Tag: USC-F1.2-k0.15

Watching Designed Proteins Assemble with High-Speed AFM

How the surface beneath a protein can influence the structures it forms

What determines whether nanoscale building blocks assemble into an ordered structure or remain disordered?

In a study published in Nature Communications, Sakshi Yadav Schmid and colleagues investigated this question using designed protein nanorods at liquid–solid interfaces. Combining high-speed atomic force microscopy (HS-AFM), machine-learning analysis and computational modelling, they explored how the surface beneath the proteins influences their movement and organization.

The researchers observed a striking contrast: the same protein nanorods formed different arrangements on two types of mica, even though both surfaces have the same potassium-ion sublattice to which the proteins were designed to bind. Their findings reveal why understanding nanoscale assembly requires looking beyond the interactions deliberately built into the protein design.

Following protein assembly in real time

Self-assembly occurs when individual building blocks organize into larger structures through interactions with one another and their surroundings. To understand how these structures form, it helps to observe the process as it unfolds—not just examine the final arrangement.

High-speed AFM allowed the researchers to observe the protein nanorods in liquid as the assembly process unfolded. Using NanoWorld ARROW-UHFAuD and USC-F1.2-k0.15 cantilevers in their high-speed AFM experiments, they could follow how the nanorods moved and how their arrangements changed over time, rather than relying solely on images of the final structures.

The experiments compared two substrates: muscovite mica (m-mica) and fluorophlogopite mica (f-mica). Under the high-salt conditions investigated, the nanorods behaved very differently on the two surfaces.

On muscovite mica, the nanorods were highly mobile. Small groups of aligned rods appeared, grew and rearranged until a more stable, layered structure emerged. This arrangement is known as smectic order: the elongated building blocks point predominantly in the same direction and are organized into layers.

Time-lapse high-speed AFM images and corresponding nanorod identification images showing protein assembly on muscovite mica. Graphs below show the number of rods, nematic alignment and development of smectic order over time.
Figure 3. Development of smectic order on muscovite mica. Time-lapse high-speed AFM images (a) show designed protein nanorods assembling on muscovite mica. The corresponding machine-learning-based identification of the rods (b) supports quantitative measurements of the number of rods (c), nematic order (d) and smectic order (e). Together, the measurements reveal how alignment and layered organization develop over time.
Source: Yadav Schmid et al., Nature Communications 17, 2446 (2026), https://doi.org/10.1038/s41467-026-69170-0. © Battelle Memorial Institute and The Authors 2026. Licensed under CC BY 4.0. Reproduced without modification.

On fluorophlogopite mica, the rods showed translational and rotational movement but were less mobile overall. Their orientations followed three directions associated with the substrate’s potassium-ion sublattice. As more rods accumulated, they formed a high-density disordered assembly, rather than the layered structure seen on muscovite mica.

Time-lapse high-speed AFM images and corresponding nanorod identification images showing protein assembly on fluorophlogopite mica. Graphs below show rod counts and comparatively low nematic and smectic order parameters over time.
Figure 4. High-density disordered assembly on fluorophlogopite mica. Time-lapse high-speed AFM images (a) and corresponding machine-learning-based identification of the rods (b) document protein nanorods on fluorophlogopite mica. Measurements of the number of rods (c), nematic order (d) and smectic order (e) show that the assembly remains comparatively disordered.
Source: Yadav Schmid et al., Nature Communications 17, 2446 (2026), https://doi.org/10.1038/s41467-026-69170-0. © Battelle Memorial Institute and The Authors 2026. Licensed under CC BY 4.0. Reproduced without modification.

The difference was particularly intriguing because the potassium-ion patterns that guide protein binding are identical on the two substrates. Something beyond that shared pattern was influencing the outcome.

Measuring how order develops

Watching nanorods move provides a view of self-assembly in action. But how can researchers measure the transition from individual movements to collective organization?

The team used machine-learning methods to identify nanorods in the high-speed AFM images and determine their positions and orientations. From these measurements, they could track surface coverage and calculate how the degree of order changed over time.

Two quantities were especially useful. Nematic order describes how strongly elongated objects align in a common direction. Smectic order additionally captures their organization into layers.

On muscovite mica, the nanorods aligned relatively quickly, while their layered organization developed more gradually as surface coverage increased. On fluorophlogopite mica, the rods accumulated without developing comparable long-range alignment or smectic order.

