Atypical β-strand insertion mediates the noncovalent cross-linking in amyloid aggregates

Domain: chemistry
Published: 2026-01-06
Editorial outcome: major_revision

Abstract
β-Strand motifs are essential recognition modules in protein-protein interactions (PPIs), which govern cellular signaling networks and regulate molecular pathway dynamics. Herein we present an unexpected discovery of a previously uncharacterized β-strand insertion mechanism termed as cross-β-strand linking, wherein β-strands within the β-sheet-rich aggregates form inter-β-sheet connections through insertion into adjacent β-sheets. These cross-β-strand linkers comprise <15% of the total β-strands in the amyloidogenic aggregates, but they can mediate a significant proportion of intermolecular interactions, operating as dynamic molecular adapters that regulate the inter-β-sheet packing geometry. Crucially, these linkers exist as conformational ensembles of heterogeneous substates, bestowing remarkable structural diversity to the aggregates. Through promiscuous engagement with multiple conformational substates, cross-β-strand linkers enable the aggregates to balance order and disorder. In this work, we provide a perspective on how low-abundance structural elements can orchestrate complex molecular architectures in assembly systems.

Introduction
PPIs are foundational to cellular communications and to orchestrate molecular pathways through precise recognition events1–3. These interactions are also critical targets for the therapeutic interventions of diseases4–6. While the classical paradigm of interprotein recognition emphasizes shape and chemical complementarity at the interfaces, i.e., the solvent-excluded regions that contribute to binding specificity and affinity7,8. However, our current understanding of PPI mechanisms remains far from complete. Over 95% of human PPIs have not been structurally characterized so far9. Moreover, native proteins usually exist as conformational ensembles comprising multiple populations10,18–20. Obviously, this ensemble-averaged attribute in these techniques fundamentally obscures the characterization of transient and/or low-abundance conformational substates, which are critical to PPI regulation10,17,21.

Short peptide motifs frequently act as recognition elements in PPI, functioning through burial in binding partner cavities, positioning within pockets/grooves, or β-sheet integration at protein interfaces6,22,23. Among PPIs involving peptide recognition elements, β-strand addition emerges as an important mechanism governing interprotein recognition23–25. This process involves the donation of ligand β-strand to receptors through three canonical classes (Fig. 1a): (1) β-sheet augmentation via edge-to-edge interactions between a ligand β-strand and a partner β-sheet; (2) β-strand complementation, where a ligand β-strand is embedded into its complementary receptor; and (3) β-strand zippering through antiparallel alignment of complementary flexible regions in both interactors23,26–30. Herein, we describe a previously unrecognized β-strand insertion mechanism: β-strands within the β-sheet-rich aggregates bridge adjacent β-planar sheets by their insertion into neighboring β-sheets (Fig. 1b). We therefore term it a cross-β-strand linking mechanism.

In this work, a cross-β-strand linking mechanism is identified in amyloid aggregation. Amyloid aggregation plays a crucial role in many protein misfolding-related diseases, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis31–33. Amyloid fibrils are mainly composed of β-sheet structures stabilized by extensive intermolecular hydrogen bonding and exhibit remarkable structural stability34. A typical feature of amyloid aggregates is conformational heterogeneity, namely, identical peptide sequences can adopt distinct conformational substates coexisting within a heterogeneous conformational ensemble. Such conformational heterogeneity is closely linked to their pathological functions17,21. However, due to the inherent heterogeneity of amyloid structures, conventional structural techniques (e.g., X-ray crystallography, NMR, and cryo-EM) remain limited in resolving coexisting conformations. To directly visualize the local structural features within amyloid fibrils, we employed scanning tunneling microscopy (STM) under ambient conditions. This technique provides exceptional resolution down to the single-molecule level, offering a complementary perspective on the heterogeneous conformational ensemble of amyloid fibrils. STM relies on electron tunneling and requires a conductive, flat substrate; therefore, highly oriented pyrolytic graphite (HOPG) was used as the supporting substrate. Unlike more reactive STM substrates such as Cu(001) or Cu(110) — which may form covalent bonds with adsorbates—HOPG is chemically inert to interact with adsorbates35. Although STM imaging was performed under nominally dry conditions, a thin residual water film on the HOPG surface helped maintain local hydration, thereby preventing dehydration-induced denaturation36. We use STM-based imaging to detect non-periodic structural signals with submolecular precision and reveal the structural basis of cross-β-strand linking. Despite constituting a minor population (<15%) of the total β-strands in aggregates, these cross-β-strand linkers exhibit conservation across diverse amyloid peptides, promoting the structural order of amyloid fibril architecture through inter-sheet cross-linking.

Results
STM imaging provides direct visualization of β-strand insertion in amyloid β-sheet aggregates
We initiated our investigation with the aggregates formed by the 225–250 segment of galectin-3 (gal-3 225–250), which participates in the PPIs between gal-3 molecules and their phase separation37,38. This peptide undergoes spontaneous self-assembly into amyloid-like fibrils in aqueous solution, as confirmed by transmission electron microscopy (TEM) imaging (Supplementary Fig. 1a). Fourier transform infrared (FTIR) spectroscopy confirms the conformational transition of the fibrils, as evidenced by the increased β-sheet (1632 cm−1) bands relative to its monomeric state (Supplementary Fig. 1b). Time-lapse thioflavin-T (ThT) fluorescence assays show a sigmoidal increase in fluorescence intensity, characteristic of amyloid fibril formation kinetics (Supplementary Fig. 1c). The structure-informed machine learning analysis using ZipperDB [https://zipperdb.mbi.ucla.edu/] predicts39 a high aggregation propensity for gal-3 225–250 with Rosetta energy below −23 kcal/mol, which is well aligned with experimental observations (Supplementary Fig. 1d). These multiscale validation approaches collectively establish gal-3 225–250 as a reliable model system for studying β-sheet-driven self-assembly processes.

To investigate the intermolecular interfaces of β-sheet aggregates, we performed STM imaging of the gal-3 225–250 aggregates after equilibrium incubation (36 h). Our group recently developed an STM-based single-molecule imaging platform, which bypasses the limitation of ensemble averaging and enables direct characterization of conformational ensembles17,21,40,41. The samples were deposited onto freshly cleaved HOPG substrates, a standard choice for ambient STM studies due to its atomically flat surface and chemical inertness42. The STM tips with an atomically sharp radius were homemade from platinum-iridium alloys via the combined processes of mechanical and electric pulsing. Distinct molecular conformations on the HOPG surface result in the change of the local density of electron states, which consequently modulate tunneling current magnitudes. By scanning the tip across the surface, we obtained real-space topographical maps reflecting the molecular structures, dispensing with structural reconstructions.

The single-molecule imaging of gal-3 225–250 aggregates reveals signature β-sheet features (Supplementary Fig. 1e and Supplementary Table 1). The molecules appear as periodic bright linear patterns forming lamellar structures, with protrusions in the local density of electron states corresponding to peptide bonds and aromatic residues21,42,43. The intermolecular spacing between adjacent molecules (4.2±0.1 Å) matches the characteristic β-sheet interstrand distance42,44 (Supplementary Fig. 1f). Individual β-strands within the β-sheet assemblies exhibit an orientation perpendicular to the direction of sheet extension. We also attempted STM imaging of the sample before aggregation (0 h) (Supplementary Fig. 2). However, the result shows that they lack sufficient intermolecular and substrate interactions to form stabilized, ordered structures under ambient conditions, because the unassembled molecules on the HOPG surface undergo rapid thermal diffusion, which precludes the acquisition of single-molecule images45.

More detailed analysis of gal-3 225–250 aggregates via STM imaging uncovers an unprecedented structural feature: stochastic cross-β-strand linkers acting as molecular mortise-and-tenon joints between two β-sheets (Fig. 1c–f). These exceptional β-strand linkers protrude from one β-sheet domain, traverse through the inter-sheet gap, and integrate seamlessly into an adjacent β-sheet domain without any structural discontinuity. Two features are observed for the β-strand linkers. First, the occurrence frequency is low: there are only ~5 instances observed per 15×15 nm area containing ~100 molecules. The second one is the conformational plasticity. Individual cross-β-strand linkers exhibit a pronounced length variability (3.0–7.3 nm), indicative of their dynamic conformational flexibility. Our findings represent direct visualization of stochastic β-strand-mediated inter-sheet connections in amyloid aggregates, challenging the conventional models of β-sheet aggregation that typically emphasize the highly ordered arrangement of β-strands46–48.

Cross-β-strand linkers form interstrand connections with distinct conformational substates in β-sheet aggregates
Single-molecule imaging allows us to decode the structural organization of cross-β-strand linkers with a nanoscale resolution. In the gal-3 225–250 aggregates, β-strands exhibit length variations from 1.6 to 7.3 nm (average: 4.2±1.2 nm), while cross-β-strand linker lengths span from 3.0 to 7.3 nm. Given the periodicity of 3.25 Å between consecutive residues in the β-strands, we established a quantitative relationship between strand length and amino acid sequence: N=L/3.25 Å, where N is the number of residues and L is β-strand length49. Detailed discussion is provided in Supplementary Note 1.1. This relationship allows us to distinguish the distinct conformational substates based on their residue counts (Supplementary Fig. 3a).

As shown in Fig. 2a, this analysis reveals a highly heterogeneous conformational ensemble in the β-sheet aggregates: 18 coexisting conformational substates (labeled I–XVIII) were identified, corresponding to 5–22 residues. The most abundant substate (IX) contains 13 residues and accounts for 14.1% of total β-strands (Fig. 2b). Among these 18 substates, 14 substates (V–XVIII) participate in the formation of cross-β-strand linkers (Fig. 2b). Importantly, the cross-β-strand linker represents a minor structural element within individual conformational substates (Supplementary Table 2).

In a pleated β-sheet, adjacent β-strands are interconnected laterally through a hydrogen bond network that stabilizes the structure47,49,50. The conformational diversity of β-strands enhances the heterogeneity in the interstrand recognition. Within the gal-3 225–250 aggregates, we identified 139 distinct inter-β-strand interaction types among the 18 conformational substates, with 62 interactions (accounting for ~45% of total interaction types with a population only around ~14%) involving the cross-β-strand linkages (Fig. 2c, d and Supplementary Fig. 3b). The disproportionate contributions of the cross-β-strand linkers to the interactions indicate that they are critical mediators of interstrand interactions. Cross-β-strand linkers exhibit no preferential selection bias toward specific conformational substates (Supplementary Fig. 4), as all conformational substates in the aggregates participate in the interactions with cross-β-strand linkers.

Cross-β-strand linkers are prevalent across diverse β-sheet aggregates
With the cross-β-strand linkers observed, one question naturally arises: Is it unique to gal-3 225–250 aggregates or prevalent in the β-sheet-rich aggregates? To answer this question, we employed STM to characterize a series of aggregation structures formed by β-sheet-containing peptides, including the 206–231 segment of galectin-3 (gal-3 206–231), the 129–154 segment of galectin-3 (gal-3 129–154), amyloid-β42 (Aβ42), amyloid-β42 with an N-terminal extension of six residues (−5Aβ42), the 8–37 segment of human islet amyloid polypeptide (hIAPP 8–37), hIAPP 8–37 with a replacement of Ser-to-Gly at site 20 (hIAPP S20G 8–37), and the 341–357 segment of the transactive response DNA binding protein 43 (TDP-43 341–357). Under the same incubation conditions used for STM imaging, the results of TEM, ThT fluorescence, and FTIR analyses coherently confirm that these aggregates are enriched in β-sheets and exhibit characteristic amyloid-like properties51, while AFM imaging further confirms their amyloid-like morphology on the HOPG surfaces (Supplementary Fig. 1a and Supplementary Figs. 5–9). Comparative FTIR spectra of the different peptide aggregates are shown in Supplementary Tables 3 and 4 and a detailed discussion is provided in Supplementary Note 1.2. The STM results confirm that all these peptide aggregates exhibit hallmarks of β-sheet assembly characterized by periodic electron-dense streaks arranged in a lamellar pattern with an orientation perpendicular to the direction of sheet extension (Fig. 3a–h and Supplementary Fig. 10, Supplementary Table 1).

Besides, the occurrence of cross-β-strand linkers exhibits significant molecular specificity. Linker-positive aggregates (Fig. 3a–e) include gal-3 206–231 (12.6% of total β-strand population), KRT-R (4.2%), Aβ42 (4.1%), hIAPP 8–37 (3.0%), and hIAPP S20G 8–37 (2.1%), while linker-negative aggregates (Fig. 3f–h) comprise −5Aβ42, TDP-43 341–357, and gal-3 129–154. We also scanned STM images of Aβ42 aggregates at 80 μM and consistently observed cross-β-strand linkers (Supplementary Fig. 11). Further analysis also confirms the two features shared by the cross-β-strand linkers across different molecular systems. First, the cross-β-strand linkers in all the systems account for a low population but participate in the interaction types in a disproportionate manner, which is similar to the case of the gal-3 225–250 aggregates above (Supplementary Fig. 12, and Supplementary Table 5). Second, these linkers demonstrate exceptional conformational plasticity, with quantitative analysis revealing distinct conformational substates: 20 substates for gal-3 206–231, 14 for Aβ42, 19 for hIAPP 8–37, 19 for hIAPP 8–37, 13 for KRT-R, and 19 for hIAPP S20G 8–37 (Supplementary Figs. 13–17). Collectively, these findings reveal the wide distribution of cross-β-strand linkers in diverse β-sheet assemblies, implying their structural and functional significance across protein aggregation systems. We note that STM imaging can only detect peptide segments within approximately 1 nm of the HOPG substrate, owing to the exponential decay of electron tunneling probability with distance, while regions beyond this range remain invisible. Consequently, STM primarily resolves molecular features in close proximity to the surface. Importantly, STM does not rely on three-dimensional reconstruction but instead directly visualizes the distribution of electronic states on the two-dimensional surface. Detailed discussion is provided in Supplementary Note 1.3.

