , Berkeley, California , United States of America 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 A ), 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. - Funding: This work was supported by the Office of Science, Office of Basic Energy Sciences of the United States Department of Energy (contract no. DE-AC0205CH11231) and partially supported by the William Myron Keck Foundation (#011808). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The dynamic character of proteins dictates their functions and ultimately represents an accurate portrayal of their many personalities [1,2]. A snapshot of proteins frozen in crystals reveals a single, unique structure that is often used as a blueprint for studies in structurefunction relationships. However, these structures fail to encompass the dynamic nature of proteins in solution. Protein dynamics involves both equilibrium fluctuations that regulate biological function and other non-equilibrium effects of biological motors, which convert chemical energy to mechanical energy. Although the experimental approach to determine the dynamic structure at atomic-resolution level is not available, molecular dynamics (MD) simulations have been widely used to link structure and dynamics by enabling the exploration of the conformational energy landscape accessible to protein molecules [1,2]. MD simulations could provide detailed dynamics and function in structure, but one of the major obstacles of MD is a potential energy barrier to determine global protein conformations and equilibrium fluctuations. Cryo-electron tomography (cryoET) is an experimental approach to provide a structural snapshot of a single-instance biological object from a series of tilted viewing angles [3,4]. This method has been rapidly adopted and applied to reveal the threedimensional (3D) structure of cells, bacteria, and even proteins. Unfortunately, the resolution of 3D density maps rarely goes beyond 30 A using conventional ET reconstruction methods [5], and is generally insufficient to determine domain information of single-instance protein. An alternate cryoET approach to improve the resolution of protein structure is a 3D classification and averaging method in which hundreds to thousands of 3D subvolumes are selected from a large-volume, low-resolution 3D reconstruction [6]. This highly used method can reduce noise and improve the 3D subvolume reconstruction resolution up to 20 A when the protein has a high symmetry, such as GroEL and nuclear pores [7]. However, when the protein has no symmetry, but with multiple-conformational structures, such as a human IgG antibody and the high-density lipoprotein (HDL), the classification and orientation determination of subvolumes are challenging. Moreover, the average of hundreds of different conformational structures could be detrimental to elucidate the structural equilibrium fluctuations of a protein. We believe, there are adverse effects from image distortion and measuring tilt-errors in conventional tomography reconstruction methods, which play a major role in limiting the resolution of the large-size whole micrograph reconstruction. The image distortions (introduced by lens astigmatism [8], energy filter [9,10], radiationinduced deformations [11] and defocus-related distortion) can generate the displacement (translational errors) and measuring tilterrors (including tilt-axis and tilt-angle errors). Since measuring tilt-angle is usually performed by tracking the movements of gold fiducial markers between the tilt micrographs, the inconsistency of displacement introduced by image distortion results in the inconsistency of movement of markers during tilting, therefore resulting in an inconsistency of tilt-angles. The final determined tilt-angle is actually an average of tilt-angles suggested from different areas of the micrograph. Greater distortion results in greater displacement, thus producing a larger tilt-angle error. Image distortion is generally a large-scale deformation/displacement, in which different areas within a micrograph present a different amount of displacement. Mathematically, large-scale deformation/distortion can be represented by a combination of local displacement ( (...truncated)