This quantitative analysis showed not only that the final structures differed, but also how the two assembly processes followed different paths.

Why the surface matters

What could explain such different behavior on substrates with identical potassium-ion sublattices?

An important clue lies in the water next to the solid surface. The atomic structures of muscovite and fluorophlogopite mica differ in ways that influence the arrangement of nearby water molecules. These interfacial hydration structures can, in turn, affect the interactions experienced by the protein nanorods.

To investigate this explanation, the researchers used Monte Carlo simulations that considered the mobility of rod-shaped building blocks and the influence of preferred orientations imposed by the substrate. When the model introduced a directional preference representing the broken symmetry associated with muscovite mica, smectic ordering could emerge. Without that preference, the model reproduced behavior consistent with disordered assembly.

The distinction between the methods is important: high-speed AFM revealed how the nanorods moved and assembled, machine-learning analysis quantified the developing order, and the simulations helped explain how surface-related effects could produce the observed differences.

The findings highlight a factor that is easy to overlook when designing protein assemblies: the surrounding liquid and the structure of the underlying surface can influence the outcome, even when the intended protein–surface binding pattern is the same.

By revealing these effects as assembly happens, high-speed AFM provides an experimental window into processes that may be difficult to understand from finished structures alone. Combined with quantitative analysis and modelling, such observations can help researchers develop a more complete understanding of how designed proteins organize at interfaces.

Full citation

Yadav Schmid, S.; Helfrecht, B.; Stegmann, A.; Legg, B. A.; Pyles, H.; Chen, J.; Edison, J. R.; Ziatdinov, M.; Preisler, Z.; Dollar, O.; Whitelam, S.; Kalinin, S.; Baker, D.; Mundy, C. J.; Zhang, S.; De Yoreo, J. J.
Impact of solvent forces and broken symmetry on the assembly of designed proteins at a liquid-solid interface.
Nature Communications 2026, 17, 2446.
https://doi.org/10.1038/s41467-026-69170-0

Creative Commons Attribution 4.0 International (CC BY 4.0)

https://creativecommons.org/licenses/by/4.0/

Decoding the Ubiquitin Code: How the RQT Complex Clears Colliding Ribosomes

Matsuo et al., recently published a landmark study in Nature Communications (vol. 14, article 79, Jan 10 2023). , looking at how cells recognize and resolve ribosome collisions—a critical event in obeying translational fidelity and avoiding protein quality control failure.

The researchers employed a combination of molecular genetics, biochemical assays, ubiquitin-binding studies, and advanced imaging. Key steps included mutational deletion of ubiquitin‐binding domains in RQT subunits, affinity assays for K63-linked ubiquitin chains, and highspeed atomic force microscopy (HSAFM) to visualize complex behavior at the molecular level. Notably, they used intrinsically disordered regions of Rqt4 mapped by realtime HSAFM.

The study shows that Cue3 and Rqt4 of the RQT complex interact with the K63-linked ubiquitin chain and facilitate the recruitment of the RQT (ribosome-associated quality control trigger) complex to ubiquitinated colliding ribosomes. Deletion of either domain abolished RQT’s ability to dissociate colliding ribosomes. Crucially, HSAFM revealed that Rqt4’s flexible disordered segments expand the interaction radius, enabling effective engagement with the ubiquitin chain. This expanded search capability enhances timely RQT recruitment and ribosome splitting before rogue collisions build up.
These findings elucidate a molecular “decoding” mechanism—how RQT interprets the ubiquitin code (specifically K63 ubiquitination) and transforms it into mechanical action, splitting ribosomal subunits to facilitate quality control. This work provides a mechanistic link between ubiquitin signaling and translational rescue pathways in the cell NanoWorld’s USC-F1.2-k0.15, designed for resonance frequencies of 1.2 MHz and tip radii below 10 nm, were used in the high-speed AFM studies that were crucial to this investigation.

