Cryo-EM structure of renal AL amyloid fibrils from a patient with λ1 light chain amyloidosis

Domain: neuroscience
Published: 2025-12-17
Editorial outcome: major_revision

Abstract
Systemic light-chain amyloidosis (AL) is characterized by the misfolding and aggregation of immunoglobulin light chains (LCs) into amyloid fibrils, leading to multiorgan deposition and dysfunction, with the kidneys being one of the most commonly involved organs. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of AL amyloid fibrils from the kidney of a male patient with renal AL amyloidosis. Two distinct polymorphic fibril structures, polymorph A and polymorph B, were identified, both featuring ordered cores (Gln16-Ser95) with β-sheet-rich architectures stabilized by interchain hydrogen bonds and salt bridges. Notably, six mutations in the IGLV1-44*01 gene sequence, including Gln39His and Tyr37Phe, were identified within the fibril core. These mutations influence fibril stability and aggregation by altering intramolecular and intermolecular interactions, such as CH-π stacking and salt bridge formation. Comparative analysis with previously reported heart-derived IGLV1-44 fibrils reveals structural variations linked to light-chain sequence differences. Our findings provide critical insights into the molecular determinants of fibril assembly and organ tropism in AL amyloidosis.

Introduction
Systemic light-chain amyloidosis (AL) is a disorder characterized by the abnormal aggregation of immunoglobulin light chains (LCs) into oligomers and amyloid fibrils, which subsequently deposit in various organs and tissues, leading to progressive organ dysfunction. The disease is associated with clonal plasma cell dyscrasia1. As of 2018, an estimated 73,567 cases had been reported globally over the preceding two decades, with the prevalence continuing to rise over time2. The heart and the kidney are the most frequently affected organs in systemic AL amyloidosis, although virtually all organs—except the brain—can be involved3. Renal involvement typically manifests as heavy proteinuria or nephrotic syndrome; however, the lag between the characteristic pathological changes and the onset of clinical symptoms contributes to an early diagnosis rate of less than 30%4. The prognostic situation of the patient is primarily determined by the extent of myocardial amyloid infiltration, with a current median overall survival of 12–18 months5. These clinical challenges underscore the urgent need for advances in understanding the molecular pathogenesis of the disease and the mechanisms underlying organ-tropism amyloid deposition.

Among all types of systemic amyloidosis, AL amyloidosis exhibits the greatest clinical heterogeneity. This variability arises from the genetic recombination of variable (V), joining (J), and constant (C) gene segments, somatic hypermutation, and junctional diversity at the V/J interface during V/J recombination6–8. As a result, the LC sequences derived from either the λ or κ loci are nearly unique to each individual patient9,10. Previous studies have shown that LCs assemble into homodimers, with each monomer comprising two immunoglobulin domains. The N-terminal variable domain (VL), in particular, exhibits a high degree of sequence variability11,12. The polymorphic amyloid fibril proteins predominantly originate from the VL domain of the LC precursor13. Somatic mutations in the immunoglobulin lambda variable (IGLV) gene segments, which encode the LC variable domains, reduce the thermodynamic stability of the protein, thereby facilitating amyloid fibril formation1,14.

IGLV1-44 is a germline gene of the immunoglobulin lambda (λ) LC variable (V) region, primarily functioning in immune responses. It encodes a protein component of the immunoglobulin complex, typically localized to the extracellular space and plasma membrane. As an LC gene, IGLV1-44 plays a critical role in generating antibody diversity, enabling immunoglobulins to recognize and bind a wide range of antigens. Previous studies have demonstrated a significant association between IGLV1-44 and cardiac AL amyloidosis, with this gene being markedly overrepresented among λ LCs implicated in cardiac involvement, compared to other germline genes15,16. This association suggests that IGLV1-44 may have a unique role in amyloid fibril formation, possibly due to mutations that reduce protein stability and thereby promote amyloidogenic propensity17. More recent studies, however, have shown that IGLV1-44 is also the most frequently used IGLV subfamily among patients with predominant renal involvement. This indicates a potential genetic predisposition or pathophysiological mechanism linking IGLV1-44 to renal AL amyloidosis. Structural features or specific amino acid sequences encoded by this gene may render the LCs more prone to misfolding and aggregation, facilitating amyloid fibril deposition within renal tissue18.

