Abstract Insects rely on odorant receptors (ORs), tetrameric ligand-gated ion channels, to detect and discriminate a wide range of chemical cues. Within the insect OR family, composed of hundreds of OR subtypes, a conserved subunit termed odorant receptor co-receptor (Orco) is present in insect olfactory neurons. While Orco and some ORs can assemble into homo-tetramers, odor-activated channels are hetero-tetramers comprising Orco and an odor-specific OR. Cryogenic electron microscopy (cryo-EM) structures from heterologous expression systems revealed that Orco and OR assembled with a 3:1 (Orco:OR) stoichiometry. In these complexes, the OR subunit is solely responsible for ligand-recognition, whereas the Orco subunits form the majority of the ion conduction pathway and are key for assembly and membrane trafficking. Because Orco and OR can assemble in diverse stoichiometries, of which Orco (4) and OR (4) homo-tetramers, as well as Orco (3) /OR (1) hetero-tetramers, have been experimentally documented, we sought to investigate assembly and conformational dynamics of insect Orco/OR complexes. Here, using high-speed atomic force microscopy (HS-AFM), unique in providing simultaneous structural and temporal resolution, we find that Orco/OR complexes can reversibly transition into stable non-tetrameric oligomeric states, such as trimers and pentamers, through membrane-diffusive oligomer exchange. Furthermore, while all cryo-EM structures showed a fourfold symmetric or pseudo-symmetric conformation, we observed that tetrameric assemblies frequently adopt a symmetry-broken ‘pinched’ configuration, in addition to the canonical symmetric ‘square’ arrangement. Notably, ligand-binding modulates the ‘pinching’ kinetics in Orco/OR hetero-tetramers. Overall, these findings reveal key aspects of assembly and conformational dynamics of the insect OR family.

Scanning probe microscopy (SPM) distinguishes itself from light and electron microscopy by sensing surface interactions with a nanoscale probe, rather than relying on the detection of particles or waves; and has evolved into a versatile tool across several fundamental and applied research fields. SPM uses raster-scanning for image formation, comprising trace (left-to-right) and retrace (right-to-left) scans - this spatial redundancy is however usually not fully taken advantage of. Here, we introduce a self-supervised deep learning framework for Scanning Probe microscopy Image DEnoising and Restoration (SPIDER), by utilizing trace and retrace information. SPIDER improves the signal-to-noise ratio up to 3-fold and accelerates imaging speed up to 6-fold, circumventing the need for a large training set and a ground truth. We further demonstrate that SPIDER enables imaging acceleration by utilizing spatial information from the fast-scan axis to reconstruct missing spatial information in the slow-scan axis in a self-supervised manner. The self-supervised reconstruction is competitive with supervised learning methods. We anticipate that SPIDER will improve SPM imaging and inspire further applications. The authors present self-supervised Scanning Probe microscopy Image DEnoising and Restoration (SPIDER), a deep learning framework that denoises scanning probe microscopy images, enabling imaging acceleration and anisotropic resolution restoration.

High-speed atomic force microscopy (HS-AFM) has emerged as a powerful tool for the investigation of the dynamic behavior of membrane proteins in near-native environments. Unlike traditional structural techniques, HS-AFM allows for real-time imaging of individual protein molecules at nanometer resolution under physiological conditions. This capability enables direct visualization of conformational changes, intermolecular interactions, and functional cycles of membrane proteins, such as channels, transporters, and receptors, without the need for labeling or extensive sample preparation. HS-AFM provides insights into the structural plasticity and mechanistic pathways of membrane proteins that are often averaged out or missed by ensemble techniques. Its ability to capture dynamic events at sub-second timescales has significantly advanced our understanding of membrane protein conformational dynamics, single-molecule kinetics, as well as about diffusion, clustering, and interactions within lipid bilayers. As the technology continues to evolve, HS-AFM holds great promise for bridging structural and functional studies, offering a unique window into the real-time molecular mechanisms of membrane-associated biological processes. In this chapter, we detail protocols from membrane protein expression, purification, and reconstitution to final HS-AFM experiments.