Fig. 3: The dynamics of the RQT complex
licensed under a Creative Commons Attribution 4.0 International License
Matsuo, Y., Uchihashi, T. & Inada, T. Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex. Nat Commun 14, 79 (2023). https://doi.org/10.1038/s41467-022-35608-4
Decoding the Ubiquitin Code: How the RQT Complex Clears Colliding Ribosomes

a HS-AFM image of Slh1. Two major particles were indicated as Class1 and Class2. b The pseudo-AFM images of Slh1 belonging to Class1 and Class2 particles, which were simulated using predicted Slh1 structure lacking N-terminal region by Alphafold2. c The HS-AFM images and schematized molecular features of Slh1. d Classification of Slh1 particles. All particles used for the classification were presented in the supplementary Fig. 4. e HS-AFM images of Slh1 lacking N-terminal region (Slh1∆N). f HS-AFM image of Cue3. g HS-AFM image of Rqt4. h, i HS-AFM images of Slh1/Cue3 complex. j, k HS-AFM images of Slh1/Rqt4 complex. l The time-lapse HS-AFM images of the RQT complex. All experiments were performed at least twice with highly reproducible results.

This article beautifully merges state-of-the-art structural biology with nanotechnology tools to reveal how molecular collisions are detected and resolved. The incorporation of ultra-fast AFM probe technology makes it possible to observe RQT in action, delivering new insight into cellular quality control.

Real-time multistep asymmetrical disassembly of nucleosomes and chromatosomes visualized by high-speed atomic force microscopy

During replication, expression, and repair of the eukaryotic genome, cellular machinery must access the DNA wrapped around histone proteins forming nucleosomes. These octameric protein·DNA complexes are modular, dynamic, and flexible and unwrap or disassemble either spontaneously or by the action of molecular motors. Thus, the mechanism of formation and regulation of subnucleosomal intermediates has gained attention genome-wide because it controls DNA accessibility.*

In the article  “Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy” Bibiana Onoa, César Díaz-Celis, Cristhian Cañari-Chumpitaz, Antony Lee and Carlos Bustamante describe how they imaged nucleosomes and their more compacted structure with the linker histone H1 (chromatosomes) using high-speed atomic force microscopy to visualize simultaneously the changes in the DNA and the histone core during their disassembly when deposited on mica.*

Furthermore, Bibiana Onoa et al. trained a neural network and developed an automatic algorithm to track molecular structural changes in real time. *

The authors’ results show that nucleosome disassembly is a sequential process involving asymmetrical stepwise dimer ejection events. The presence of H1 restricts DNA unwrapping, significantly increases the nucleosomal lifetime, and affects the pathway in which heterodimer asymmetrical dissociation occurs. *

Bibiana Onoa et al.  observe that tetrasomes are resilient to disassembly and that the tetramer core (H3·H4)2 can diffuse along the nucleosome positioning sequence. Tetrasome mobility might be critical to the proper assembly of nucleosomes and can be relevant during nucleosomal transcription, as tetrasomes survive RNA polymerase passage. These findings are relevant to understanding nucleosome intrinsic dynamics and their modification by DNA-processing enzymes. *

To characterize the nucleosomes dynamics in 2D, individual molecules were observed in buffer using an Ando-type high speed atomic force microscope together with NanoWorld Ultra-Short Cantilevers for HS-AFM of the USC-F1.2-K0.15 AFM probe type ( typical spring constant 0.15 N/m, typical resonance frequency in air 1200 kHz, resonance frequency 500–600 kHz in liquid). *

The AFM data presented in the article allow the authors to directly visualize the dynamics of DNA and histones during nucleosome and chromatosome disassembly, providing a simultaneous observation of DNA unwrapping and histone dissociation. *

The experimental and analytical strategy presented shows that real-time HS-AFM is a robust and powerful tool for studying single nucleosomes and chromatin dynamics. *

graphical abstract from Bibiana Onoa et al 2024 "Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy" - NanoWorld Ultra-Short Cantilevers of the USC-F1.2-k0.15 AFM probe type were used for the high-speed atomic force microscopy
graphical abstract from Bibiana Onoa et al 2024 “Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy”

*Bibiana Onoa, César Díaz-Celis, Cristhian Cañari-Chumpitaz, Antony Lee and Carlos Bustamante
Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy
ACS Central Science 2024, 10, 1, 122–137
DOI: https://doi.org/10.1021/acscentsci.3c00735

Open Access The article “Real-Time Multistep Asymmetrical Disassembly of Nucleosomes and Chromatosomes Visualized by High-Speed Atomic Force Microscopy” by Bibiana Onoa, César Díaz-Celis, Cristhian Cañari-Chumpitaz, Antony Lee and Carlos Bustamante is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.