Structural Information, Resolution, and Noise in High-Resolution Atomic Force Microscopy Topographs

Abstract

AFM has developed into a powerful tool in structural biology, providing topographs of proteins under close-to-native conditions and featuring an outstanding signal/noise ratio. However, the imaging mechanism exhibits particularities: fast and slow scan axis represent two independent image acquisition axes. Additionally, unknown tip geometry and tip-sample interaction render the contrast transfer function nondefinable. Hence, the interpretation of AFM topographs remained difficult. How can noise and distortions present in AFM images be quantified? How does the number of molecule topographs merged influence the structural information provided by averages? What is the resolution of topographs? Here, we find that in high-resolution AFM topographs, many molecule images are only slightly disturbed by noise, distortions, and tip-sample interactions. To identify these high-quality particles, we propose a selection criterion based on the internal symmetry of the imaged protein. We introduce a novel feature-based resolution analysis and show that AFM topographs of different proteins contain structural information beginning at different resolution thresholds: 10 A (AqpZ), 12 A (AQP0), 13 A (AQP2), and 20 A (light-harvesting-complex-2). Importantly, we highlight that the best single-molecule images are more accurate molecular representations than ensemble averages, because averaging downsizes the z-dimension and "blurs" structural details.

Atomic force microscopy of the bacterial photosynthetic apparatus: plain pictures of an elaborate machinery

“…Indeed it seems likely that often single molecule images provide a more faithful view than averages (Fechner et al 2009). In contrast to the results provided by techniques that analyze purified LH complexes and require the averaging of signals from many molecules, such as X-ray crystallography, n.m.r.…”

“…In principle, the AFM can provide atomic resolution on solid crystalline surfaces such as mica or graphite (Binning et al 1987(Binning et al , 1986. On membranes in buffer solution, the imaging resolution of 10 Å is most probably limited due to protein fluctuation at room temperature (Fechner et al 2009). In contact-mode high-resolution AFM, images are acquired at scan speeds around 1 ms/nm.…”

Noninvasive Protein Structural Flexibility Mapping by Bimodal Dynamic Force Microscopy

“…Atomic force microscopy (AFM) [1] has enabled molecular resolution images of packed arrays of biomolecules [2][3][4][5], sub-2-nm images of individual biomolecules [6], and studying biomolecular interactions in liquid [7]. Single force spectroscopy measurements are already an established tool to measure intermolecular and intrabiomolecule forces [8]; however, those measurements are not compatible with high resolution imaging.…”

Sampling Protein Form and Function with the Atomic Force Microscope

“…The imaging resolution of the atomic force microscope is comparable with that of electron microscopes, and it has the special capability to image samples in a variety of environments such as in vacuum, air, or liquid, which therefore enables studying biological specimens in their native environments (i.e. in buffer solutions) (3,4). In addition, its ability to "touch" the sample gives it the advantage to manipulate single particles/molecules and probe their mechanical properties (5)(6)(7)(8).…”

“…27), label-free detection and counting of single proteins (28, 29), and force spectroscopy measurements of binding and unbinding events (30, 31). In structural biology, AFM has shown to be a powerful tool for high resolution imaging of proteins in near native conditions (3,6) and structural studies of supramolecular assemblies like protein filaments and viruses by nanoindentation methods (32, 33). These experiments show the potential of AFM to study both "form" and "function" of proteins, thereby resolving questions in proteomics and structural biology quasi-simultaneously.…”