Amyloid deposits are typically persistent and resistant to degradation, making structural elucidation of amyloid fibrils a critical avenue for understanding their in vivo aggregation mechanisms and guiding therapeutic development. In general, AL fibrils exhibit the characteristic structural features of amyloid fibrils, such as the cross-β architecture and an average width of approximately 15nm13,19. However, since amyloid fibrils often derive from natively folded proteins under physiological conditions, structures of fibrils generated in vitro under denaturing conditions may not fully represent those found in patient-derived amyloids20,21. Cryo-electron microscopy (cryo-EM) combined with three-dimensional (3D) reconstruction has emerged as the preferred method for determining the molecular structure of ex vivo amyloid fibrils. To date, the structures of fibrils extracted from the hearts of five patients with advanced cardiac amyloidosis have been reported. These include λ6-AL55, λ3-FOR005, λ1-FOR001, λ1-FOR006, FOR103, FOR010, and λ3-AL59, each exhibiting distinct folding conformations17,22–26. Notably, fibrils extracted from both the heart and the kidney of the same patient (AL55) display nearly identical folding patterns, highlighting the immunoglobulin LC sequence as a key determinant of fibril structure27. It is important to emphasize that although the kidney is one of the most frequently affected organs in AL amyloidosis—with involvement observed in 60–70% of cases28—there is still a lack of systematic structural characterization of amyloid fibrils specifically from patients with isolated renal involvement. Current understanding of AL fibril structure remains limited in scope across different patient subgroups with organ-specific involvement, underscoring the need for broader and more targeted structural studies.

In this study, we employed cryo-EM to determine the high-resolution 3D structure of LC amyloid fibrils extracted from the kidney tissue of a patient with AL amyloidosis. Negative-stain transmission electron microscopy (TEM) analysis revealed the presence of two distinct structural polymorphs, designated as polymorph A and polymorph B, with corresponding cryo-EM reconstructions reaching resolutions of 2.9Å and 3.0Å, respectively. The fibril widths were measured at approximately 13nm and 14nm for polymorphs A and B. Further structural analysis demonstrated that the fibril core is composed of the Glu16–Ser95 segment of the LC protein. This region assembles into a highly ordered protofilament architecture through antiparallel β-sheet stacking, forming the structural backbone of the fibrils.

Results
Extraction and sequence analysis of amyloid fibrils from a patient’s renal biopsy
To investigate the structural organization of native amyloid fibrils, we selected a 66-year-old male patient diagnosed with renal AL amyloidosis and nephrotic syndrome as the study subject. Renal tissue samples were obtained via percutaneous kidney biopsy, and AL amyloid fibrils were extracted from the tissue using an optimized protocol for isolating tissue-derived amyloid fibrils19. To analyze the molecular composition of the isolated fibrils, we employed a proteomics-based approach. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was used to separate the fibrillar proteins, followed by liquid chromatography–tandem mass spectrometry of the gel bands to accurately determine the amino acid sequence characteristics of the fibril-forming proteins (Supplementary Fig. 1). Previous studies have demonstrated that the immunoglobulin light chain variable region (IGLV) is associated with organ tropism29. Bioinformatic analysis revealed that the core component of the deposited fibrils in this patient corresponds to a germline-derived sequence encoded by the IGLV1-44*01 gene, consistent with earlier findings linking IGLV1-44 fragments to predominant renal involvement18. Compared to the native germline sequence, the fibril protein exhibited six mutations within the IGLV1-44 region—Ser26Thr, Ser31Gly, Tyr37Phe, Gln39His, Gly65Ala, and Asp86His—likely arising from B cell clone-specific somatic hypermutation30. These mutations may reduce the stability of the LC and thereby promote its misfolding, facilitating amyloid fibril formation and aggregation.

Cryo-EM structural determination of the renal AL fibril
To investigate the structure of the extracted fibrils, we first performed negative-staining TEM. This initial analysis revealed the presence of fibrils with the characteristic helical symmetry of amyloid but was insufficient to resolve high-resolution details (Supplementary Fig. 2). To determine the atomic structures, we subsequently employed cryo-EM. Following cryo-EM data collection, we used RELION for helical reconstruction of this fibril species. 3D classification from helical image processing of 5786 filaments revealed two major polymorphs of twisted fibrils, herein designated as polymorphs A and B (Supplementary Fig. 3). Based on the 0.143 Fourier shell correlation (FSC) criterion, the reconstructions were refined to spatial resolutions of 2.9Å for polymorph A and 3.0Å for polymorph B (Supplementary Fig. 4). The density maps showed that each fibril consists of two protofilaments (Fig. 1a, b). Consistent with the density map, polymorph A fibril displays a (pseudo)2₁ screw symmetry, characterized by a helical twist of −179.58° and a rise of 2.46Å. Polymorph B exhibits C1 helical symmetry, characterized by a helical twist of 0.87° and a helical rise of 4.92Å for the winding dimer (SI Table 1). Both polymorphs form right-handed helices, with polymorph A measuring approximately 14nm in width and a half-pitch of ~106nm, while polymorph B has a width of ~13nm and a half-pitch of ~102nm. The ordered fibril core extends from Gln16 to Ser95 and contains an intramolecular disulfide bond formed between Cys22 and Cys89 (Fig. 2a).

