Novel experimental strategy for high resolution AFM imaging of membrane-associated bacterial toxins

Bacterial pore-forming toxins （PFTs） are essential virulence factors of many human pathogens. Knowl- edge of their structure within the membrane is critical for an understanding of their function in pathogenesis and for the development of useful therapy. Atomic force microscopy （AFM） has often been employed to structurally interrogate many membrane proteins, including PFTs, owing to its ability to produce sub-nanometer resolution images of samples under aqueous solution. However, an absolute prerequisite for AFM studies is that the samples are single-layered and closely-packed, which is frequently challenging with PFTs. Here, using the prototypical member of the cholesterol-dependent cytolysin family of PFTs, perfringolysin O （PFO）, as a test sample, we have developed a simple, highly robust method that routinely produces clean, closely-packed samples across the entire specimen surface. In this approach, we first use a small Teflon well to prepare the supported lipid bilayer, remove the sample from the well, and then directly apply the proteins to the bilayer. For reasons that are not clear, bilayer preparation in the Teflon well is essential. We anticipate that this simple method will prove widely useful for the preparation of similar samples, and thereby enable AFM imaging of the greatest range of bacterial PFTs to the highest possible resolution.     

Direct resolution of the pitch of DNA on positively charged lipid bilayers by frequency-modulation AFM

High resolution structural studies of DNA and DNA binding proteins by atomic force microscopy (AFM) require well-bound samples on suitably flat substrates. Adsorbing the DNA onto a positively charged supported lipid bilayer has previously been shown to be a potentially effective strategy for structural studies with AFM. Here, using our home-built frequency-modulation AFM (FM-AFM), we show that these bilayer substrates are only maximally effective for high resolution AFM when the samples are short, linear DNA, compared with circular plasmid DNA. We find that, with the former sample, the measured width of the DNA is about 2 nm, the known DNA diameter, and there is a clear height modulation along the length of the DNA with a periodicity of about 3.4 nm, in excellent agreement with the known pitch of the double helix. This sample preparation strategy is expected to enable higher resolution studies of DNA and DNA binding proteins with FM-AFM than that can presently be achieved.     

系统电磁兼容性软件仿真

随着电子设备和电子系统的飞速发展，系统的电磁兼容性问题越来越受到重视，本文介绍一种用于解决系统电磁兼容性问题的软件仿真技术。