Cross-β-strand linkers in amyloidal aggregates promote the structural order
The mortise-and-tenon-like cross-β-strand linkers may function as hierarchical stabilizers that modulate the assembly order of β-sheet aggregates. To quantitatively characterize the structural order of the aggregate morphologies, we conducted the short-time Fourier transform (STFT) analysis of the STM images. The original STM images were processed through binary segmentation to generate 500×500-pixel square regions, which were subsequently analyzed via the STFT with a 10-pixel sampling interval (Fig. 4a, Supplementary Figs. 18 and 19). The periodic stripe patterns of β-sheet aggregates manifest as distinct frequency-domain features in the power spectral density (PSD) image obtained via the STFT: (1) the orientation angle (θ) reflecting the directionality of β-sheet alignment, and (2) the spatial frequency (q) quantifying the reciprocal of periodicity (i.e., width/spacing) of β-sheets. For example, the STFT analysis of the red- and green-boxed regions in the STM image revealed distinct local frequency-domain characteristics, with corresponding (q, θ) values of (0.02 nm−1, 45°) for the red-boxed region and (0.01 nm−1, 63.4°) for the green-boxed region (Fig. 4a). Through systematic spatial windowing, color-coded parameter maps were generated to visualize the morphometric value distributions of θ and q across discrete window positions (Fig. 4b, c).

Quantitative assessment of morphometric parameters indicates the spatial heterogeneity in aggregate structural ordering. Gal-3 225–250 β-sheets exhibit a multimodal θ distribution with two main peaks at 54° and 107°, yielding a coefficient of variation (CV) of 0.54 (Fig. 4d). Meanwhile, the q value displays a distribution ranging from 0.02 nm−1 to 0.28 nm−1, peaking at 0.12 nm−1 with a CV of 0.29 (Fig. 4e). Correlation analysis between cross-β-strand linker frequency and morphometric parameters shows a clear dependence on peptide type. Circular visualization employs diameter encoding for cross-β-strand proportion and chromatic coding for sample discrimination, with gray dashed circle denoting linker-negative aggregates (Fig. 4f). Notably, the frequency of cross-β-strand linkers exhibits a significant negative correlation with the CV of q (Pearson’s correlation coefficient = −0.7, P=0.03), suggesting that these linkers promote ordered packing in β-sheet periodic assemblies (Supplementary Fig. 20). Conversely, no statistically significant association can be drawn between linker density and the CV of θ.

Methods
Reconstitution of peptide assemblies in solution
The peptides were obtained from Anhui Guoping Pharmaceutical Co., Ltd, after synthesis and purification, with a confirmed purity of over 98% through high-performance liquid chromatography and mass spectrometry analysis. All lyophilized peptide powders were initially dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Innochem), a fluorinated solvent known for efficient disruption of secondary structures. After complete evaporation of HFIP, gal-3 129–154, gal-3 206–231, and gal-3 225–250 were re-dissolved in 30 mM Tris buffer (pH 7.2) containing 2% (v/v) DMSO at a final concentration of 400 μM. The solutions were incubated at 37 °C with shaking at 210 rpm for 36 h. Aβ42, hIAPP 8–37, hIAPP S20G 8–37, −5Aβ42, and TDP-43 341-357 were dissolved in Milli-Q water containing 2% (v/v) DMSO to a final concentration of 40 μM. These samples were incubated under the same conditions (37 °C, 210 rpm) for 72 h. KRT-R was dissolved in Milli-Q water containing 2% (v/v) DMSO to a final concentration of 1 mM and incubated at 37 °C with shaking at 210 rpm for 48 h. hIAPP and hIAPP S20p were re-dissolved in 100 μM TEA (pH 6.0) containing 2% (v/v) DMSO to a final concentration of 500 μM, and incubated at 37 °C with shaking at 210 rpm for 72 h17,21,41,52.

Time-lapse ThT fluorescence assays for monitoring peptide aggregation
To monitor the aggregation kinetics of peptides, a ThT fluorescence assay was performed. ThT stock solution was formulated in deionized water and passed through a 0.22 μm membrane filter to eliminate particulate matter. The final reaction mixtures (200 μL total volume) contained 50 μM ThT and one of the following peptides: 400 μM gal-3 129–154, 400 μM gal-3 206–231, 400 μM gal-3 225–250, 40 μM Aβ42, 40 μM hIAPP 8–37, 1 mM KRT-R, 40 μM hIAPP S20G 8–37, 40 μM −5Aβ42, or 40 μM TDP-43 341–357.

The gal-3 129–154, gal-3 206–231, and gal-3 225–250 peptides were dissolved in 30 mM Tris buffer (pH 7.2), while the remaining peptides were dissolved in Milli-Q water. Each mixture was transferred to black, clear-bottom 96-well plates and sealed with optically clear adhesive film to prevent evaporation during incubation. Fluorescence readings were acquired using a Synergy H1 microplate reader with an excitation wavelength of 450 nm and an emission wavelength of 485 nm. The plates were incubated at 37 °C with moderate shaking, and fluorescence intensity was measured hourly for 48 h. Control samples lacking peptide were prepared under identical conditions to account for background ThT fluorescence.

Peptide concentration determination
The concentration of the peptide in a 6 M guanidinium chloride solution was quantified through UV-Vis spectroscopic analysis (PerkinElmer, USA). The peptide concentration was calculated using the Beer-Lambert equation53:

A=εLc (1)

where A represents the UV absorbance at 280 nm, ε is the molar extinction coefficient of the peptide, L is the optical path length of the cuvette, and c is the peptide concentration.

TEM measurements
For TEM analysis, 10 μL of the assembled peptide solution was dropped onto a 200-mesh formvar carbon-coated copper grid and incubated at room temperature for 2 min. After removing the excess solution, the sample was air-dried completely at room temperature, followed by negative staining with 1% phosphotungstic acid for 30 s. The images were acquired using a Hitachi HT7700 transmission electron microscope (Hitachi, Tokyo, Japan).

FTIR analysis of peptide samples
FTIR spectra were acquired using a PerkinElmer Spectrum One FTIR Spectrometer (Waltham, Massachusetts, USA). 40 μL aliquot of each peptide solution was deposited onto a CaF₂ window and allowed to dry completely before measurement. Spectra were recorded three times over the range of 400–4500 cm−1 at a resolution of 2 cm−1. All data were processed using PeakFit software (Version 4.12, San Jose, CA, USA). Each spectrum was baseline-corrected, smoothed using a Gaussian instrumental response function, and subjected to second-order derivative calculation, followed by peak fitting on the derivative spectra. Figures of the processed spectra were generated using Prism software (Version 8.01, San Diego, CA, USA).

STM-based single-molecule imaging
For single-molecule imaging experiments using STM, 10 μL of peptide solution was applied to the surface of freshly cleaved HOPG and incubated at 37 °C for 20 min. Following incubation, the excess peptide solution was removed from the substrate in an ambient environment. Single-molecule imaging was performed using a Nanoscope IIIa SPM system (Bruker, USA) with STM tips made from Ir/Pt wires (20/80). To ensure reproducibility, each imaging experiment was repeated with different STM tips and samples. To analyze the β-strands in STM images, we used NanoScope Analysis software (version 1.9, Bruker Corporation, USA).

Interpeptide interaction analysis
The measurement of peptide strand lengths in STM images was performed using Gwyddion software (v2.31, Czech Metrology Institute, Czech Republic). Conformational substates were categorized by dividing β-strands into 3.25 Å stepwise length bins. Adjacent conformational substates were then identified from STM images. The distribution of conformational substates was quantified by counting discrete instances of each category.

Thermal stability assay
The thermal stability of 500 μM hIAPP and hIAPP S20p aggregates was assessed using the integrated UNcle stability platform (Unchained Labs, Norton, MA). Static light scattering was monitored at an excitation wavelength of 266 nm as the temperature increased from 25 to 95 °C at a rate of 0.25 °C per minute.

Chemical denaturation assay
The 500 μM hIAPP and hIAPP S20p aggregates were analyzed using a Zetasizer Nano ZS system (Malvern, UK). Corresponding volume of 6 M guanidine hydrochloride was sequentially added to the aggregates and thoroughly mixed to obtain the concentration gradient of guanidine hydrochloride. The samples were then irradiated with a 4 mW, 633 nm laser, and the scattering intensity was measured at a 90° angle. A digital signal processor was used as a photodetector to record the scattering intensity of the samples.

2D STFT analysis
The STFT was employed to analyze periodic patterns in STM images. Raw STM images were first binarized through threshold segmentation to isolate regions of interest for subsequent analysis. A square window of size N×N (500×500) pixels was translated across the preprocessed image with a step size of 10 pixels to ensure full spatial coverage. For each localized subregion, the 2D discrete Fourier transform was computed as54,55:

F(qx, qy) = 1/N^2 * Σ_{x=0}^{N-1} Σ_{y=0}^{N-1} w(x-xw, y-yw)f(x,y)e^{-i2π(qxx+qyy)/N} (2)

where f(x,y) represents the binarized image, and the square window function w(x-xw, y-yw) is defined as54,55:

w(x-xw, y-yw) = 1 for |x-xw| ≤ N/2 and |y-yw| ≤ N/2, 0 otherwise: (3)

The PSD was calculated as54,55:

PSD(qx, qy) = |F(qx, qy)|^2 (4)

To resolve orientation-specific periodic features, the PSD was converted from Cartesian coordinates qx, qy to polar coordinates p(q, θ) via54,55:

qx = q cos θ, qy = q sin θ (5)

where q = sqrt(qx^2 + qy^2) represents spatial frequency (inverse of periodicity) and θ = arctan(qy/qx) denotes orientation angle. The normalized joint probability density function was derived as54,55:

p(q, θ) = PSD(q, θ) / RR PSD(q, θ) dq dθ (6)

Marginal distributions p(q) and p(θ), quantifying the dominant spatial frequencies and preferential orientations of β-sheet aggregates, were obtained by integrating over the complementary variable54,55

p(q) = ∫_{0}^{π} p(q, θ) dθ, p(θ) = ∫_{0}^{qmax} p(q, θ) dq (7)

The spatial frequency (q) and azimuthal angle (θ) distributions were visualized through heatmaps to statistically highlight dominant periodic features and their orientation preferences.

AFM imaging
Incubated peptide solutions (10 μL) were deposited on the freshly cleaved HOPG substrates, followed by air-drying, ddH₂O rinsing, and then air-drying again. Aggregate morphology was examined using a Dimension FastScan AFM (Bruker, Billerica, MA) equipped with ScanAsyst Air probes (Bruker, Camarillo, CA; resonance frequency 70 kHz, spring constant 0.4 N/m). All images were acquired at a resolution of 512×512 pixels and analyzed using NanoScope Analysis software.

