In order to increase our understanding of structural dynamics of biomolecules at the single-molecule level, they would need to be captured at the sub-nanometer scale and in physiologically relevant conditions. There are few techniques currently available to do that. Now, scientists have developed a computational technique that greatly increases the resolution of atomic force microscopy (AFM). The method reveals atomic-level details on proteins and other biological structures under normal physiological conditions, opening a new window into the world of biology.

In a study titled, “Localization atomic force microscopy,” published in Nature, the investigators describe the new technique, which is based on a strategy used to improve resolution in light microscopy.

While X-ray crystallography and cryo-electron microscopy can determine molecular structures down to the resolution of individual atoms, they do so on molecules that are either scaffolded into crystals or frozen at ultra-cold temperatures, possibly altering them from their normal physiological shapes. AFM can analyze biological molecules under normal physiological conditions, but the resulting images have been blurry and low resolution.

“Atomic force microscopy can easily resolve atoms in physics, on solid surfaces of silicates and on semiconductors, so it means that in principle the machine has the precision to do that,” said Simon Scheuring, PhD, professor of physiology and biophysics in anesthesiology at Weill Cornell Medicine. “The technique is a bit like if you were to take a pen and scan over the Rocky Mountains, so that you get a topographic map of the object. In reality, our pen is a needle that is sharp down to a few atoms and the objects are single protein molecules.”

However, AFM’s resolution limits the assessment of conformational details of biomolecules. To address that problem, Scheuring and his colleagues adapted a concept from light microscopy called super-resolution microscopy. “Theoretically it wasn’t possible by optical microscopy to resolve two fluorescent molecules that were closer together than half the wavelength of the light,” he said. However, by stimulating the adjacent molecules to fluoresce at different times, microscopists can analyze the spread of each molecule and pinpoint their locations with high precision.

Instead of stimulating fluorescence, Scheuring’s team noted that the natural fluctuations of biological molecules recorded over the course of AFM scans yield similar spreads of positional data. First author George Heath, PhD, who was a postdoctoral associate at Weill Cornell Medicine at the time of the study and is now a faculty member at the University of Leeds, engaged in cycles of experiments and computational simulations to understand the AFM imaging process in greater detail and extract the maximum of information from the atomic interactions between tip and sample.

Using a method like super-resolution analysis, they were able to extract much higher resolution images of the moving molecules. In this work, the group presents localization AFM (LAFM), a technique that can overcome current resolution limitations. By applying localization image reconstruction algorithms, to peak positions in high-speed AFM and conventional AFM data, the authors write, they “increase the resolution beyond the limits set by the tip radius, and resolve single amino acid residues on soft protein surfaces in native and dynamic conditions.”

Because previous AFM studies have routinely collected the necessary data, the new technique can be applied retroactively to the blurry images the field has generated for decades. As an example, the new paper includes an analysis of an AFM scan of an aquaporin membrane protein, originally acquired during Scheuring’s doctoral thesis. The reanalysis generated a much sharper image that matches X-ray crystallography structures of the molecule closely. “You basically get quasi-atomic resolution on these surfaces now,” said Scheuring. To showcase the power of the method, the authors provided new high-resolution data on annexin, a protein involved in cell membrane repair, and on a proton-chloride antiporter of which they also report structural changes related to its function.

Besides allowing researchers to study biological molecules under physiologically relevant conditions, the new method has other advantages. For example, X-ray crystallography and cryo-electron microscopy rely on averaging data from large numbers of molecules, but AFM can generate images of single molecules. “Instead of having observations of hundreds of molecules, we observe one molecule a hundred times and calculate a high-resolution map,” said Scheuring.

Imaging individual molecules as they carry out their functions could open entirely new types of analysis. “Let’s say you have a [viral] spike protein that’s in one conformation and then it gets activated and goes into another conformation,” said Scheuring. “You would in principle be able to calculate a high-resolution map from that same molecule as it transits from one conformation to the next, not from thousands of molecules in one or the other conformation.” Such high-resolution single-molecule data could provide more detailed information and avoid the potentially misleading results that can occur when averaging data from many molecules. Furthermore, the map might reveal new strategies for precisely redirecting or interrupting such processes.

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