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Self-Assembling Protein Nanomaterials – Design, Production and Characterization
Published in Klaus D. Sattler, st Century Nanoscience – A Handbook, 2020
Bhuvana K. Shanbhag, Victoria S. Haritos, Lizhong He
Transmission electron microscopy (TEM) and atomic force microscopy (AFM) are the most widely used microscopy methods for the characterization of protein nanomaterials. TEM produces high-resolution images due to the smaller wavelength of the electrons. The final assembled structures of protein nanomaterials can be visualized using TEM, by subjecting the protein sample to a negative staining procedure using uranyl acetate/uranyl formate. An example of a TEM image of assembled VLP and protein nanoparticles formed by BCA-P114 is shown in Figure 16.11a and b, respectively. For higher resolution images, 2D and 3D reconstruction of protein nanostructures can be performed by subjecting the electron micrographs to computer-aided image processing and refining processes in an iterative fashion. This procedure is often used to validate the experimental structure of the protein assembly with the computational model (King et al., 2012, 2014). Cryo-EM, a variation of TEM, is also used to view protein assemblies (Liu et al., 2018) where the sample is prepared using liquid nitrogen allowing visualization of the biological structures close to their native state.
Recombinant expression and characterization of yeast Mrc1, a DNA replication checkpoint mediator
Published in Preparative Biochemistry & Biotechnology, 2020
About 3 μL of aliquots were applied to a freshly glow-discharged carbon-coated 400-mesh copper-electron microscopy specimen grid, and then was preserved by staining with 0.75% (w/w) uranyl formate (UF) solution. Images were recorded at a magnification of ×62,000 on a 4096 × 4096 charge-coupled device (CCD) detector (FEI Eagle) with a Tecnai F20 electron microscope (FEI) operating at an acceleration voltage of 200 kV. Images were recorded by using low-dose procedures at ∼1.0–1.2 μm under focus. Two-fold pixel binning of the original CCD images resulted in a final pixel size of 3.01 Å per pixel. Particles were manually or automatically picked and were montaged for interactive screening.[21] Totally, 23,152 Mrc1 (287-1096AA) particles and 18,930 Mrc1 (287-1096AA)-DNA particles were picked after manual cleaning. Iterative alternating rounds of supervised multi-reference alignment and classification as well as reference-free alignment to improve the homogeneity of the image classes were run with SPIDER[22] and SPARX.[23]