Visualization
Scientific visualization workflows were implemented in Python, employing Pandas and NumPy frameworks for preprocessing pipelines that enforced data integrity through rigorous cleansing and format standardization prior to visualization. Visual representations were produced using Matplotlib’s computational graphic tools.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript “Atypical beta-Strand Insertion Mediates the Noncovalent Cross-Linking in Amyloid Aggregates” the\nAuthors described the detection and identification of β-strands that form inter-β-sheet connections through insertion into\nadjacent β-sheets in protein aggregates.\nThis discovery is potentially interesting if the Authors can demonstrate that these atypical cross-β-strand linkers genuinely\nexist in amyloid aggregates of different proteins (i.e., that their presence is not merely a result of the sample preparation\nprocedures for scanning tunneling microscopy (STM)-based imaging).\n1.In the STM images, the aggregates appear as protein monolayers on the substrate surface, lacking the typical morphology\nof amyloid aggregates. The reason for this peculiar apparent morphology should be better explained by comparing it to\nstandard approaches used to display typical amyloid morphology.\n2.Figure S1B. Please also show the absorption spectra and a supplementary table indicating the peak positions of the\nAmide I components with their assignment to protein secondary structures. Did the gal-3 225-250 aggregates, obtained after\n36 hours of incubation, show the characteristic downshift of the beta-sheet peak compared to gal-3 225-250 monomers (doi:\n10.1110/ps.041024904)?\nFTIR spectroscopy (or other experimental approaches capable of detecting beta-sheet structures) must be applied to\ncompare all the investigated proteins and peptides in their pre-aggregation conformation and in their post-aggregation state\n(doi: 10.1110/ps.041024904).\n3.The Authors investigated the gal-3 225-250 sample using STM imaging after 36 hours of incubation. This investigation\nmust also be performed on the gal-3 225-250 sample before incubation to demonstrate that the observed cross-β-strand\nlinkers are genuinely present in the aggregates formed during the 36-hour incubation and are not solely due to the protocol\nemployed for STM sample preparation. The same STM imaging must be performed on the other model systems before and\nafter amyloid aggregation (if possible for all systems; for instance, it is very difficult to analyze Abeta monomers).\n4.The Authors must demonstrate (for all the investigated model systems) that the polypeptides investigated by STM imaging\nare indeed in an amyloid-like conformation. That is, they must be characterized by all three hallmarks of the amyloid-like\nstate: fibrillar morphology (whether typical or atypical), β-sheet structure, and tinctorial properties\n(https://doi.org/10.1146/annurev-biochem-061516-045115). The appearance of these properties should be demonstrated\nunder the very same incubation conditions employed for the samples characterized by STM imaging. In other words, the\nauthors must unequivocally demonstrate that the observed aggregates are indeed amyloid aggregates and not randomly\ngenerated by the sample preparation procedure.\n5. The preparation procedure and incubation conditions for all investigated model systems should be provided in the\nmanuscript, even if they refer to protocols already published elsewhere.\n6. Was the ThT fluorescence assay performed under the exact same conditions as the sample incubation before STM\nimaging?", "reviewer_2": "Mo et al. studied amyloid aggregation of different peptides. They claim to have discovered a novel β-strand insertion\nmechanism called cross-β-strand linking, which connects β-sheets across filaments (this is not the word the authors used; in\nfact they nowhere really defined what superstructures beyond beta-sheets within fibrils are supposed to be linked). The\nauthors further speculate that these linkers are rare, but they play a key role in the packing and orientation of β-sheets, and\nadding structural diversity.\nThis work raises more questions than it answers.\nI will explain my criticism using Abeta42 as an example. There are many fibril structures for Abeta42 published in the PDB.\nSo if the authors claim to have discovered a new method for assembling Abeta42 filaments into a fibril, they would have to\nexplain this mechanism using such fibril models. However, this was not done. In fact, when looking at Fig. 3B, I find it difficult\nto reconcile this with all the fibril structures that have been elucidated for Abeta42.\nIt could be that the results for the other peptides studied in this work are more reliable,\nbut the case of Abeta42 is sufficient for me to advise against publishing this work.\nFurthermore, the biological relevance of the current results has not been explained.\nThe “Methods” section is quite short; not even the peptide concentrations were specified. The ThT curves for all peptides\nshould be provided.\nIt may be that the manuscript may become publishable in Nat. Commun. after adding further results and better explaining the\nresults, especially their significance. At this stage, however, publication appears premature.", "reviewer_3": "In the article \"Atypical β-Strand Insertion Mediates the Noncovalent Cross-Linking in Amyloid Aggregates\" the authors\ndescribe the assembly of the short peptide galectin-3 (gal-3 225-250) and found that within the 2D assembly, the peptide\nparticipates by bridging two β-sheets. The authors primarily use STM images and 2D STFT analysis to support the peptide's\nparticipation and report that it promotes structural order in the assemblies.\nThe work shows a new class of interaction that had not been described in previous work by the same research group.\nThe work is original and significant for the field because it shows that β-sheet connection can occur in peptides with a\ntendency to form fibers. The work is a continuation of a well-established line of research and shows novel results.\nThe work is based solely on the analysis of STM images, which are of high quality and the analyses well executed.\nHowever, extrapolation to interactions in native proteins requires further study; for example, it could be complemented with\ninteraction modeling to support the experimental results.\nThere are no flaws in the data analysis, interpretation, and conclusions. However, the comparison with what was reported in\nproteins with native structure needs to be supported in more detail.\nThe methodology was well executed, and the results and analyses meet the expected standards.\nThe methodology is sufficient to be reproduced.\nThe work was well executed and the STM analyses are of high quality. However, some points need to be clarified before\npublication:\n1. The authors mention three canonical pathways for the donation of β-strands to receptors in proteins with native structure,\nand the authors extrapolate their results from denatured short peptides on HOPG as a fourth mechanism. The authors could\nexplain in a more detailed way how to extrapolate these results to native proteins in a cellular environment.\n2. The authors could specify whether the assemblies are observed only at 40 μM.\n3. The authors should explain what they mean by saying that β-strand fragments are “operating as dynamic molecular\nadapters that regulate the inter-β-sheet packing geometry and orientation.” This is because they mentioned that “no\nstatistically significant association can be drawn between linker density and the CV of θ.”\n4. The authors must provide a reference for the following statement: “Our group recently developed an STM-based single-\nmolecule imaging platform, which bypasses the limitation of ensemble averaging and enables direct characterization of\nconformational ensembles.”\n5. Because STM is the primary technique used, we consider it important to provide detailed and referenced support for the\nfollowing statement: “The molecules appear as periodic bright linear patterns forming lamellar structures, with protrusions in\nthe local density of electron states corresponding to peptide bonds and aromatic residues.”\n6. The authors should explain the relationship between the length of the cross-β-strand linkers and the dynamic\nconformational flexibility.\n7. If the peptide used has 25 amino acids, how does this explain the presence of shorter fragments attached to the cross-β-\nstrand linkers, for example? Figure 2 A.\n8. The authors must provide a reference for the following statement: “In a pleated β-sheet, adjacent β-strands are\ninterconnected laterally through a hydrogen bond network that stabilizes the structure”.\n9. The authors should explain the following statement: “Through promiscuous interactions with diverse conformational\nstates.” How do the authors know they are not specific?\n10. How can the role of B-Strand in the assembly of ordered 2D structures be transferred to 1D structures as the authors\nstate “cross-serve as molecular \"stitchers\" that integrate multiple assembly intermediates into functional fibrils\"."}, "author_response": "Response: We thank the reviewer for recognizing the novelty of our work and for the\nvaluable input into our manuscript. Please find our point-by-point response below.\nQuestion 1: In the STM images, the aggregates appear as protein monolayers on the\nsubstrate surface, lacking the typical morphology of amyloid aggregates. The reason\nfor this peculiar apparent morphology should be better explained by comparing it to\nstandard approaches used to display typical amyloid morphology.\nResponse: We thank the reviewer for this great comment. First of all, we would like to\nemphasize that the \"monolayer-like\" appearance of the aggregates in our STM images\nstems from the fundamental principles of STM imaging and it does not mean our\nsamples lack amyloid characteristics in our samples. In STM imaging, due to the\nexponential decay of electron tunneling probability with distance, it can only detect\nmolecular features within approximately 1 nm of the conductive HOPG substrate\nsurface. This imaging mechanism thus reveals the portions of assemblies in close\nproximity to the HOPG surface, while structures beyond this tunneling distance\nthreshold remain undetectable.\nFollowing the reviewer’s advice, we performed additional characterizations to\nvalidate the structural features of these assemblies in our experiments. We performed\ncomplementary transmission electron microscopy (TEM) and atomic force microscopy\n(AFM) under identical aggregation conditions. Both measurements confirm the\nformation of typical amyloid fibrillar structures. Furthermore, consistent with literature\nresults, our ThT fluorescence assays show a significantly gradually increased\nfluorescence signal, which indicates the formation of β-sheet–rich amyloid aggregates.\nCollectively, these results confirm that the assemblies observed by STM exhibit\ncharacteristic amyloid fibril morphology.\nFigure R1. Characterization of gal-3 129-154 aggregate formation by (A) AFM,\n(B)\nTEM, and (C) a ThT fluorescence assay. The ThT fluorescence intensity is plotted over\ntime. Data represent the mean of three independent replicates. The shaded area indicates\nthe experimental data range, and the solid line represents the fitted curve.\nTo reflect the above-mentioned points, we have included Figure R1 as Supplementary\nFigure 8 into the revised supplementary information and have revised the manuscript\naccordingly:\n(page 8, line 197) “We note that STM imaging can only detect peptide segments within\napproximately 1 nm of the HOPG substrate, owing to the exponential decay of electron\ntunneling probability with distance, while regions beyond this range remain invisible.”\n(page 7, line 170) “Under the same incubation conditions used for STM imaging,\nthe\nresults of TEM, ThT fluorescence, and FTIR analyses coherently confirm that these\naggregates are enriched in β-sheets and exhibit characteristic amyloid-like properties44,\nwhile AFM imaging further confirms their amyloid-like morphology on the HOPG\nsurfaces (Supplementary Fig.1A and Figs. 5-8).”\n(page 14, line 365) “AFM imaging. Incubated peptide solutions (10 μL) were deposited\non the freshly cleaved HOPG substrates, followed by air-drying, ddH₂O rinsing, and\nthen air-drying again. Aggregate morphology was examined using a Dimension\nFastScan AFM (Bruker, Billerica, MA) equipped with ScanAsyst Air probes (Bruker,\nCamarillo, CA; resonance frequency 70 kHz, spring constant 0.4 N/m). All images were\nacquired at a resolution of 512 × 512 pixels and analyzed using NanoScope Analysis\nsoftware.”\nQuestion 2: Figure S1B. Please also show the absorption spectra and a supplementary\ntable indicating the peak positions of the Amide I components with their assignment to\nprotein secondary structures. Did the gal-3 225-250 aggregates, obtained after 36\nhours of incubation, show the characteristic downshift of the beta-sheet peak compared\nto gal-3 225-250 monomers (doi: 10.1110/ps.041024904)?\nFTIR spectroscopy (or other experimental approaches capable of detecting beta-sheet\nstructures) must be applied to compare all the investigated proteins and peptides in\ntheir pre-aggregation conformation and in their post-aggregation state (doi:\n10.1110/ps.041024904).\nResponse: We thank the reviewer for the great comment. Following the reviewer’s\nsuggestion, we have analyzed the β-sheet characteristic peak in our data in reference to\nthe methods mentioned in the referenced article (doi: 10.1110/ps.041024904). We have\nsupplemented the revised manuscript with FTIR spectra and the corresponding peak\npositions of the Amide I components for all the samples, along with a supplementary\ntable assigning these peaks to the respective protein secondary structures.\nThe results show that only gal-3 225-250, gal-3 206-231, and gal-3 129-154 exhibit\na slight downshift (red shift) of the β-sheet peak after aggregation, while no significant\npeak shift was observed in the other samples. Furthermore, except for KRT-R\ndisplaying a clear conformational transition, no significant changes in the β-sheet\ncontent were observed between the pre- and post-aggregation states of the other\nsamples (Figure R2, corresponding to Supplementary Figure 7 in the supplementary\ninformation).