Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Abstract

Structural determination of proteins is rather challenging for proteins with molecular masses between 40 – 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 – 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high#resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high#throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

Calsyntenin-3 Molecular Architecture and Interaction with Neurexin 1α

“…We used a unique combination of electron microscopy and tomography (34) to obtain unprecedented structural information on a multidomain and apparently flexible protein, intractable by other methods. The structures of Cstn3 were studied by optimized negative stain EM, a validated method that has proven successful for relatively small proteins (28), rather than conventional negative stain EM or cryo-EM. As with any negative stain EM technique, we cannot exclude potential artifacts as a result of its chemical characteristics, which might impact the resolution limit, the shape, and/or the conformation of the observed macromolecules.…”

“…An aliquot (ϳ4 l) of sample was placed on a glow-discharged (15 s) thin carbon-coated 200-mesh copper grid (CF200-Cu, EMS). After ϳ1 min of incubation, excess solution was blotted with filter paper, and the grid was stained for ϳ1 min by submersion in a drop (ϳ35 l) of 1% (w/v) uranyl formate (27,28) before nitrogen air drying at room temperature.…”

“…EM Data Acquisition and Image Preprocessing-The negative stain micrographs were acquired at room temperature on a Gatan UltraScan 4Kϫ4K CCD by a Zeiss Libra 120 transmission EM (Carl Zeiss NTS) operating at 120 kV at ϫ125,000 magnification (0.94 Å/pixel) under low defocus (Ϫ0.1 to ϳϪ0.6 m) by following a strategy developed for imaging small and asymmetric proteins (28). The micrograph defocus and astigmatism were examined by ctffind3 (FREALIGN software package) (29).…”

A facile method for isolation of recombinant human apolipoprotein A-I from E. coli

“…A freshly prepared untagged apoA-I rHDL sample was processed for negative staining as previously described [15 – 17]. In brief, the sample was diluted to 5 μg/mL protein with Dulbecco’s PBS.…”

Image Processing and Cryo-Transmission Electron Microscopy; Example of Human Proteasome

“…The samples were taken in a dry environment because the level of contrast in the biological sample can be improved using the negative-staining method with metal stains. Several staining solutions, including uranyl acetate and uranyl formate and staining techniques such as the carbon sandwich method, have been adopted to compensate for the structural distortions caused by sample drying (Ohi et al, 2004;Rames et al, 2014). In addition, a random conical tilt method for 3D reconstruction has been applied to perform a detailed structural analysis (Booth et al, 2011;Radermacher et al, 1987).…”