இயற்பியல் வேதியியல் & உயிர் இயற்பியல் இதழ்
திறந்த அணுகல்

சுருக்கம்

High-Resolution Single-Molecule Structure Revealed by Electron Microscopy and Individual Particle Electron Tomography

Lei Zhang and Gang Ren

The protein is naturally dynamic and heterogeneous 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. However, a single, unique structure of protein determined from X-ray crystal and conventional single-particle electron microscopy is insufficient to encompass the dynamic nature of proteins in solution. Structure determination of dynamic and heterogeneous protein is essentially required the determination of each individual particle of protein. Recently, Drs. Gang Ren and Lei Zhan published the first single molecule threedimensional (3D) EM images of individual proteins ever obtained with enough clarity to determine their structure, an IgG antibody (14 Å resolution) and a 17nm HDL (36 Å resolution). These results depended upon four innovations: i) improved cryo-electron microscopy (cryoEM) sample preparation and Electron microscopy (EM) operation conditions resulted in the successful imaging of a 17 nm HDL particle (120-200kDa) by cryo-electron tomography (cryoET); ii) developed an optimized NS (OpNS) protocol that eliminates the rouleau artifact that has plagued EM research for three decades. This OpNS protocol provides high-contrast single lipoprotein images with similar size (<5%) and shape (<5%) to that seen by cryoEM; iii) developed a high-resolution and high contrast sample preparation protocol, cryo-positive-staining (cryoPS) that allows direct visualization of the secondary structure of a small protein, such as the β-strands in CETP and the helical double belt of apoA-I in spherical HDL; iv) developed a robust tomography reconstruction method, Individual Particle Electron Tomography (IPET) that is a high-resolution, high throughput reconstruction method that, to the best of our knowledge, is the only method for determining an individual protein structure. Remarkably, IPET went against the conventional wisdom that a single protein can NOT be reconstructed by EM and this opens a door for the study of protein dynamics via a particle-byparticle structural comparison.