\nAdditionally, we note that the referenced literature suggests that natural proteins\ntypically exhibit broader half-peak widths. To investigate this, we performed statistical\nanalysis of the half-peak widths of the β-sheet peaks before and after aggregation. The\nresults show that, except for KRT-R and gal-3 129-154, the half-peak widths become\nnarrower after aggregation, which is consistent with the trends described in the\nliterature.\nSupplementary Table 3: FTIR amide I region peak positions and secondary structure\nassignments for pre- and post-aggregation states.\nPeak1 Peak2 Peak3 Peak4\n1617 cm-1 1632 cm-1 1654 cm-1 1670 cm-1\npre-aggregation\n(β-sheet 13.8%) (β-sheet 66.5%) (α-helix 13.2%) (β-turn 6.4%)\ngal-3 225-250\n1607 cm-1 1617 cm-1 1631 cm-1\npost-aggregation\n(β-sheet 8.2%) (β-sheet 14.8%) (β-sheet 77.0%)\n1630 cm-1 1661 cm-1 1683 cm-1\npre-aggregation\n(β-sheet 68.0%) (α-helix 19.2%) (β-turn 12.8%)\ngal-3 206-231\n1609 cm-1 1629 cm-1 1670 cm-1 1683 cm-1\npost-aggregation\n(β-sheet 21.7%) (β-sheet 40.2%) (β-turn 16.3%) (β-turn 21.7%)\n1628 cm-1 1648 cm-1 1665 cm-1 1683 cm-1\npre-aggregation\n(β-sheet 64.5%) (Random coil 13.5%) (β-turn 14.3%) (β-turn 7.7%)\nAβ42\n1633 cm-1 1648 cm-1 1665 cm-1 1683 cm-1\npost-aggregation\n(β-sheet 40.6%) (Random coil 17.3%) (β-turn 27.3%) (β-turn 9.4%)\n1628 cm-1 1648 cm-1 1665 cm-1 1683 cm-1\npre-aggregation\n(β-sheet 60.0%) (Random coil 9.7%) (β-turn 21.9%) (β-turn 8.4%)\nhIAPP 8-37\n1628 cm-1 1648 cm-1 1665 cm-1 1683 cm-1\npost-aggregation\n(β-sheet 39.4%) (Random coil 16.3%) (β-turn 24.4%) (β-turn 19.9%)\n1629 cm-1 1655 cm-1 1675 cm-1\npre-aggregation\n(β-sheet 15.6%) (α-helix 63.6%) (β-turn 20.8%)\nKRT-R\n1630 cm-1 1657 cm-1 1675 cm-1\npost-aggregation\n(β-sheet 55.6%) (α-helix 22.7%) (β-turn 21.7%)\n1628 cm-1 1652 cm-1 1670 cm-1\npre-aggregation\n(β-sheet 63.2%) (α-helix 17.3%) (β-turn 19.5%)\nhIAPP S20G 8-37\n1627 cm-1 1648 cm-1 1668 cm-1\npost-aggregation\n(β-sheet 63.3%) (α-helix 15.8%) (β-turn 20.9%)\n1626 cm-1 1648 cm-1 1670 cm-1 1688 cm-1\npre-aggregation\n(β-sheet 56.1%) (Random coil 18.7%) (β-turn 17.3%) (β-turn 7.8%)\n-5Aβ42\n1626 cm-1 1648 cm-1 1670 cm-1 1688 cm-1\npost-aggregation\n(β-sheet 55.5%) (Random coil 19.5%) (β-turn 15.7%) (β-turn 9.3%)\n1620 cm-1 1640 cm-1 1661 cm-1 1683 cm-1\npre-aggregation\n(β-sheet 41.1%) (Random coil 16.0%) (α-helix 34.4%) (β-turn 8.4%)\nTDP-43 341-357\n1620 cm-1 1640 cm-1 1661 cm-1 1683 cm-1\npost-aggregation\n(β-sheet 45.0%) (Random coil 15.0%) (α-helix 32.6%) (β-turn 7.5%)\n1631 cm-1 1670 cm-1\npre-aggregation\n(β-sheet 75.8%) (β-turn 24.2%)\ngal-3 129-154\n1629 cm-1 1665 cm-1\npost-aggregation\n(β-sheet 72.7%) (β-turn 27.3%)\nSupplementary Table 4: Comparison of half-peak widths of β-sheet peaks between\npre- and post-aggregation states.\nFWHM\npre-aggregation 18.1 cm-1\ngal-3 225-250\npost-aggregation 13.5 cm-1\npre-aggregation 22.2 cm-1\ngal-3 206-231\npost-aggregation 16.9 cm-1\npre-aggregation 23.3 cm-1\nAβ42\npost-aggregation 21.0 cm-1\npre-aggregation 24.7 cm-1\nhIAPP 8-37\npost-aggregation 23.1 cm-1\npre-aggregation 25.7 cm-1\nKRT-R\npost-aggregation 27.2 cm-1\npre-aggregation 24.6 cm-1\nhIAPP S20G 8-37\npost-aggregation 24.0 cm-1\npre-aggregation 23.4 cm-1\n-5Aβ42\npost-aggregation 23.2 cm-1\npre-aggregation 19.5 cm-1\nTDP-43 341-357\npost-aggregation 18.9 cm-1\npre-aggregation 29.0 cm-1\ngal-3 129-154\npost-aggregation 32.1 cm-1\nFigure R2. Secondary structure composition analysis of peptide samples pre- and post-\naggregation, determined by FTIR spectroscopy of the amide I region. Black dashed\nlines, experimental curves; gray solid lines, fitting curves; the peaks in red, blue, green,\nand yellow, correspond to β-sheet, random coil, β-turn, and α-helix component,\nrespectively.\nWe have added discussion to the revised supplementary information:\n“1.2 FTIR Analysis of Amide I Components and Secondary Structure\nAssignments Pre and Post Aggregation\nFTIR spectra of all samples were analyzed, and the corresponding β-sheet peak\npositions and secondary structures are shown in Supplementary Tables 3 and 4,\nSupplementary Figure 7. Only gal-3 225–250, gal-3 206–231, and gal-3 129–154\nexhibit a slight red shift in the β-sheet peak after aggregation, while no significant shift\nwas observed in the other samples. Additionally, except for KRT-R, which displays a\nclear conformational transition, no major changes in β-sheet content were observed\nbetween the pre- and post-aggregation states. Statistical analyses of the half-peak\nwidths reveal that, except for KRT-R and gal-3 129–154, the widths become narrower\nafter aggregation, consistent with the results in natural proteins2.”\nWe have added the following references to the revised supplementary information:\n“2. Zandomeneghi, G., Krebs, M. R. H., McCammon, M. G. & Fändrich, M. FTIR\nreveals structural differences between native beta-sheet proteins and amyloid fibrils.\nProtein Sci. Publ. Protein Soc. 13, 3314–3321 (2004).”\nTo reflect the above-mentioned points, we have included Figure R2 as\nSupplementary Figure 7 into the revised supplementary information, and the following\ntext into the revised manuscript:\n(page 7, line 175) “Comparative FTIR spectra of the different peptide aggregates are\nshown in Supplementary Tables 3 and 4 and detailed discussion is provided in\nSupplementary 1.2.”\nQuestion 3: The Authors investigated the gal-3 225-250 sample using STM imaging\nafter 36 hours of incubation. This investigation must also be performed on the gal-3\n225-250 sample before incubation to demonstrate that the observed cross-β-strand\nlinkers are genuinely present in the aggregates formed during the 36-hour incubation\nand are not solely due to the protocol employed for STM sample preparation. The same\nSTM imaging must be performed on the other model systems before and after amyloid\naggregation (if possible for all systems; for instance, it is very difficult to analyze Abeta\nmonomers).\nResponse: We appreciate the reviewer's valuable suggestion regarding STM imaging\nof the non-incubated gal-3 225-250 sample. While we have attempted to visualize the\npeptide in its unaggregated state, none of the features in the peptide can be observed.\nThis can be attributed to the inherent molecular mobility under ambient conditions.\nUnassembled molecules on the HOPG surfaces experience rapid thermal diffusion, as\nthey lack sufficient intermolecular and adsorbate-substrate interactions to achieve the\nimmobilization required for high-resolution STM imaging - a phenomenon well-\ndocumented in previous studies (J. Am. Chem. Soc., 2000, 122, 5550-5556).\nImportantly, only upon forming stable self-assembled structures that suppress thermal\ndiffusion do the peptides become amenable to STM characterization. This explains why\nthe cross-β-strand linker features observed exclusively emerge after 36 hours of\nincubation, confirming they represent genuine structural characteristics of the\naggregates rather than preparation artifacts.\nWe have supplemented Figure R3 with STM images of the sample at 0 hours\n(before aggregation) as Supplementary Figure 2 in the supplementary information.\nFigure R3. STM images of all samples before aggregation. Tunneling conditions: I =\n198.4 pA, V = 549.9 mV (gal-3 225-250); I = 198.4 pA, V = 549.9 mV (gal-3 206-\nbias bias\n231); I = 198.4 pA, V = 499.9 mV (Aβ42); I = 198.4 pA, V = 479.7 mV (hIAPP\nbias bias\n8-37); I = 198.4 pA, V = 549.9 mV (KRT-R) ; I = 198.4 pA, V = 549.9 mV (hIAPP\nbias bias\nS20G 8-37) ; I = 198.4 pA, V = 449.8 mV (-5Aβ42) ; I = 198.4 pA, V = 549.9 mV\nbias bias\n(TDP-43 341-357) ; I = 198.4 pA, V = 549.9 mV (gal-3 129-154). Scale bars, 10 nm.\nbias\nWe have included the following text into the revised manuscript:\n(page 5, line 111) “We also attempted STM imaging of the sample before aggregation\n(0 hour) (Supplementary Fig. 2). However, the result shows that they lack sufficient\nintermolecular and substrate interactions to form stabilized, ordered structures under\nambient conditions, because the unassembled molecules on the HOPG surface undergo\nrapid thermal diffusion, which precludes the acquisition of single-molecule images38.”\nWe have added the following references to the revised manuscript:\n“38. Qiu, X. et al. Alkane-assisted adsorption and assembly of phthalocyanines and\nporphyrins. J. Am. Chem. Soc. 122, 5550–5556 (2000).”\nQuestion 4: The Authors must demonstrate (for all the investigated model systems) that\nthe polypeptides investigated by STM imaging are indeed in an amyloid-like\nconformation. That is, they must be characterized by all three hallmarks of the amyloid-\nlike state: fibrillar morphology (whether typical or atypical), β-sheet structure, and\ntinctorial properties\n(https://doi.org/10.1146/annurev-biochem-061516-045115). The appearance of these properties should be demonstrated\nunder the very same incubation conditions employed for the samples characterized by STM imaging. In other words,\nthe authors must unequivocally demonstrate that the observed aggregates are indeed\namyloid aggregates and not randomly generated by the sample preparation procedure.\nResponse: We thank the reviewer for the insightful comment. Similar to our response\nto Q1 above, we have supplemented the revised manuscript with TEM, ThT\nfluorescence, and FTIR spectroscopy data obtained under exactly the same incubation\nconditions as those used for STM imaging. TEM images reveal typical or atypical\nfibrillar morphologies (Figure R4), ThT fluorescence shows a significant increase after\naggregation (Figure R5), and FTIR spectra demonstrate that the aggregates are\nprimarily β-sheet rich (Figure R2). These three hallmarks of amyloid aggregates (doi:\n10.1146/annurev-biochem-061516-045115) confirm that the observed aggregates\nexhibit amyloid-like properties.\nFigure R4. TEM images of gal-3 206-231 (A), Aβ42 (B), hIAPP 8-37 (C), KRT-R (D),\nhIAPP S20G 8-37 (E), -5Aβ42 (F), TDP-43 341–357 (G) and gal-3 129-154 (H).\nPeptides were incubated at 37 °C with shaking at 210 rpm under the following\nconditions: gal-3 fragments at 400 μM for 36 h; Aβ42, hIAPP 8–37, hIAPP S20G 8-37,\n-5Aβ42, and TDP-43 341-357 at 40 μM for 72 h; KRT-R at 1 mM for 48 h Scale bars\nare indicated in each panel. Scale bars, 200 nm.\nFigure R5. ThT fluorescence kinetics curves of gal-3 225-250 (A), gal-3 206-231 (B),\nAβ42 (C), hIAPP 8-37 (D), KRT-R (E), hIAPP S20G 8-37 (F), -5Aβ42 (G), TDP-43\n341-357 (H), and gal-3 129-154 (I). Experimental data are shown as mean ± SEM from\nthree replicates (red solid line, gray shaded area), and buffer control data are shown as\nmean ± SEM (blue solid line, gray shaded area). The shaded area indicates the\nexperimental data range, and the solid line represents the fitted curve.\nFigure R2. Secondary structure composition of samples before and after aggregation\nbased on FTIR amide I region. Black dashed lines, experimental curves; gray solid lines,\nfitting curves; the peaks in red, blue, green, and yellow, correspond to β-sheet, random\ncoil, β-turn, and α-helix component, respectively.\nWe have included Figure R4, R5, and R2 as Supplementary Figures 5-7 into the\nrevised supplementary information, and included the following text to the revised\nmanuscript:\n(page 7, line 170) “Under the same incubation conditions used for STM imaging,\nthe\nresults of TEM, ThT fluorescence, and FTIR analyses coherently confirm that these\naggregates are enriched in β-sheets and exhibit characteristic amyloid-like properties44,\nwhile AFM imaging further confirms their amyloid-like morphology on the HOPG\nsurfaces (Supplementary Fig.1A and Figs. 5-8).”\nWe have added the following references to the revised manuscript:\n“44. Chiti, F. & Dobson, C. M. Protein misfolding, amyloid formation, and human\ndisease: a summary of progress over the last decade. Annu. Rev. Biochem. 86, 27–68\n(2017).”\nQuestion 5: The preparation procedure and incubation conditions for all investigated\nmodel systems should be provided in the manuscript, even if they refer to protocols\nalready published elsewhere.\nResponse: Following the reviewer’s suggestion, we have added the procedure in the\n“The preparation procedure and incubation conditions for all investigated model\nsystems” section in the revised manuscript. The revised text reads as:\n(page 11, line 270) “All lyophilized peptide powders were initially dissolved in\n1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Innochem), a fluorinated solvent known for\nefficient disruption of secondary structures. After complete evaporation of HFIP, gal-3\n129-154, gal-3 206-231, and gal-3 225-250 were re-dissolved in 30 mM Tris buffer\n(pH 7.2) containing 2% (v/v) DMSO at a final concentration of 400 μM. The solutions\nwere incubated at 37 °C with shaking at 210 rpm for 36 hours. Aβ42, hIAPP8-37, hIAPP\nS20G8-37, -5Aβ42, and TDP-43 341-357 were dissolved in Milli-Q water containing\n2% (v/v) DMSO to a final concentration of 40 μM. These samples were incubated under\nthe same conditions (37 °C, 210 rpm) for 72 hours. KRT-R was dissolved in Milli-Q\nwater containing 2% (v/v) DMSO to a final concentration of 1 mM and incubated at\n37 °C with shaking at 210 rpm for 48 hours17,21,45.”\nQuestion 6: Was the ThT fluorescence assay performed under the exact same\nconditions as the sample incubation before STM imaging?\nResponse: Yes, ThT fluorescence assay was conducted under the exact same conditions\nas the sample incubation prior to STM imaging.\nWe have included the following text to the revised manuscript:\n(page 7, line 170) “Under the same incubation conditions used for STM imaging,\nthe\nresults of TEM, ThT fluorescence, and FTIR analyses coherently confirm that these\naggregates are enriched in β-sheets and exhibit characteristic amyloid-like properties44,\nwhile AFM imaging further confirms their amyloid-like morphology on the HOPG\nsurfaces (Supplementary Fig.1A and Figs. 5-8).”\n------------------------------------------------------------------------\nReviewer: 2\nMo et al. studied amyloid aggregation of different peptides. They claim to have\ndiscovered a novel β-strand insertion mechanism called cross-β-strand linking, which\nconnects β-sheets across filaments (this is not the word the authors used; in fact they\nnowhere really defined what superstructures beyond beta-sheets within fibrils are\nsupposed to be linked). The authors further speculate that these linkers are rare, but\nthey play a key role in the packing and orientation of β-sheets, and adding structural\ndiversity.