High-speed atomic force microscopy (HS-AFM) movies have contributed significantly to our understanding of biomolecular dynamics, because these experiments have resolved conformational changes at submolecular resolution in aqueous environments. However, the resolution of HS-AFM images and AFM images in general critically depend on the sharpness of the tip. Focused electron beam-induced deposition (FEBID) in a scanning electron microscope (SEM) yields AFM tips with high aspect ratio and small tip radius, formed by deposition of metal-organic precursors such as ferrocene, producing amorphous carbon deposits, permissive for the reproducible acquisition of high-resolution images. Although FEBID tips are essential for HS-AFM image quality, it is common to rely on commercial cantilevers, because of limited SEM access or perceived technical barriers. Here, we showcase the use of a commercial benchtop SEM for the fabrication of FEBID tips for high-resolution AFM. To facilitate the fabrication process, we developed a cantilever and sample holder that minimize drift and mechanical vibrations during SEM operation and enable efficient and reproducible fabrication of sharp AFM tips. The shape of the FEBID tips was evaluated as a function of beam defocus, deposition time, acceleration voltage and beam current. We describe an optimized workflow for FEBID tip fabrication (eight tips can be fabricated within 1-2 h) and demonstrate high-resolution and long-term stable HS-AFM imaging of annexin V and plasmid DNA using the FEBID tips fabricated in this study. We anticipate that this protocol may be useful for a wide range of AFM practitioners and facilitate the reproducible acquisition of high-resolution AFM data.

Ion channel inactivation is a key modulatory mechanism that shapes action potentials and cellular excitability. In N-type (ball-and-chain) inactivation, a tethered N-terminal domain occludes the open pore. The prokaryotic MthK channel, a homolog of BK channels, undergoes such inactivation via its N-terminus. Notably, MthK inactivation was observed in liposome assays but not in decane-containing planar bilayer recordings, suggesting membrane dependence. We found that MthK inactivation progressively slowed with increasing bilayer thickness in liposomes composed of varying acyl-chain length lipids. Pore size was not a determining factor, as cryo-EM structures and molecular dynamics (MD) simulations showed similar pore dimensions across conditions, and block of a non-inactivating mutant by a peptide mimicking the N-terminal domain was largely bilayer thickness-independent. Instead, MD simulations, later confirmed with mutagenesis, revealed that N-terminal arginines form stronger interactions with lipid phosphates in thicker bilayers, thus limiting the access of the N-terminus to the pore and slowing inactivation.

The cantilever mediates tip-sample interaction detection in all atomic force microscopes (AFMs). Canonical cantilevers are beams, where length, width, and thickness define the physical properties such as stiffness and resonant frequency, that also mediate laser-reflection to report on cantilever deflection. High-speed AFM (HS-AFM) demands miniaturized cantilevers that are soft and fast, but miniaturized beams reduce laser signal quality. Here, we present a seesaw cantilever with a rigid reflective board oscillating over torsional hinges separating the laser-reflective and mechanical functions. Finite element analysis verified the seesaw mechanism. The board can be optimized for laser-reflection and the shortened distance between tip and hinges enhances the angular sensitivity, while the stiffness is tunable via the hinge dimensions. We detail seesaw cantilever design, fabrication, tip addition, physical equations, and sub-molecular imaging of biological samples. We propose that seesaw cantilevers offer a promising alternative to traditional beam cantilevers for diverse AFM applications.