Salt bridges and hydrophobic interactions mediate stability in the fibril protein fold
The 3D cryo-EM map was fitted with contiguous peptide segments corresponding to residues Gln16-Ser95 of the IGLV1-44 fibril protein (Fig. 2b, c). The N- and C-termini of the protein were surrounded by diffuse density (Supplementary Fig. 3), indicating structural disorder in the first and last ~15 residues (Fig. 2c). Within the fibril core, β-strand conformations were identified at residues Gln16–Ser21, Thr26–Gly30, Asn32–Asn35, Gly42–Tyr50, Asn53–Val59, Ser64–Ser71, Ser73–Ile76, Glu84–Tyr88, and Ala90–Asp93, which we designated as β1 through β9, respectively (Fig. 2c, d). These β-strands interact along the fibril z-axis via backbone hydrogen bonding and side-chain interactions, including aromatic stacking and polar residue contacts.

Both polymorphs A and B are composed of two protofilaments entwined together; however, the protofilament interaction interface differs significantly. Polymorph A’s interface is stabilized by two pairs of inter-chain aromatic residue interactions, specifically asymmetric CH-π stacking between His39-Pro41 and Pro41-His39, markedly enhancing interface stability (Fig. 2a). In contrast, polymorph B maintains protofilament interactions through a single CH-π interaction between His39 and Pro41, with an interface topology exhibiting asymmetric features. Notably, both polymorphs display an identical β-sheet layering pattern within the core of individual protofilaments, which is underscored by a very low Cα RMSD of 0.001Å between the two protofilaments. This indicates that structural variations arise from differences in inter-protofilament interface regulation rather than conformational rearrangements within protofilaments.

The fibril surface exhibits a pronounced charge distribution pattern, with a molecular surface highly enriched in charged and polar amino acid residues. The N-terminal region is dominated by positive electrostatic potential (blue), whereas the C-terminal region exhibits a negative potential (red) (Fig. 3a). Protein folding creates two major cavities, labeled A and B. Cavity A’s inner surface is primarily composed of hydrophobic side chains such as Leu, Ile, and Phe, conferring pronounced hydrophobicity; in contrast, cavity B is enriched with polar residues like Ser and charged side chains including Asp and Lys, resulting in a hydrophilic character (Fig. 3a, b). Notably, compared to cardiac fibrils (Supplementary Fig. 5a, b), our kidney-derived fibrils display a more abundant and complex surface charge pattern, which is likely an adaptation to the charge-rich microenvironment of the kidney.

Implication of patient-specific mutations for fibril morphology
Compared to the germline IGLV1-44 amino acid sequence, the amyloidogenic LC contains six mutations (Fig. 3c, d), which are widely recognized as drivers of amyloidosis in affected patients31,32. Among these, the Gln39His mutation introduces a charge polarization effect via the histidine side chain, enabling a cross-chain CH-π stacking interaction with the adjacent Pro41 residue of a neighboring protofilament subunit. This interaction facilitates the formation of a unique fibril interface, leading to a dimer architecture distinct from other amyloid fibrils. It thereby constitutes a core structural basis for IGLV1-44 fibril assembly and enhances the aggregation kinetics of the protein subunits compared to their native or non-amyloidogenic states. The Tyr37Phe mutation (Fig. 3b) may strengthen the van der Waals forces and hydrophobic synergism between β-sheet layers of the fibril, further stabilizing the fibril. Additionally, the Asp86His mutation (Fig. 3b) can form a salt bridge with Glu84, reinforcing the internal fibril architecture. In contrast, the Ser26Thr, Ser31Gly, and Gly65Ala mutations appear to have minimal impact on fibril structure. Collectively, these distinct mutation types may contribute to organ-tropism deposition patterns, such as cardiac or renal involvement. Detecting mutation profiles in patient LCs could therefore guide the selection of targeted therapies, providing a rationale for first-line treatment options in clinical practice. For instance, if a mutation profile closely aligns with that characteristic of cardiac-derived fibrils, a cardiotropic treatment strategy would be prioritized. Conversely, the detection of a mutation profile associated with renal amyloidosis would justify a renally targeted approach. However, it is important to note that the existing maps of organ-specific mutational signatures remain incomplete. This evolving framework for mutational profiling nonetheless allows for a more personalized and biologically rational selection of first-line therapies, ultimately aiming to improve treatment outcomes.