\nResponse: We thank the reviewer for your incredible input into our manuscript. Please\nfind below our point-to-point response.\nQuestion 1: I will explain my criticism using Abeta42 as an example. There are many\nfibril structures for Abeta42 published in the PDB. So if the authors claim to have\ndiscovered a new method for assembling Abeta42 filaments into a fibril, they would\nhave to explain this mechanism using such fibril models. However, this was not done.\nIn fact, when looking at Fig. 3B, I find it difficult to reconcile this with all the fibril\nstructures that have been elucidated for Abeta42.\nIt could be that the results for the other peptides studied in this work are more reliable,\nbut the case of Abeta42 is sufficient for me to advise against publishing this work.\nResponse: We thank the reviewer for this insightful question. In terms of Aβ42 in Fig.\n3B, first of all, we would like to emphasize that cryo-EM and STM rely on\nfundamentally distinct imaging principles. Cryo-EM resolves the three-dimensional\nstructure of protein fibrils through averaging of periodic signals and 3D helical\nreconstruction, making it particularly suitable for high-resolution characterization of\nrigid, well-ordered fibrillar architectures. In contrast, STM directly visualizes\nmolecular structures via tunneling current between a probe and a conductive substrate,\nwithout the need for reconstruction or signal averaging. This enables STM to detect\nflexible aggregates with limited long-range order and provides high sensitivity to local\nconformational variations and low-population substates. Consistent with previous\nstudies, TEM confirmed that our Aβ42 aggregates adopt typical amyloid-like fibrillar\nmorphologies (Figure R6), supporting that the samples used for STM imaging represent\ncanonical amyloid architectures. Furthermore, while cryo-EM analysis primarily\nreveals the well-ordered β-sheet core regions within the fibril structures. Beyond these\ncores, there typically exist diffuse density regions surrounding the fibril cores, which\ncorrespond to the disordered protein assembly or non-fibrillar components (Science,\n2022, 375, 167; Nat. Commun., 2025, 16, 5815; Proc. Natl. Acad. Sci. U. S. A., 2015,\n112, 11858). Due to their dynamic and heterogeneous nature, these regions often evade\nhigh-resolution structural determination by cryo-EM. STM, however, can capture\nspatial information from both the ordered cores and adjacent disordered regions,\noffering a complementary view of fibril organization. Therefore, direct comparison\nbetween STM results and PDB-derived cryo-EM structures may not be appropriate.\nNevertheless, key structural parameters obtained from our STM measurements\nalign well with those from established techniques. The β-strand spacing measured by\nSTM (~4.0-4.8 Å) is consistent with PDB data, supporting the reliability of our\napproach (Supplementary Figure 9B). Moreover, in our previous STM study of the 25-\nresidue β-sheet peptide KRT-R (Sci. Adv. 2023, 9, eadg7943), we found that—unlike\nmulti-strand Aβ—KRT-R adopts a simple fold with one β-strand motif. FTIR analysis\nindicated a secondary structure composition of 55.6% β-sheet, 21.7% β-turn, and 22.7%\nα-helix. STM-derived β-sheet domains averaged 13 ± 2 residues, corresponding to ~52\n± 8% of the total sequence, in agreement with FTIR data and demonstrating the ability\nof STM to quantify β-sheet content. It should be noted that in longer peptides with\nmultiple β-motifs, segments situated beyond ~1 nm from the HOPG surface fall outside\nthe detectable tunneling range of STM. Overall, these findings validate STM as a robust\ntool for structural analysis of β-sheet-rich fibrils.\nFigure R6. TEM images of Aβ42 aggregates. Peptides were incubated at 37 °C with\nshaking at 210 rpm under the following conditions: 40 μM for 72 h. Scale bar, 200 nm.\nWe have included the following text to the revised manuscript:\n(page 8, line 200) “Consequently, STM primarily resolves molecular features in close\nproximity to the surface. Importantly, STM does not rely on three-dimensional\nreconstruction but instead directly visualizes the distribution of electronic states on the\ntwo-dimensional surface. Detailed discussion is provided in Supplementary 1.3.”\nWe have added the following text as Supplementary 1.3 to the revised\nsupplementary information:\n“1.3 Comparative Analysis of Cryo-EM and STM in Structural\nCharacterization of Protein Fibrils\nCryo-EM and STM rely on different imaging principles. Cryo-EM uses signal\naveraging and 3D helical reconstruction to resolve high-resolution structures of well-\nordered fibrils, primarily capturing β-sheet core regions3–6. However, disordered and\ndiffuse regions around these cores, which are often associated with non-fibrillar\ncomponents, are difficult to resolve due to their dynamic nature.\nIn contrast, STM directly visualizes molecular structures using tunneling current,\nallowing it to detect flexible aggregates with limited long-range order. STM is\nparticularly sensitive to local conformational changes and can capture both ordered β-\nsheet cores and adjacent disordered regions, providing a more comprehensive view of\nfibril organization. As a result, direct comparisons between STM and cryo-EM data\nfrom PDB structures are not always straightforward.\nDespite these differences, STM-derived parameters, such as β-strand spacing\n(~4.0-4.8 Å), align well with established techniques, confirming the reliability of STM\nfor fibril analysis (Supplementary Figure 9B). In a previous study of the 25-residue β-\nsheet peptide KRT-R7, STM quantified β-sheet content, with domains averaging 13 ± 2\nresidues (~52 ± 8% of the total sequence), consistent with FTIR data. However, for\nlonger peptides with multiple β-strand motifs, segments more than ~1 nm from the\nHOPG surface fall outside the STM tunneling range, limiting its resolution. These\nresults support STM as a valuable tool for structural analysis of β-sheet-rich fibrils.”\nWe have added the following references to the revised supplementary information:\n“3. Yang, Y. et al. Cryo-EM structures of amyloid-β 42 filaments from human brain.\nScience 375, 167–172 (2022).\n4. Arseni, D. et al. TDP-43 forms amyloid filaments with a distinct fold in type a\nFTLD-TDP. Nature 620, 898–903 (2023).\n5. Wei, Q. et al. An O-glycopeptide participates in the formation of distinct Aβ42\nfibril structures and attenuates Aβ42 neurotoxicity. Nat. Commun. 16, 5815 (2025).\n6. Schmidt, M. et al. Peptide dimer structure in an aβ(1-42) fibril visualized with cryo-\nEM. Proc. Natl. Acad. Sci. U. S. A. 112, 11858–11863 (2015).\n7. Zhang, W. et al. Single-molecule visualization determines conformational substate\nensembles in β-sheet–rich peptide fibrils. Sci. Adv. 9, eadg7943 (2023).”\nQuestion 2: Furthermore, the biological relevance of the current results has not been\nexplained.\nResponse: Again, we thank the reviewer for this insightful comment. We fully agree\nwith the reviewer that the biological relevance of the insertion mechanism has not been\nvalidated under physiological conditions, and this is actually one of the most central\nissues that we are still pursuing in our lab. Despite so, we can still formulate some\nreasonable hypotheses based on the established knowledge of amyloid aggregates.\nAmyloid fibrils are known to exhibit considerable structural polymorphism and\nheterogeneity. While cryo-EM and NMR have successfully resolved many rigid, well-\nordered fibrillar architectures, a substantial population of flexible fibrils remains\nunresolved or is often categorized as disordered (Science, 2022, 375, 167; Nat.\nCommun., 2025, 16, 5815.). Gaining structural insights into the supramolecular\norganization of these flexible fibrils—though underrepresented in conventional\nmodels—may reveal functionally important characteristics, particularly in the context\nof neurodegenerative diseases and other pathological conditions.\nThe cross-β-strand linker mechanism, albeit constituting a minor fraction of\namyloid aggregates, could play a critical regulatory role at the molecular level by\nmodulating β-sheet arrangement and spatial organization. Acting as polymorphic\nmolecular adaptors, these linkers confer structural flexibility during aggregate assembly,\nfacilitating more sophisticated intermolecular interactions and enhancing adaptability\nto environmental changes. This plasticity may enable amyloid aggregates to\naccommodate varying physiological or pathological conditions, thereby influencing\nfibril formation, stability, and overall aggregation propensity. Although further\nexperimental confirmation is required, these hypotheses offer a novel perspective on\nthe structural diversity and adaptive flexibility of amyloid aggregates in biological\nsystems and specifically on how non-canonical structural features may regulate\naggregate functionality and pathological impact. This mechanism could provide\nvaluable implications for future research into the biological roles of protein aggregation\nand its contribution to disease mechanisms.\nWe have included the following text to the revised manuscript:\n(page 10, line 258) “Such structural plasticity may represent biologically relevant states,\nenabling amyloid aggregates to adapt their stability and aggregation propensity under\nphysiological and pathological conditions (Supplementary 1.5).”\nWe have added the following discussion as Supplementary 1.5 to the revised\nsupplementary information:\n“1.5 Biological Relevance of Cross-β-strand Linkers in Amyloid Aggregation\nAmyloid fibrils are structurally polymorphic and heterogeneous. While cryo-EM\nand NMR have elucidated many rigid, well-ordered fibrils, a substantial fraction of\nflexible fibrils remains unresolved or is labeled as disordered3,5. STM provides\ncomplementary structural insights into these flexible assemblies, which may represent\nbiologically relevant states. Within such aggregates, cross-β-strand linkers, though\nminor in abundance, modulate β-sheet arrangement and spatial organization. Acting as\npolymorphic adaptors, they could confer structural flexibility during assembly, promote\ncomplex intermolecular interactions, and enhance adaptability to environmental\nconditions. This plasticity may enable amyloid aggregates to adjust their formation,\nstability, and aggregation propensity under physiological or pathological contexts,\noffering perspectives on how non-canonical structural features contribute to disease\nmechanisms.”\nWe have added the following references to the revised supplementary information:\n“3. Yang, Y. et al. Cryo-EM structures of amyloid-β 42 filaments from human brain.\nScience 375, 167–172 (2022).\n5. Wei, Q. et al. An O-glycopeptide participates in the formation of distinct Aβ42\nfibril structures and attenuates Aβ42 neurotoxicity. Nat. Commun. 16, 5815 (2025).”\nQuestion 3: The “Methods” section is quite short; not even the peptide concentrations\nwere specified. The ThT curves for all peptides should be provided.\nResponse: We thank the reviewer for this great suggestion. Following the reviewer’s\nsuggestion, we have revised the “Methods” section to include detailed experimental\nconditions, including the concentrations of all peptides used. In addition, we have\nprovided the ThT fluorescence curves for all peptides (Figure R5), and included it as\nSupplementary Figure 6 in the supplementary information.\nMethods\n(page 11, line 267) “Reconstitution of peptide assemblies in solution. The peptides\nwere obtained from Anhui Guoping Pharmaceutical Co. Ltd., after synthesis and\npurification, with a confirmed purity of over 98% through high performance liquid\nchromatography and mass spectrometry analysis. All lyophilized peptide powders were\ninitially dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Innochem), a\nfluorinated solvent known for efficient disruption of secondary structures. After\ncomplete evaporation of HFIP, gal-3 129-154, gal-3 206-231, and gal-3 225-250 were\nre-dissolved in 30 mM Tris buffer (pH 7.2) containing 2% (v/v) DMSO at a final\nconcentration of 400 μM. The solutions were incubated at 37 °C with shaking at 210\nrpm for 36 hours. Aβ42, hIAPP8-37, hIAPP S20G8-37, -5Aβ42, and TDP-43 341-357\nwere dissolved in Milli-Q water containing 2% (v/v) DMSO to a final concentration of\n40 μM. These samples were incubated under the same conditions (37 °C, 210 rpm) for\n72 hours. KRT-R was dissolved in Milli-Q water containing 2% (v/v) DMSO to a final\nconcentration of 1 mM and incubated at 37 °C with shaking at 210 rpm for 48\nhours17,21,45.”\n(page 11, line 283) “Time-lapsed ThT fluorescence assays for monitoring peptide\naggregation. To monitor the aggregation kinetics of peptides, a ThT fluorescence assay\nwas performed. ThT stock solution was formulated in deionized water and passed\nthrough a 0.22 μm membrane filter to eliminate particulate matter. The final reaction\nmixtures (200 μL total volume) contained 50 μM ThT and one of the following peptides:\n400 μM gal-3 129–154, 400 μM gal-3 206–231, 400 μM gal-3 225–250, 40 μM Aβ42,\n40 μM hIAPP8–37, 1 mM KRT-R, 40 μM hIAPP S20G8–37, 40 μM -Aβ42, or 40 μM\nTDP-43 341–357.\nThe gal-3 129–154, gal-3 206–231, and gal-3 225–250 peptides were dissolved in\n30 mM Tris buffer (pH 7.2), while the remaining peptides were dissolved in Milli-Q\nwater. Each mixture was transferred to black, clear-bottom 96-well plates and sealed\nwith optically clear adhesive film to prevent evaporation during incubation.\nFluorescence readings were acquired using a Synergy H1 microplate reader with an\nexcitation wavelength of 450 nm and an emission wavelength of 485 nm. The plates\nwere incubated at 37 °C with moderate shaking, and fluorescence intensity was\nmeasured hourly for 48 hours. Control samples lacking peptide were prepared under\nidentical conditions to account for background ThT fluorescence.”\n(page 12, line 310) “TEM Measurements. For TEM analysis, 10 μL of the assembled\npeptide solution was dropped onto a 200-mesh formvar carbon-coated copper grid and\nincubated at room temperature for 2 minutes. After removing the excess solution, the\nsample was air-dried completely at room temperature, followed by negative staining\nwith 1% phosphotungstic acid for 30 seconds. The excess stain was removed, and the\ngrid was thoroughly dried again at room temperature. TEM imaging was then\nperformed using a Hitachi HT7700 transmission electron microscope (Hitachi, Tokyo,\nJapan).”