The solvent of membrane proteins is the membrane lipids in which they are embedded. Therefore, the nature of the lipids that surround membrane proteins impacts their dynamics and interactions. Unfortunately, how membrane proteins dynamically interact is difficult to study, and little is experimentally known how membrane proteins interplay in a membrane at the molecular scale. Herein, high‐speed atomic force microscopy (HS‐AFM) is used to dynamically image a well‐controlled bottom‐up system consisting of two aquaporin‐fold membrane proteins, pentameric FocA and tetrameric GlpF, that interact in membranes composed of varying amounts of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) and E. coli lipids. It is found that the lipid environment significantly influences membrane protein mobility and interaction, where increased E. coli lipid content reduces protein movement, while DOPC‐rich environments promote mobility. Furthermore, the supramolecular structures of the membrane proteins and protomer interactions in clusters are also lipid modulated, where E. coli lipids favor specific protein–protein interactions, whereas greater interaction variability is found in DOPC. These findings highlight the role of lipids in regulating protein diffusion and interactions and suggest that lipid–protein interaction energetics play a significant role in controlling membrane protein interactions and supramolecular assembly.

Cryogenic electron microscopy (cryo-EM), X-ray crystallography, and nuclear magnetic resonance (NMR) contribute structural data that are interchangeable, cross-verifiable, and visualizable on common platforms, making them powerful tools for our understanding of protein structures. Unfortunately, atomic force microscopy (AFM) has so far failed to interface with these structural biology methods, despite the recent development of localization AFM (LAFM) that allows extracting high-resolution structural information from AFM data. Here, we build on LAFM and develop a pipeline that transforms AFM data into 3D-density files (.afm) that are readable by programs commonly used to visualize, analyze, and interpret structural data. We show that 3D-LAFM densities can serve as force fields to steer molecular dynamics flexible fitting (MDFF) to obtain structural models of previously unresolved states based on AFM observations in close-to-native environment. Besides, the .afm format enables direct 3D or 2D visualization and analysis of conventional AFM images. We anticipate that the file format will find wide usage and embed AFM in the repertoire of methods routinely used by the structural biology community, allowing AFM researchers to deposit data in repositories in a format that allows comparison and cross-verification with data from other techniques.

Bcs1 is a AAA-ATPase that transports the Rieske iron-sulfur protein (ISP) across the inner mitochondrial membrane. Bcs1 is a particular molecular machine in many regards: In contrast to canonical, hexameric, soluble AAA-ATPases (i) Bcs1 is heptameric, (ii) Bcs1 is a transmembrane protein with each subunit featuring a transmembrane helix, (iii) Bcs1 transports ISP in its folded state, and (iv) Bcs1 works using a concerted mechanism and not a hand-over-hand or stochastic mechanism. How Bcs1 binds and transports folded ISP is unknown. Here we used high-speed atomic force microscopy (HS-AFM) single-molecule analysis and report that Bcs1 subunits are conformationally fully coupled: When Bcs1 is exposed to a mixture of AMP-PNP and ADP where the probability to be in the AMP-PNP or the ADP conformation is equal, all Bcs1 ring complexes are either in AMP-PNP or ADP state, and none forms a hetero-conformer ring. When Bcs1 is exposed to a mixture of AMP-PNP and ATP, traces of AMP-PNP inhibit Bcs1 action showing that all subunits in the ring must be compatible to hydrolyze ATP and undergo a conformational change for function. Furthermore, ISP binds exclusively to the matrix cavity of apo-conformation AAA-domains. Finally, ISP-Bcs1 binding is long enough to outlast the apo-conformation lifetime in ATP-turnover conditions, assuring high transport efficiency. Our single-molecule structural and kinetic data reveals that Bcs1 works according to a unique mechanism so far unknown for any AAA-ATPase.

TRPV3 belongs to the large superfamily of tetrameric transient receptor potential (TRP) ion channels. Recently, using high-speed atomic force microscopy (HS-AFM), we discovered a rare and transient pentameric state for TRPV3 that is in equilibrium with the tetrameric state, and, using cryo-EM, we solved a low-resolution structure of the TRPV3 pentamer, in which, however, many residues were unresolved. Here, we present a higher resolution and more complete structure of the pentamer, revealing a domain-swapped architecture, a collapsed vanilloid binding site, and a large pore. Molecular dynamics simulations and potential of mean force calculations of the pentamer establish high protein dynamics and permeability to large cations. Subunit interface analysis, together with thermal denaturation experiments, led us to propose a molecular mechanism of the tetramer-to-pentamer transition, backed experimentally by HS-AFM observations. Collectively, our data demonstrate that the TRPV3 pentamer is in a hyper-activated state with unique, highly permissive permeation properties.

