To avoid the influence of image distortion and tilt angle errors, the reconstruction algorithm (called focused electron tomography reconstruction (FETR)) reduces the reconstruction size for precisely aligning the tilt images under units of dynamic filters and soft-boundary masks through local refinement iteration [31]. IPET, 3D structure of IgG, antibody dynamics, FTI-277 HCl antibody engineering, homodimer antibody, structure of bispecific IgG1 1. Introduction Antibodies, or immunoglobulins FTI-277 HCl (Ig), are glycoproteins that make up the humoral portion of the adaptive immune system and fight against pathogens such as viruses, bacteria, parasites, and diseased cells [1]. Antibodies share more than 90% of their identity in their main sequences; they can be divided into five major classes including IgA, IgD, IgE, IgG, and IgM [2] based on their heavy chains, which differ in physicochemical and serologic properties as well as in their behavior as antigens themselves [3,4]. About 10C20% of proteins in the blood are IgG proteins. Due to their enormous variability, plasma antibodies have more than 10 billion types, which can be classified into four subclasses: IgG1, IgG2, IgG3, and IgG4 [5], in the order of decreasing large quantity in serum [2]. IgG1, for example, primarily responds to soluble and membrane protein antigens [6]. Antibodies contain two fragment antigen binding arms (Fab) that are created from an N-terminal heavy chain variable domain name (VH) and a light chain variable domain name (VL) (Physique 1A). The VH and VL are linked together to form a monomer (H2L2 Tbx1 via inter- or intra-chain disulfide bonds (S-S)) [7,8]. Each heavy FTI-277 HCl chain contains one VH and three to four constant domains (CH1, CH2, and CH3 or C1, C2, and C3). These VH and VL models lie together at the antigen-binding cleft, and the hinge region between CH1 and CH2 allows antibodies (IgGs) to display their flexibility upon antigen binding. The lower hinge region between CH2 and CH3 compose the fragment crystalline domain name (Fc), which is responsible for IgG-Fc binding (FcR, effector function), C1q (match activation), and the neonatal Fc receptor (FcRn, homeostasis and placental transport, except for IgG2) [9]. Glycosylation of IgG1 mainly occurs FTI-277 HCl on Asn-297 of the CH2 domains [10]. Open in a separate windows FTI-277 HCl Physique 1 Structure and dynamics of antibodies. (A) The crystal structure of mouse IgG2a (PDB access: 1IGT) shows that an antibody consists of two identical heavy protein chains (blue and reddish) combined with two identical light chains (green and yellow), which are composed of 7 (for constant domains) to 9 (for variable domains) -strands (Copyright? Wikipedia). (B,C) However, the crystal structures of mouse IgG1 and human IgG1 (PDB entries: 1IGY and 1HZH) are different, especially for the Fab domains location and orientation. (D) The crystal structure of the full-length of the IgG4 antibody was also determined by the crystal structure and the hinge SS was deleted (PDB access 5DK3). (E) Those structures are also different from the IgG revealed by atomic pressure microscopy (AFM) (Copyright? 2016 The Royal Society of Chemistry) [17], (F) small-angle X-ray scattering (SAXS), and (G) neutron scattering (Copyright? 2012 Elsevier Ltd.). This variance is also different from that of (H) IgD in answer revealed by SAXS (Copyright? 2005 Elsevier Ltd.) or (I) the structure and fluctuation of IgG1 obtained by negative-staining electron tomography (NS-ET) and individual-particle electron tomography (IPET) 3D reconstructions [16]. Copyright? 2015 the Authors, managed by Nature Publishing Group. Monoclonal antibody production has shifted biological and pharmaceutical research as well as clinical therapeutics [11]. In 1975, K?hler and Milstein first used hybridoma technology to develop a mouse monoclonal antibody for combating kidney transplant rejection [12]. So far, more than 60 therapeutic monoclonal antibody products have been approved in the US or Europe, while nearly 100 antibodies are currently being tested in clinical trials [13,14]. First seen with the development of Humira (adalimumab) for treating rheumatoid arthritis, fully humanized antibodies have been generated by assembling lymphocyte V-region genes cloned to display Fab fragments on bacteriophage surfaces [15]. Today, fully humanized antibodies significantly improve the security and efficacy of monoclonal antibody drug-based therapeutics and diagnostics, including antibody-conjugated drug delivery systems, oncoprotein-targeted malignancy therapies, and immunotherapies for cardiovascular, neurodegenerative, and other diseases [16]. In the biopharmaceutical industry, ten of the fifteen top-selling drugs are protein biologics, and of these, five are monoclonal antibodiesthe largest class of biotherapeuticswhich represent at least 40% of the drugs in development today. A key limiting factor in the biopharmaceutical industry is the failure of current methods to characterize the structure of protein-based drugs fully and efficiently with sufficient precision and accuracy. Thus, we review several major imaging techniques for characterizing antibody structure and dynamics. 2. Methods.