\nFigure R5. ThT fluorescence kinetics curves of gal-3 225–250 (A), gal-3 206–231 (B),\nAβ42 (C), hIAPP 8–37 (D), KRT-R (E), hIAPP S20G 8–37 (F), −5Aβ42 (G), TDP-43\n341–357 (H), and gal-3 129–154 (I). Experimental data are shown as mean ± SEM from\nthree replicates (red solid line, gray shaded area), and buffer control data are shown as\nmean ± SEM (blue solid line, gray shaded area). The shaded area indicates the\nexperimental data range, and the solid line represents the fitted curve.\nReviewer: 3\nIn the article \"Atypical β-Strand Insertion Mediates the Noncovalent Cross-Linking in\nAmyloid Aggregates\" the authors describe the assembly of the short peptide galectin-3\n(gal-3 225-250) and found that within the 2D assembly, the peptide participates by\nbridging two β-sheets. The authors primarily use STM images and 2D STFT analysis\nto support the peptide's participation and report that it promotes structural order in the\nassemblies.\nThe work shows a new class of interaction that had not been described in previous work\nby the same research group.\nThe work is original and significant for the field because it shows that β-sheet connection can occur in peptides with a\ntendency to form fibers. The work is a continuation of a well-established line of research and shows novel results.\nThe work is based solely on the analysis of STM images, which are of high quality and the analyses well executed.\nHowever, extrapolation to interactions in native proteins requires further study; for example, it could be complemented with\ninteraction modeling to support the experimental results.\nThere are no flaws in the data analysis, interpretation, and conclusions. However, the comparison with what was reported in\nproteins with native structure needs to be supported in more detail.\nThe methodology was well executed, and the results and analyses meet the expected standards.\nThe methodology is sufficient to be reproduced.\nThe work was well executed and the STM analyses are of high quality. However, some points need to be clarified before\npublication:\nResponse: We thank the reviewer for the very positive comment on the novelty and\nquality of our work and for the incredible input into our manuscript. Please find below\nour point-to-point response.\nQuestion 1: The authors mention three canonical pathways for the donation of β-\nstrands to receptors in proteins with native structure, and the authors extrapolate their\nresults from denatured short peptides on HOPG as a fourth mechanism. The authors\ncould explain in a more detailed way how to extrapolate these results to native proteins\nin a cellular environment.\nResponse: We thank the reviewer for this insightful question. We fully agree with the\nreviewer that the biological relevance of the insertion mechanism has not been validated\nunder physiological conditions, and this is actually one of the most central issues that\nwe are still pursuing in our lab. Despite so, we can still formulate some reasonable\nhypotheses based on the established knowledge of amyloid aggregates.\nFirst, we would like to emphasize that the donation of β-strands observed in our\nstudy occurs specifically for pathological protein aggregates, which are distinct from\nnatively folded proteins. These pathological short proteins or peptides exist naturally\nunder both physiological and pathological conditions. When they aggregate, they could\nexert direct pathogenic effects and act as central drivers of the associated disease\nprocesses.\nSecond, it is important to clarify that the aggregation of peptides/proteins occurs in\nsolution. We highlight a key development in cryo-EM sample preparation: coating TEM\ngrids with graphene—a surface chemically analogous to the HOPG—has been shown\nto mitigate denaturation caused by the air–water interface (J Struct Biol, 2008, 163, 214;\nSmartMat, 2021, 2, 202; Nano Lett, 2021, 21, 9587; Nat Methods, 2023, 20, 123). This\napproach enables effective protein adsorption while shielding samples from interfacial\ndenaturation, thereby better preserving native conformations. These findings imply that\nHOPG surface in our STM experiments would help preserve native conformations.\nFurthermore, unlike more reactive substrates such as Au(111) or Cu(110)—which often\nform covalent bonds with adsorbates (Nanoscale, 2019, 11, 17235)—HOPG exhibits\npredominantly weak van der Waals interactions with adsorbed molecules. As a result,\nthe HOPG substrate is expected to have negligible impact on fibril conformation (Sci\nAdv, 2023, 9, eadg7943).\nFinally, we recognize that high-resolution structural characterization of proteins\nwithin a cellular environment remains a significant challenge for classical biophysical\ntechniques, such as cryo-EM, NMR, and X-ray crystallography. Although methods like\ncryo-electron tomography (cryo-ET) allow imaging under near-native cellular\nconditions (Cell, 2025, 188, 2094; Nat. Commun. 2024, 15, 8831), they currently lack\nthe resolution necessary to resolve the heterogeneously conformational ensembles\ncharacteristic of protein or peptide aggregates.\nWe have included the following text to the revised manuscript:\n(page 10, line 254) “The β-strand donation mechanism observed by STM occurs\nspecifically in pathological protein or peptide aggregates, rather than in natively folded\nproteins. There is good reason to assume that these aggregates naturally exist in vivo\nand drive pathological processes (Supplementary 1.4).”\nAnd we have added the following discussion as Supplementary 1.4 to the revised\nsupplementary information:\n“1.4 Biological Relevance of Aggregates Observed on the HOPG surfaces\n“The aggregation of peptides and proteins occurs in solution and is not induced by\nthe HOPG surface. To address concerns regarding potential substrate-induced\nconformational artifacts, we highlight an important advance in cryo-EM sample\npreparation: the use of graphene-coated grids—chemically analogous to the HOPG—\nhas been shown to mitigate denaturation caused by the air–water interface8–11. This\nmethod facilitates effective protein adsorption while shielding samples from\ndenaturation at the air–water interface, thereby aiding in the preservation of native\nconformations. These findings imply that HOPG surface in our STM experiments\nwould help preserve native conformations. Moreover, unlike more reactive substrates\nsuch as Au(111) or Cu(110)—which can form covalent bonds with adsorbates12—\nHOPG primarily engages in weak van der Waals interactions with adsorbed molecules.\nConsequently, the HOPG substrate is expected to have a negligible impact on fibril\nconformation7. We also note that high-resolution structural characterization of proteins\nwithin a cellular environment remains challenging for classical biophysical techniques\nsuch as cryo-EM, NMR, and X-ray crystallography. Although methods like cryo-\nelectron tomography (cryo-ET) allow imaging under near-native cellular conditions13,14,\nthey currently lack the resolution required to resolve the heterogeneous conformational\nensembles characteristic of protein or peptide aggregates.”\nWe have added the following references to the revised supplementary information:\n“7. Zhang, W. et al. Single-molecule visualization determines conformational substate\nensembles in β-sheet–rich peptide fibrils. Sci. Adv. 9, eadg7943 (2023).\n8. Taylor, K. A. & Glaeser, R. M. Retrospective on the early development of\ncryoelectron microscopy of macromolecules and a prospective on opportunities for the\nfuture. J. Struct. Biol. 163, 214–223 (2008).\n9. Xu, J., Cui, X., Liu, N., Chen, Y. & Wang, H.-W. Structural engineering of graphene\nfor high-resolution cryo-electron microscopy. Smartmat 2, 202–212 (2021).\n10. Zhang, J. et al. Hydrophilic, clean graphene for cell culture and cryo-EM imaging.\nNano Lett. 21, 9587–9593 (2021).\n11. Zheng, L. et al. Uniform thin ice on ultraflat graphene for high-resolution cryo-EM.\nNat. Methods 20, 123–130 (2023).\n12. Engelbrekt, C. et al. Chemistry of cysteine assembly on au(100): electrochemistry,\nin situ STM and molecular modeling. Nanoscale 11, 17235–17251 (2019).\n13. Klumpe, S. et al. In-cell structure and snapshots of copia retrotransposons in intact\ntissue by cryo-ET. Cell 188, 2094-2110.e18 (2025).\n14. Marcink, T. C. et al. How a paramyxovirus fusion/entry complex adapts to escape\na neutralizing antibody. Nat. Commun. 15, 8831 (2024).”\nQuestion 2: The authors could specify whether the assemblies are observed only at 40\nμM.\nResponse: The concentrations of the samples used in our study were as follows:\n400 μM for gal-3 129-154, gal-3 206-231, and gal-3 225-250; 40 μM for Aβ42,\nhIAPP8-37, hIAPP S20G8-37, -5Aβ42, and TDP-43 341-357; and 1 mM for KRT-R.\nThe concentration for the corresponding peptide was chosen as the minimal\nconcentration to make the peptide aggregation readily occurs under the experimental\nconditions. At lower concentrations, peptide aggregation does not produce detectable\nfibrils by STM, TEM, or ThT assays.\nBesides 40 μM for Aβ42, we also present STM images of Aβ42 at 80 μM\n(aggregated for 72 h), demonstrating that cross-β-strand linkers remain clearly\nobservable at this higher concentration (Figure R7).\nTo reflect the above-mentioned points, we have included Figure R7 as\nSupplementary Figure 10 into the revised supplementary information, and the\nfollowing text into the revised manuscript:\n(page 8, line 185) “We also scanned STM images of Aβ42 aggregates at 80 μM and\nconsistently observed cross-β-strand linkers (Supplementary Fig. 10).”\nFigure R7. STM image of Aβ42 fibrils incubated for 72 h at a concentration of 80\nμM. Scale bar, 5 nm. Tunneling conditions: I = 198.4 pA, V = 449.8 mV.\nbias\nQuestion 3: The authors should explain what they mean by saying that β-strand\nfragments are “operating as dynamic molecular adapters that regulate the inter-β-sheet\npacking geometry and orientation.” This is because they mentioned that “no\nstatistically significant association can be drawn between linker density and the CV of\nθ.”\nResponse: We thank the reviewer for this question. Actually, this point was overlooked\nin our original manuscript. To address this ambiguity, we deleted the “orientation” part,\nand have now revised the manuscript accordingly to address this issue.\n(page 2, line 25) “These cross-β-strand linkers comprise <15% of the total β-strands in\nthe amyloidogenic aggregates, but they can mediate a significant proportion of\nintermolecular interactions, operating as dynamic molecular adapters that regulate the\ninter-β-sheet packing geometry”\nQuestion 4: The authors must provide a reference for the following statement: “Our\ngroup recently developed an STM-based single-molecule imaging platform, which\nbypasses the limitation of ensemble averaging and enables direct characterization of\nconformational ensembles.”\nResponse: We thank the reviewer for this kind advice. Following the reviewer’s advice,\nwe have added the following references to the revised manuscript:\n“17. Zhang, W. et al. Single-molecule visualization determines conformational substate\nensembles in β-sheet–rich peptide fibrils. Sci. Adv. 9, eadg7943 (2023).\n21. Yu, L. et al. Experimental insights into conformational ensembles of assembled β-\nsheet peptides. ACS Cent. Sci. 9, 1480–1487 (2023).\n33. Yu, L. et al. Molecular recognition of human islet amyloid polypeptide assembly\nby selective oligomerization of thioflavin T. Sci. Adv. 6, eabc1449 (2020).\n34. Wang, R. et al. Submolecular resolution of β-sheet plasticity: decoding mutations\nand PTMs in protein aggregation disorders. ACS Cent. Sci. 11, 927–937 (2025).”\nQuestion 5: Because STM is the primary technique used, we consider it important to\nprovide detailed and referenced support for the following statement: “The molecules\nappear as periodic bright linear patterns forming lamellar structures, with protrusions in\nthe local density of electron states corresponding to peptide bonds and aromatic\nresidues.”\nResponse: We thank the reviewer for this great suggestion. Following the reviewer’s\nadvice, we have included the following references in the revised manuscript to support\nthis description.\n“21. Yu, L. et al. Experimental insights into conformational ensembles of assembled β-\nsheet peptides. ACS Cent. Sci. 9, 1480–1487 (2023).\n35. Mao, X. et al. Beta structure motifs of islet amyloid polypeptides identified through\nsurface-mediated assemblies. Proc. Natl. Acad. Sci. U. S. A. 108, 19605–19610 (2011).\n36. Ma, X. et al. Amyloid β (1–42) folding multiplicity and single-molecule binding\nbehavior studied with STM. J. Mol. Biol. 388, 894–901 (2009).”\nQuestion 6: The authors should explain the relationship between the length of the\ncross-β-strand linkers and the dynamic conformational flexibility.\nResponse: We thank the reviewer for the valuable suggestion. Following the reviewer’s\nsuggestion, we have provided further clarification and additional discussion in the\nrevised manuscript and they are copied below:\n(page 6, line 139) “Detailed discussion is provided in Supplementary 1.1.”\nWe have added the following discussion as Supplementary 1.1 to the revised\nsupplementary information:\n“1.1 Cross-β-Strand Linker Length and Conformational Flexibility\nPrevious studies have reported that the distance between two neighboring residues\nin a β-strand is approximately 0.325 nm1. Based on this structural parameter, we\nestimated that the cross-β-strand linker length observed in our STM imaging (3.0–7.3\nnm) corresponds to approximately 9–22 amino acid residues. This result suggests that\nthese linkers are not of fixed length, but may comprise variable numbers of residues,\nthereby exhibiting substantial structural flexibility and dynamic variability. This\nvariation in linker length reflects the intrinsic conformational flexibility of the cross-β-\nstrand linkers.”\nWe have added the following references to the revised supplementary information:\n“1. Garrett, R. H. & Grisham, C. M. Biochemistry, 2nd Ed. (Saunders College\nPublishing, 1999).”\nQuestion 7: If the peptide used has 25 amino acids, how does this explain the presence\nof shorter fragments attached to the cross-β-strand linkers, for example? Figure 2 A.