Negative‐Stain Transmission Electron Microscopy of Molecular Complexes for Image Analysis by 2D Class Averaging

Abstract

Negative-stain transmission electron microscopy (EM) is a technique that has provided nanometer resolution images of macromolecules for about 60 years. Developments in cryo-EM image processing have maximized the information gained from averaging large numbers of particles. These developments can now be applied back to negative-stain image analysis to ascertain domain level molecular structure (10 to 20Å) more quickly and efficiently than possible by atomic resolution cryo-EM. Using uranyl acetate stained molecular complexes of influenza hemagglutinin bound to Fab 441D6, we describe a simple and efficient means to collect several hundred micrographs with SerialEM. Using RELION, we illustrate how tens of thousands of complexes can be auto-picked and classified to accurately describe the domain level topology of this unconventional hemagglutinin head-domain epitope. By comparing to the cryo-EM density map of the same complex, we show that questions about epitope mapping and conformational heterogeneity can readily be answered by this negative-stain method. C 2019 The Authors.Keywords: electron microscopy r image analysis r image processing r negative stain

Moving in the mesoscale: Understanding the mechanics of cytoskeletal molecular motors by combining mesoscale simulations with imaging

“…Negative stain EM (nsEM) has historically provided a wide range of information on conformational states of motor proteins, 20 insight into how motors interact with their tracks, 21 mechanical properties, 22,23 and even binding kinetics. 24 However, the structural resolution of nsEM is limited to $10 Å, 25 while that F I G U R E 1 Schematic representation of a super-macromolecular array of dyneins within a primary cilium (left), and myosin 5a (Myo5a) working independently to move a cargo along the actin network (right) of cryoEM (Figure 2c,d) is now beginning to rival X-ray crystallography (currently 1.22 Å best resolution 14 ) as a result of better detectors and advanced processing techniques. 26 CryoEM has now been used successfully to resolve the structure of a full-length motor protein for the first time, 27 motors bound to their tracks, [28][29][30] and the organization of actin and myosin in muscle myofibrils, 31 none of which would have been possible by X-ray crystallography.…”

Nano Milk Protein-Mucilage Complexes: Characterization and Anticancer Effect

“…Doxorubicin HCl (4 µg/mL) was used as positive control, while cells treated with unsupplemented medium were taken as negative control. The plates were incubated at 37 • C for 24 h and the anti-cancer activity of the samples was determined using neutral red uptake assay according to Repetto et al [54]. Briefly, after the incubation period, the medium was replaced with 150 µL of neutral red dye (100 mg/mL) dissolved in serum-free medium, and the cells were incubated at 37 • C for 3 h. Next, the cells were rinsed with PBS, and 150 µL of elution medium (EtOH/AcCOOH, 50:1) was added followed by gentle shaking for 10 min to ensure complete dissolution.…”

A Fast Image Alignment Approach for 2D Classification of Cryo-EM Images Using Spectral Clustering

“…After alignment, the cryo-EM projection images with nearly the same projection directions are grouped in the 2D classification step. Well-aligned cryo-EM projection images with correct in-plane rotations and translational shifts in the x-axis and y-axis directions can improve the accuracy of the 2D classification [39]. Correctly classifying the cryo-EM projection images into homogeneous groups renders the satisfactory determination of the preliminary 3D structures [40].…”