3D structure determination of protein using TEM single particle analysis

Proteins play important roles in cell functions such as enzymes, cell trafficking, neurotransmission, muscle contraction and hormone secretion. However, some proteins are very difficult to be crystallized and their structures are undetermined. Several techniques have been developed to elucidate the structure of macromolecules; X-ray or electron crystallography, nuclear magnetic resonance spectroscopy, and high-resolution electron microscopy. Among them, electron microscopy based single particle reconstruction (SPA) technique is a computer-aided structure determination method. This method reconstructs the 3D structure from projection images of dispersed protein. A large number of two-dimensional particle images are picked up from EM films, aligned and classified to generate 2D averages, and used to reconstruct the 3D structure by assigning the Euler angle of each 2D average. Due to the necessity of elaborate collaboration between the classical biology and the innovative information technology including parallel computing, scientists needed to break unseen barriers to get a start of this analysis. However, recent progresses in electron microscopes, mathematical algorithms, and computational abilities greatly reduced the height of barriers and expanded targets that are considered to be primarily addressable using single particle analysis. Membrane proteins are one of these targets to which the single particle analysis is successfully applied for the understanding of their 3D structures. For this purpose, we have developed various SPA methods [1-5] and applied them to different proteins [6-8].Here, we introduce reconstructed proteins, and discuss the availability of this technique. The intramembrane-cleaving proteases (I-CLiPs) that sever the transmembrane domains of their substrates have been identified in a range of organisms and play a variety of roles in biological conditions. I-CLiPs have been classified into three groups: serine-, aspartyl- and metalloprotease-type. Signal peptide peptidase (SPP) is an atypical aspartic protease that hydrolyzes peptide bonds within the transmembrane domain of substrates and is implicated in several biological and pathological functions. The structure of human SPP was determined by SPA at a resolution of 22 Å [8]. SPP forms a slender, bullet-shaped homotetramer with dimensions of 85 x 85 x 130 Å. The SPP complex has four concaves on the rhombus-like sides, connected to a large chamber inside the molecule. For the tetrameric assembly, the N-terminal region of SPP was found to be sufficient. Moreover, when N-terminal region was overexpressed, the formation of the endogenous SPP tetramer was inhibited, which suppressed the proteolytic activity within cells. From these data, the N-terminal region is considered to work as the structural scaffold.Transmembrane (TM) translocation of newly synthesized secretion proteins and membrane proteins are carried out by a Sec translocon protein complex. The polypeptide-conducting pore is formed by the SecYEG-SecA complex in bacteria, and the membrane protein SecDF is necessary for the efficient transport of proteins. However the molecular mechanism how SecDF realized efficient transport is not clear. A previous X-ray structural study of the whole protein and subdomain suggest that SecDF has at least two conformational variants, which could reflect molecular dynamics of this protein. To confirm this hypothesis, we analyzed the 3D structure of SecDF using dark field STEM electron tomography and single particle reconstruction. We determined two different whole SecDF protein structures which well explains the X-ray data. From these data, we would like to propose the possible molecular mechanism of SecDF during polypeptide translocation.