\nResponse: We thank the reviewer for this insightful question. We would like to clarify\nthat in our STM imaging, only the portion of the adsorbed species that is close enough\nto the HOPG surface, i.e. ~ 1 nm, to allow tunneling electrons to pass through the energy\nbarrier is visible in the STM images, while the portion of a peptide with a distance\ngreater than the tunneling distance (such as the non-assembled fragments ) are not\ndetectable. Therefore, the STM images provide a representation of the peptides that are\nin immediate proximity to the surface, while those located further away appear invisible\nin the images.\nTo address this ambiguity, we have revised the manuscript accordingly:\n(page 8, line 197) “We note that STM imaging can only detect peptide segments within\napproximately 1 nm of the HOPG substrate, owing to the exponential decay of electron\ntunneling probability with distance, while regions beyond this range remain invisible.”\nQuestion 8: The authors must provide a reference for the following statement: “In a\npleated β-sheet, adjacent β-strands are interconnected laterally through a hydrogen\nbond network that stabilizes the structure”.\nResponse: We thank the reviewer for this kind suggestion. Following the reviewer’s\nsuggestion, we have added the following references to the revised manuscript:\n“40. Riek, R. & Eisenberg, D. S. The activities of amyloids from a structural perspective.\nNature 539, 227–235 (2016).\n42. Garrett, R. H. & Grisham, C. M. Biochemistry, 2nd ed. Saunders College\nPublishing (1999).\n43. Nelson, R. et al. Structure of the cross-β spine of amyloid-like fibrils. Nature 435,\n773–778 (2005).”\nQuestion 9: The authors should explain the following statement: “Through\npromiscuous interactions with diverse conformational states.” How do the authors\nknow they are not specific?\nResponse: We thank the reviewer for this great question. In our manuscript, the term\n“promiscuous interactions” was used to indicate that the cross-β-strand linker can\ninteract with multiple conformational substates within the aggregate, highlighting the\ndiversity of its binding modes rather than restriction to a single specific interaction. To\nillustrate this, we constructed a two-dimensional interaction matrix for the cross-β-\nstrand linker, which demonstrates its ability to engage with diverse conformational\nsubstates without a clear preferential binding mode. Given these observations, we\ninterpret these interactions as non-specific.\nTo reflect the above-mentioned points, we have added Figure R8 as\nSupplementary Figure 4 into the revised supplementary information and have revised\nthe manuscript accordingly:\n(page 7, line 157) “Cross-β-strand linkers exhibit no preferential selection bias toward\nspecific conformational substates (Supplementary Fig. 4), as all conformational\nsubstates in the aggregates participate in the interactions with cross-β-strand linkers.”\nFigure R8. The two-dimensional probability matrix illustrates interactions mediated by\nthe cross-β-strand linker. The distribution of interactions between the cross-β-strand\nlinker conformational substates (X axis) and the aggregate conformational substates (Y\naxis) is shown. The β-strand length of each substate is indicated in amino acid residues\n(AAs). Colors range from dark to light blue to represent interaction probability, with\nthe lightest blue corresponding to a probability of 0.\nQuestion 10: How can the role of B-Strand in the assembly of ordered 2D structures\nbe transferred to 1D structures as the authors state “cross-serve as molecular\n\"stitchers\" that integrate multiple assembly intermediates into functional fibrils\".\nResponse: We thank the reviewer for pointing this out. Although mature fibrils\nultimately adopt a one-dimensional morphology, their assembly process is not strictly\nunidirectional. Rather, fibril formation involves protofilaments undergoing face-to-face\nstacking and twisting to form higher-order structures. Our results further suggest that\nmature fibrils can undergo additional cross-linking between adjacent protofilaments.\nImportantly, cross-β-strand linkers represent only a minor component among the β-\nstrands within the aggregates, and fibril elongation remains primarily governed by axial\none-dimensional stacking along the fibril axis. We acknowledge that the “stitcher”\nanalogy may have been imprecise. These linkers in fact function as localized, “patch-\nlike” connectors that modulate discrete inter-protofilament interaction sites and serve\nas structural focal points, rather than acting as the dominant drivers of large-scale fibril\nremodeling. We have revised the relevant statements in the manuscript to convey this\nmeaning more accurately and clearly.\n(page 10, line 250) “Through diverse and dynamic interactions with multiple\nconformational substates, cross-β-strand linkers may serve as molecular connectors that\nintegrate multiple assembly intermediates into functional fibrils.”"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The Authors responded to my concerns fairly satisfactorily, but they still need to address a few more points before the\nmanuscript can be published.\nPlease verify the completeness of the \"Materials and Methods\" section (how were the FTIR measurements conducted and\nhow were the spectra analyzed?).\nIn addition to the second derivatives, I suggest to also report the IR absorption spectra.", "reviewer_2": "The authors carefully addressed all comments.\nThe manuscript is now more convincing than the original submission. However, I remain unconvinced that the reported\nresults genuinely originate from biological or in vitro samples. In particular, the concern raised by Reviewer 1 (\"... that these\natypical cross-β-strand linkers genuinely exist in amyloid aggregates of different proteins, i.e., that their presence is not\nmerely a result of the sample preparation procedures for scanning tunneling microscopy (STM)-based imaging\") still applies.\nThe authors conducted additional experiments to demonstrate that amyloid fibrils formed—an aspect I did not doubt—but\nthese do not sufficiently prove that the effects observed in STM images are not artifacts of the HOPG substrate. I understand\nthat proving this with alternative methods may be challenging, but reporting results that only occur due to the chosen method\ndoes not strengthen the findings.", "reviewer_3": "In the manuscript “Atypical β-Strand Insertion Mediates the Noncovalent Cross-Linking in Amyloid Aggregates”, the authors\nreport the detection and identification of β-strands forming inter-β-sheet connections through insertion into adjacent β-sheets\nwithin protein aggregates on HOPG surfaces.\n1. It is recommended that the authors support the introduction by emphasizing the importance of understanding amyloid fiber\nassembly, particularly highlighting the relevance of studies conducted on van der Waals (VDW) surfaces. This is especially\npertinent given their statement: “To the best of our knowledge, this discovery represents the first direct visualization of\nstochastic β-strand-mediated inter-sheet connections in amyloid aggregates, challenging the conventional models of β-sheet\naggregation that typically emphasize the highly ordered arrangement of β-strands”\n2. The mention of a new fourth mechanism of interaction could be incorporated into the discussion section.\n3. The Section “1.4, Biological Relevance of Aggregates Observed on the HOPG Surfaces, the main interactions in HOPG\nare hydrophobic” in supplementary information should be improved because the support for the use of HOPG as substrate is\nnot clarified. It is because it is well established that proteins can become denatured when their intramolecular interactions\nare overcome by the interaction with the surface, as usually on HOPG. In contrast, graphene oxide (GO) presents functional\ngroups that enable complementary charge interactions, hydrogen bonds, and hydrophobic\ninteractions that can interact with proteins, and reduce the probability to denature. HOP and GO are different substrates."}, "author_response": "Response: We sincerely thank the reviewer for this great advice. The description of\nFTIR measurements and data analysis has been supplemented into the revised\nmanuscript and is copied as follows:\n(page 14, line 348) FTIR Analysis of Peptide Samples\nFTIR spectra were acquired using a PerkinElmer Spectrum One FTIR\nSpectrometer (Waltham, Massachusetts, USA). 40 μL aliquot of each peptide solution\nwas deposited onto a CaF₂ window and allowed to dry completely before measurement.\nSpectra were recorded three times over the range of 400-4500 cm⁻¹ at a resolution of 2\ncm⁻¹. All data were processed using PeakFit software (Version 4.12, San Jose, CA,\nUSA). Each spectrum was baseline-corrected, smoothed using a Gaussian instrumental\nresponse function, and subjected to second-order derivative calculation, followed by\npeak fitting on the derivative spectra. Figures of the processed spectra were generated\nusing Prism software (Version 8.01, San Diego, CA, USA).\nIn addition, we have included the original non-baseline-corrected IR absorption\nspectra together (Figure R1) in the revised Supplementary information as\nSupplementary Figure 8.\nFigure R1. Original FTIR absorption spectra of gal-3 225–250 (A), gal-3 206–231 (B),\nAβ42 (C), hIAPP 8–37 (D), KRT-R (E), hIAPP S20G 8–37 (F), −5Aβ42 (G), TDP-43\n341–357 (H), and gal-3 129–154 (I) pre- and post-aggregation.\nResponse: We sincerely thank the reviewer for recognizing our revision efforts and\nraising this insightful comment.\nThe HOPG surface is highly hydrophobic with aromatic planes, a property that\nfacilitates the adsorption of protein or peptide monomers via hydrophobic interactions\nand π–π stacking. Such adsorption can drive conformational changes in monomeric\npeptides and alter their aggregation kinetics. However, our experiments depart from this\nmonomer-focused context: we deposited preformed mature amyloid fibrils onto the\nHOPG surfaces. These fibrils possess highly stabilized core β-sheet structures—an\nobservation consistent with prior studies (Nat. Commun. 2017, 8, 2284; Sci. Rep. 2016,\n6, 37970)—which renders them largely impervious to substrate-induced effects.\nPrevious work has already validated that substrate exposure does not disrupt the\nstructure of preformed amyloid aggregates. For example, fibrils deposited on diverse\nsurfaces (HOPG, carbon film, mica) or observed substrate-free (via cryo-EM) retain\nlargely identical assembly structure (Nat. Commun. 2017, 8, 2284). This suggests that\nHOPG does not alter the structure of preformed amyloid fibrils. Another study\ncomparing persistence lengths of preformed fibrils on hydrophilic mica versus\nhydrophobic, aromatic HOPG surfaces found no significant correlation between fibril\npersistence length and substrate type—indicating that substrate effects on preformed\nfibrils are negligible (Sci. Rep. 2016, 6, 37970). Furthermore, although STM imaging\nwas performed under nominally “dry” conditions, a water film of varying thickness\nfrom Angstroms to several nanometers remains on the HOPG surface. Once excess\nsolution is removed, this water layer establishes a stable liquid–solid interface and\nremains intact, preserving the fibrils’ local hydration and preventing dehydration-\ninduced denaturation (ACS Nano, 2023, 17, 51). Collectively, these findings support\nour conclusion that the observed STM images faithfully capture the morphological\nfeatures of mature amyloid fibrils in a state nearly identical to their native condition.\nWe attempted to perform additional experiments to explore the potential biological\nroles of the cross-β-strand linker. We selected two peptide aggregates that have been\npreviously studied and reported, namely human islet amyloid polypeptide (hIAPP) and\nits artificial mutant carrying a post-translational modification (phosphorylation at serine\nresidue 20, hereafter referred to as hIAPP S20p) (ACS Cent. Sci. 2025, 11, 927). The\ntwo peptides differ only at residue 20, where the serine in hIAPP is phosphorylated in\nhIAPP S20p (Figure R2A). Analysis of the STM images of these two peptide aggregates\nreveals a pronounced difference in the abundance of cross-β-strand linkers: in the\nhIAPP aggregates, the distribution probability of cross-β-strand linkers was\napproximately 15.1%, whereas in the hIAPP S20p aggregates it was only about 3.5%\n(Figure R2B). To investigate the potential biological significance of the cross-β-strand\nlinker, we further examined the stability of these two aggregates.\nFigure R2. (A) Amino acid sequences of hIAPP and hIAPP S20p. S20p indicates\nphosphorylation at serine residue 20. (B) STM images of hIAPP and hIAPP S20p fibrils\nincubated for 72 h at a concentration of 40 μM and cross-β-strand linkers observed in\nthe aggregates. White boxes highlight the structure of the cross-β-strand linkers. Scale\nbars: 5 nm. Tunneling conditions: I = 198.4 pA, V = 449.9 mV.\nbias\nUsing temperature-dependent light scattering during heating, we probed structural\nstability differences between hIAPP and hIAPP S20p aggregates. The intensity of light\nscattered from their solutions declines as temperature rises: hIAPP aggregates loses ~50%\nof their scattering intensity at 50 °C, whereas hIAPP S20p aggregates loses ~87%—a\nstark contrast highlighting hIAPP aggregates’ superior stability over hIAPP S20p.\nThis\ndisparity suggests hIAPP’s greater structural stability likely stems from its higher\nproportion of cross-β-strand linkers (Figures R3A, B). Additionally, at the initial 25 °C,\nhIAPP exhibits higher scattering intensity than hIAPP S20p aggregates—further\nindicating that cross-β-strand linkers likely function as molecular connectors,\ncoalescing multiple assembly intermediates into larger fibrils (Figure R3C).\nFigure R3. Static light scattering analysis of the thermal stability of hIAPP (A) and\nhIAPP S20p aggregates (B), along with the scattering intensity measured at 25 °C (C).\nData are shown as mean ± SEM. (N = 3) *P < 0.05, **P < 0.01.\nWe further compared the stability of hIAPP and hIAPP S20p aggregates in the\npresence of a chemical denaturant. Static light scattering measurements were performed\nafter adding varying concentration of guanidinium chloride (GdmCl) to each aggregate.