Septins are cytoskeletal proteins that form filaments and higher-order structures, and remodel membranes in a variety of processes. Structural and cell biological studies provided atomic- and micro-scale details, but the understanding of septin assembly at the mesoscale is limited. Here, we used high-speed atomic force microscopy (HS-AFM) to analyze yeast septin assembly on yeast supported lipid bilayers (SLBs). We found the coexistence of three lipid phases in yeast membranes, where septin polymerized selectively on the liquid-disordered phase. Septin filaments adhered to membranes with a conserved face; and paired filaments, previously reported in less native environments, were not observed. Additionally, septin filaments exhibited lateral and longitudinal alignment. We used HS-AFM force-sweep experiments to disrupt septin structures and observe organizational recovery through self-templating. Finally, septin filaments stacked, where higher layer filament alignment was templated by the layer below. Thus, septins encode their 3D-structural organization, likely tunable by the membrane and bulk environment.

The Pyrococcus horikoshii amino acid transporter GltPh revealed, like other channels and transporters, activity mode switching, previously termed wanderlust kinetics. Unfortunately, to date, the basis of these activity fluctuations is not understood, probably due to a lack of experimental tools that directly access the structural features of transporters related to their instantaneous activity. Here, we take advantage of high-speed atomic force microscopy, unique in providing simultaneous structural and temporal resolution, to uncover the basis of kinetic mode switching in proteins. We developed membrane extension membrane protein reconstitution that allows the analysis of isolated molecules. Together with localization atomic force microscopy, principal component analysis and hidden Markov modeling, we could associate structural states to a functional timeline, allowing six structures to be solved from a single molecule, and an inward-facing state, IFSopen-1, to be determined as a kinetic dead-end in the conformational landscape. The approaches presented on GltPh are generally applicable and open possibilities for time-resolved dynamic single-molecule structural biology. Combining high-speed atomic force microscopy (AFM) with localization AFM and principal component analysis, the authors present six structures of a glutamate transporter and associate the conformational states to the molecule’s activity timeline.

Membrane proteins diffuse and interact with each other in the lipid bilayer. Thus, understanding how changes in the membrane composition and properties modulate protein-lipid and protein-protein interactions is important at the intersection between membrane biology and membrane physics. Here, the diffusion and interaction of unlabeled membrane proteins were visualized and quantified using high-speed atomic force microscopy (HS-AFM). We mixed and reconstituted the E. coli formate (FocA) and glycerol (GlpF) facilitators—FocA is a pentamer and GlpF is a tetramer of aquaporin-fold subunits. In parallel, we made small unilamellar vesicles (SUVs) of DOPC with varying proportions of E. coli lipids. Subsequently, we incubated the reconstituted protein on the mica surface and supplemented them with the SUVs and observed supported lipid bilayer (SLB) formation of the lipids and fusion with the protein patches. Following complete SLB formation with the embedded FocA and GlpF, we analyzed the proteins' interaction behavior as a function of the membrane composition. We found that with increasing proportion of E. coli lipids, the mobility of the proteins reduced significantly and protein patches were increasingly stable. We analyzed the protein patch boundary edge speed and the delta height between membrane proteins and the bilayer. The analysis indicated a strong correlation between the proportion of E. coli lipids and protein patch edge velocity, and delta height. We deduced the patch line tension, the free energy per unit length at the domain boundaries, and from the delta height value, we estimated the membrane distortion and the free energy of deformation per unit membrane area surrounding proteins. The methodology presented in this study allowed to make further progress in understanding the intricate interplay between membrane proteins and their surrounding membrane.