\nThe results show that hIAPP S20p exhibits significant dissociation upon the addition of\njust 0.6 M of guanidine hydrochloride (P＜0.001), whereas hIAPP shows no distinct\ndifference at 0.6 M (P = 0.964). hIAPP shows only slight dissociation at 1.1 M (P =\n0.015) and becomes noticeably dissociated above 1.5 M (P＜0.001). These findings\nindicate that hIAPP exhibits greater resistance to chemical denaturation and higher\nstructural stability (Figure R4).\nFigure R4. Static light scattering analysis of hIAPP (A) and hIAPP S20p aggregates\n(B) upon addition of varying concentration of GdmCl. Data are shown as mean ± SEM.\n(N = 3). *P < 0.05, **P < 0.01.\nTo reflect the above-mentioned points, we have added Figures R2-R4 as\nSupplementary Figures 21-23 into the revised supplementary information and have\nrevised the manuscript accordingly:\n(page 11, line 283) “This structural plasticity may represent biologically relevant\nstates. Our results demonstrate that aggregates with a higher content of cross-β-strand\nlinkers can better resist thermal stress and chemical denaturants (Supplementary Figs.\n21-23), thereby enabling amyloid aggregates to maintain their stability and aggregation\npropensity under physiological or pathological conditions (Supplementary 1.5).”\n(page 12, line 308) “hIAPP and hIAPP S20p were re-dissolved in 100 μM TEA\n(pH 6.0) containing 2% (v/v) DMSO to a final concentration of 500 μM, and incubated\nat 37 °C with shaking at 210 rpm for 72 hours17,21,40,51.”\n(page 15, line 377) “Thermal stability assay\nThe thermal stability of 500 μM hIAPP and hIAPP S20p aggregates was assessed\nusing the integrated UNcle stability platform (Unchained Labs, Norton, MA). Static\nlight scattering (SLS) was monitored at an excitation wavelength of 266 nm as the\ntemperature increased from 25 °C to 95 °C at a rate of 0.25 °C per minute.”\n(page 15, line 383) “Chemical denaturation assay\nThe 500 μM hIAPP and hIAPP S20p aggregates were analyzed using a Zetasizer\nNano ZS system (Malvern, UK). Corresponding volume of 6 M guanidine\nhydrochloride were sequentially added to the aggregates and thoroughly mixed to\nobtain the concentration gradient of guanidine hydrochloride. The samples were then\nirradiated with a 4 mW, 633 nm laser, and the scattering intensity was measured at a 90°\nangle. A digital signal processor was used as a photodetector to record the scattering\nintensity of the samples.”\nWe have added the following references to the revised manuscript:\n“40. Wang, R. et al. Submolecular resolution of β-sheet plasticity: decoding\nmutations and PTMs in protein aggregation disorders. ACS Cent. Sci. 11, 927–937\n(2025).”\nWe have revised Supplementary Section 1.5, and it is copied as follows:\n“1.5 Biological Relevance of Cross-β-strand Linkers in Amyloid Aggregation\nAmyloid fibrils are structurally polymorphic and heterogeneous. While cryo-EM\nand NMR have elucidated many rigid, well-ordered fibrils, a substantial fraction of\nflexible fibrils remains unresolved or is labeled as disordered3,5. STM provides\ncomplementary structural insights into these flexible assemblies, which may represent\nbiologically relevant states. We examined two previously studied peptide aggregates,\nhuman islet amyloid polypeptide (hIAPP) and its phosphorylated mutant at serine 20\n(hIAPP S20p). The peptides differ only at residue 20 (Supplementary Fig. 21A) and\nboth can form amyloid fibrils, as demonstrated in our previous study by TEM, FTIR,\nand ThT assays14. STM analysis further reveals a higher abundance of cross-β-strand\nlinkers in hIAPP (~15.1%) than in hIAPP S20p (~3.5%) (Supplementary Fig. 21B).\nTemperature-dependent light scattering shows that hIAPP aggregates loses ~50%\nof their scattering intensity at 50 °C, whereas hIAPP S20p loses ~87% (Supplementary\nFig. 22A, B). Additionally, at the initial 25 °C, hIAPP exhibits higher scattering\nintensity than hIAPP S20p aggregates—further indicating that cross-β-strand linkers\nlikely function as molecular connectors, coalescing multiple assembly intermediates\ninto larger fibrils (Supplementary Fig. 22C). In guanidinium chloride titrations, hIAPP\nS20p exhibits significant dissociation at 0.6 M, while hIAPP shows only slight\ndissociation at 1.1 M, noticeable dissociation at 1.5 M (Supplementary Fig. 23). These\nresults collectively demonstrate that aggregates with higher content of cross-β-strand\nlinkers are more resistant to thermal and chemical perturbations.”\nWe have added the following references to the revised supplementary information:\n“14. Wang, R. et al. Submolecular resolution of β-sheet plasticity: decoding\nmutations and PTMs in protein aggregation disorders. ACS Cent. Sci. 11, 927–937\n(2025).”\nResponse: We thank the reviewer for their careful reading and insightful comments on\nour manuscript.\nQuestion 1: It is recommended that the authors support the introduction by\nemphasizing the importance of understanding amyloid fiber assembly, particularly\nhighlighting the relevance of studies conducted on van der Waals (VDW) surfaces. This\nis especially pertinent given their statement: “To the best of our knowledge, this\ndiscovery represents the first direct visualization of stochastic β-strand-mediated inter-\nsheet connections in amyloid aggregates, challenging the conventional models of β-\nsheet aggregation that typically emphasize the highly ordered arrangement of β-strands”\nResponse: We thank the reviewer for this great suggestion. Following the reviewer’s\nadvice, we have strengthened this part with the following added into the revised\nmanuscript:\n(page 4, line 67) “Cross-β-strand linking mechanism is identified in amyloid\naggregation. Amyloid aggregation plays a crucial role in many protein misfolding-\nrelated diseases, such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic\nlateral sclerosis31–33. Amyloid fibrils are mainly composed of β-sheet structures\nstabilized by extensive intermolecular hydrogen bonding, and exhibit remarkable\nstructural stability34. A typical feature of amyloid aggregates is conformational\nheterogeneity, namely identical peptide sequences can adopt distinct conformational\nsubstates coexisting within a heterogeneous conformational ensemble. Such\nconformational heterogeneity is closely linked to their pathological functions17,21.\nHowever, due to the inherent heterogeneity of amyloid structures, conventional\nstructural techniques (e.g., X-ray crystallography, NMR, and cryo-EM) remain limited\nin resolving coexisting conformations. To directly visualize the local structural features\nwithin amyloid fibrils, we employed scanning tunneling microscopy (STM) under\nambient conditions. This technique provides exceptional resolution down to the single-\nmolecule level, offering a complementary perspective on the heterogeneous\nconformational ensemble of amyloid fibrils. STM relies on electron tunneling and\nrequires a conductive, flat substrate; therefore, highly oriented pyrolytic graphite\n(HOPG) was used as the supporting substrate. Unlike more reactive STM substrates\nsuch as Cu(001) or Cu(110)—which may form covalent bonds with adsorbates—\nHOPG is chemically inert to interact with adsorbates35. Although STM imaging was\nperformed under nominally dry conditions, a thin residual water film on the HOPG\nsurface helped maintain local hydration, thereby preventing dehydration-induced\ndenaturation36.”\nWe have added the following references to the revised manuscript:\n“31. Willbold, D., Strodel, B., Schröder, G. F., Hoyer, W. & Heise, H. Amyloid-\ntype protein aggregation and prion-like properties of amyloids. Chem. Rev. 121, 8285–\n8307 (2021).\n32. Teunissen, C. E. et al. Blood-based biomarkers for Alzheimer’s disease:\ntowards clinical implementation. Lancet Neurol. 21, 66–77 (2022).\n33. Wilson, D. M. et al. Hallmarks of neurodegenerative diseases. Cell 186, 693–\n714 (2023).\n34. Lührs, T. et al. 3D structure of Alzheimer’s amyloid-β (1–42) fibrils. Proc.\nNatl. Acad. Sci. U. S. A. 102, 17342–17347 (2005).\n35. Yan, X. Peptide Self-Assembly and Engineering. (Wiley-VCH, Weinheim,\nGermany., 2024).\n36. Garcia, R. Interfacial liquid water on graphite, graphene, and 2D materials.\nACS Nano 17, 51–69 (2023).”\nQuestion 2: The mention of a new fourth mechanism of interaction could be\nincorporated into the discussion section.\nResponse: We thank the reviewer for this great advice. Following the reviewer’s advice,\nwe have added the following text to the revised manuscript:\n(page 10, line 263) “Unlike the three previously reported interaction modes, this\nmechanism generates direct inter-sheet connections through the insertion of β-strands\nbetween neighboring β-sheets. Notably, this mechanism has been observed only in β-\nsheet-rich amyloid aggregates, and whether it could occur in non-amyloid protein\nassemblies remains to be further explored.”\nQuestion 3: The Section “1.4, Biological Relevance of Aggregates Observed on the\nHOPG Surfaces, the main interactions in HOPG are hydrophobic” in supplementary\ninformation should be improved because the support for the use of HOPG as substrate\nis not clarified. It is because it is well established that proteins can become denatured\nwhen their intramolecular interactions are overcome by the interaction with the surface,\nas usually on HOPG. In contrast, graphene oxide (GO) presents functional groups that\nenable complementary charge interactions, hydrogen bonds, and hydrophobic\ninteractions that can interact with proteins, and reduce the probability to denature.\nHOP and GO are different substrates.\nResponse:\nWe thank the reviewer for this insightful suggestion. We fully agree with the\nreviewer that HOPG and GO are distinct substrates. We have removed the discussion\nregarding GO and provide the following clarification on the potential effects of HOPG\non protein structure:\nThe HOPG surface is highly hydrophobic with aromatic planes, a property that\nfacilitates the adsorption of protein or peptide monomers via hydrophobic interactions\nand π–π stacking. Such adsorption can drive conformational changes in monomeric\npeptides and alter their aggregation kinetics. However, our experiments depart from this\nmonomer-focused context: we deposited preformed mature amyloid fibrils onto the\nHOPG surfaces. These fibrils possess highly stabilized core β-sheet structures—an\nobservation consistent with prior studies (Proc Natl Acad Sci U S A. 2005, 102(48), 17342).\nPrevious work has already validated that substrate exposure does not disrupt the\nstructure of preformed amyloid aggregates (Nat. Commun. 2017, 8, 2284; Sci. Rep. 2016,\n6, 37970). For example, fibrils deposited on diverse surfaces (HOPG, carbon film, mica)\nor observed substrate-free (via cryo-EM) retain identical assembly structure (Nat.\nCommun. 2017, 8, 2284). This suggests that HOPG does not alter the structure of\npreformed amyloid fibrils. Another study comparing persistence lengths of preformed\nfibrils on hydrophilic mica versus hydrophobic, aromatic HOPG surfaces found no\nsignificant correlation between fibril persistence length and substrate type—indicating\nthat substrate effects on preformed fibrils are negligible (Sci. Rep. 2016, 6, 37970).\nFurthermore, although STM imaging was performed under nominally “dry” conditions,\na water film of varying thickness from Angstroms to several nanometers remains on the\nHOPG surface. Once excess solution is removed, this water layer establishes a stable\nliquid–solid interface and remains intact, preserving the fibrils’ local hydration and\npreventing dehydration-induced denaturation (ACS Nano, 2023, 17, 51). Collectively,\nthese findings support our conclusion that the observed STM images faithfully capture\nthe morphological features of mature amyloid fibrils in a state nearly identical to their\nnative condition.\nWe have revised Supplementary Section 1.4, and it is copied as follows:\n“1.4 Biological Relevance of Aggregates Observed on the HOPG surfaces\nHOPG, characterized by its hydrophobic aromatic planes, can adsorb proteins or\npeptides via hydrophobic and π–π interactions, potentially affecting monomeric peptide\nconformation and aggregation kinetics. However, in our experiments, mature amyloid\nfibrils with highly stabilized β-sheet cores were deposited onto HOPG and were largely\nresistant to substrate effects. This is consistent with previous reports showing that fibril\nmorphology is preserved across various substrates (HOPG, carbon film, mica) or under\nsubstrate-free conditions (cryo-EM)8, and that their persistence length is independent\nof substrate type9. Although STM imaging is performed under nominally “dry”\nconditions, a thin residual water film on the HOPG surface maintains local hydration,\npreventing dehydration-induced denaturation10. Moreover, unlike more reactive\nsubstrates such as Cu(001) or Cu(110)—which can form covalent bonds with\nadsorbates11—HOPG primarily engages in weak van der Waals interactions with\nadsorbed molecules. Consequently, the HOPG substrate is expected to have a negligible\nimpact on fibril conformation7. We also note that high-resolution structural\ncharacterization of proteins within a cellular environment remains challenging for\nclassical biophysical techniques such as cryo-EM, NMR, and X-ray crystallography.\nAlthough methods like cryo-electron tomography (cryo-ET) allow imaging under near-\nnative cellular conditions12,13, they currently lack the resolution required to resolve the\nheterogeneous conformational ensembles characteristic of protein or peptide\naggregates.”\nWe have added the following references to the revised supplementary information:\n“8. Reynolds, N. P. et al. Competition between crystal and fibril formation in\nmolecular mutations of amyloidogenic peptides. Nat. Commun. 8, 1338 (2017).\n9. Makky, A., Bousset, L., Polesel-Maris, J. & Melki, R. Nanomechanical\nproperties of distinct fibrillar polymorphs of the protein α-synuclein. Sci. Rep. 6, 37970\n(2016).\n10. Garcia, R. Interfacial liquid water on graphite, graphene, and 2D materials.\nACS Nano 17, 51–69 (2023).”"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The Authors have provided satisfactory responses to the comments", "reviewer_2": "The authors carefully addressed all comments, including conducting additional experiments. The manuscript is now\nsignificantly improved, and I agree to its publication.", "reviewer_3": "The authors responded to my concerns, and I believe the manuscript can be published.\nKind regards"}, "author_response": ""}]

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