Cryo-electron microscopy (cryo-EM), X-ray crystallography, and nuclear magnetic resonance (NMR) contribute structural data that are interchangeable, cross-verifiable, and visualizable on common platforms making them powerful tools for our understanding of protein structures. Unfortunately, atomic force microscopy (AFM) has so far not found ways to interface with the other structural biology methods, because it did not produce data and files that were comparable with data from the other techniques and/or readable in the common data visualization software.

The biogenesis and maintenance of thylakoid membranes require vesicle-inducing protein in plastids 1 (VIPP1). VIPP1 is a member of the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, whose members form diverse filament-based supramolecular structures that facilitate membrane deformation and fission. VIPP1 cryo-electron microscopy (EM) structures in solution revealed helical rods and baskets of stacked rings, with amphipathic membrane-binding domains in the lumen. However, how VIPP1 interacts with membranes remains largely unknown. Here, using high-speed atomic force microscopy (HS-AFM), we show that VIPP1 assembles into right-handed chiral spirals and regular polygons on supported lipid bilayers via ESCRT-III-like filament assembly and dynamics. VIPP1 filaments grow clockwise into spirals through polymerization at a ring-shaped central polymerization hub, and into polygons through clockwise polymerization at the sector peripheries. Interestingly, VIPP1 initially forms Archimedean spirals, which upon maturation transform into logarithmic spirals through lateral annealing of strands to the outermore low-curvature spiral turns. Using high-speed atomic force microscopy, the authors reveal the polymerization mechanism of VIPP1 into ESCRT-III-like filaments that adopt spiral and polygonal supramolecular structures.

As atomic structures of membrane proteins are readily resolved and predicted due to the recent advances in biophysical methods like cryo-EM as well as computational tools like AlphaFold, there emerges an urgent need to investigate the real-time structural dynamics at the single molecule level to obtain novel insights in the membrane structural biology. Here, we present a unique sample preparation method for high-speed atomic force microscopy (HS-AFM), which enabled us to immobilize membrane proteins in an extended lipid bilayer, while preserving their dynamics. This strategy bypasses the previous requirement of the dense packing of molecules. The approach allowed to acquire HS-AFM movies of individual transporters with increased resolution and in a physiologically more relevant setting. Using GltPh, a trimeric aspartate transporter, as a model protein, we demonstrate that HS-AFM combined with the presented strategy was able to track structural dynamics of individual protomers at sub-second temporal resolution and sub-nanometer spatial resolution for tens of seconds. Recent cryo-EM studies of GltPh revealed two major states of the protein, outward-facing (OF) and inward-facing (IF) states, as well as several conformational sub-states. Real-time transitions between these states were observed in the HS-AFM data with unprecedented detail. For data analysis, we developed a principal component analysis (PCA) based method to sort protomers at each time point into different conformations according to their structural features, reconstructing a structure-time trace for each protomer of a single molecule. Using the previously developed localization AFM (LAFM) method, we calculate Angstrom-resolution GltPh surface structures of single molecules as it transits through several conformational states in the transport cycle. The presented method enables direct observation and analysis of the structure and dynamics of individual unlabeled membrane proteins, unlocking the potential of HS-AFM in the study of single molecule structural biology.

Transient receptor potential (TRP) ion channels are gated by diverse intra- and extracellular stimuli leading to cation inflow (Na+, Ca2+) regulating many cellular processes and initiating organismic somatosensation. Structures of most TRP channels have been solved. However, structural and sequence analysis showed that ~30% of the TRP channel sequences, mainly the N- and C-termini, are intrinsically disordered regions (IDRs). Unfortunately, very little is known about IDR 'structure', dynamics and function, though it has been shown that they are essential for native channel function. Here, we imaged TRPV2 channels in membranes using high-speed atomic force microscopy (HS-AFM). The dynamic single molecule imaging capability of HS-AFM allowed us to visualize IDRs and revealed that N-terminal IDRs were involved in intermolecular interactions. Our work provides evidence about the 'structure' of the TRPV2 IDRs, and that the IDRs may mediate protein-protein interactions.