Comparative analysis with cardiac fibrils and native light chain structures
The fibril structure exhibits significant differences from the native folded VL domain. Although both structures are rich in β-sheets, critical conformational rearrangements were observed around the disulfide bond region, which covalently links the N- and C-terminal regions of the protein. In the native conformation, these two domains adopt a parallel N→C orientation, whereas in the fibril assembly, they switch to an antiparallel topological arrangement (Supplementary Fig. 6). This conformational transition is attributed to an intramolecular domain rotation occurring during protein misfolding, specifically involving a 180° topological inversion of one domain around the disulfide bond axis.

A previously reported cardiac-derived AL fibril structure26, also belonging to the IGLV1-44 family, shows that hydrophobic interactions between the C-terminal extended Leu96 and N-terminal Val18 bring the protein’s N- and C-termini into close proximity, forming a stable hydrophobic core cavity together with the intramolecular disulfide bond (Fig. 4a). In contrast, our study reveals that the renal-derived amyloid fibril displays diffuse density beyond residue Ser95, resulting in a bifurcated conformation of the N- and C-termini (Fig. 4b). Further structural analysis delineates the mechanisms by which the Gln39His and Tyr37Phe mutations reshape the fibril core: Gln39His mediates inter-protofilament contacts, while Tyr37Phe causes an inward flipping of the residue. This dual rearrangement culminates in the formation of a larger internal cavity specific to the renal fibril.

Notably, the Asp86His mutation in the renal fibril forms a stable intermolecular salt bridge with the neighboring Glu84 residue, significantly enhancing fibril stability; whereas in the cardiac fibril, structural stabilization is mediated by a salt bridge interaction between Asp86 and Arg25. These findings reveal pronounced molecular assembly differences between AL amyloid fibrils derived from distinct tissues (Fig. 4c), establishing a structural basis for how mutation-driven fibril polymorphism contributes to organ tropism.

Methods
Fibril extraction from renal
Renal tissue from a 66-year-old male patient with nephrotic syndrome and AL lambda amyloidosis was obtained from the First Affiliated Hospital of Zhejiang University School of Medicine. Biological sex was obtained from medical records. Because the study involved only a single clinical sample, sex-disaggregated analysis was not possible and not applicable. Tissue was stored frozen (−80°C) without fixation until use. This stored tissue has been fully consumed for the experiments described herein. During routine diagnostic procedures, renal biopsy samples were collected via percutaneous renal needle biopsy following acquisition of informed consent for research-oriented specimen preservation and experimental applications. The presence of amyloid deposits was evaluated by Congo red staining analysis under polarized light and by electron microscopy. Amyloid typing was confirmed by immuno-electron microscopy38. Organ involvement was defined according to international criteria39. Fibrils were extracted as described by Annamalai et al.19. In brief, 250 mg of tissue were diced and washed five times with 0.5 mL Tris calcium buffer (20 mM Tris, 138 mM NaCl, 2 mM CaCl2, 0.1% NaN3, pH 8.0). Each washing step consisted of gentle vortexing and centrifugation at 3100 × g for 1 min at 4°C. The supernatant was discarded, and the pellet was resuspended in 1 mL of freshly prepared 5 mg mL−1 Clostridium histolyticum collagenase (Sigma) in Tris calcium buffer. After incubation overnight at 37°C, the tissue material was centrifuged at 3100 × g for 30 min at 4°C. The retained pellet was resuspended in 0.5 mL buffer containing 20 mM Tris, 140 mM NaCl, 10 mM ethylenediaminetetraacetic acid, 0.1% NaN3, pH 8.0, and subjected to 10 cycles of homogenization in fresh buffer and centrifugation for 5 min at 3100 × g at 4°C. The remaining pellet was homogenized in 0.5 mL ice-cold water, centrifuged for 5 min at 3100 × g at 4°C, and the fibril-containing supernatant was analyzed.

Protein sequence determination by electrospray-ionization MS
About 2 μg refolded, lyophilized, and glycosylated fibril protein was resuspended and processed by denatured protein gel electrophoresis. Afterward, the gel band of the fibril protein was cut into 1 mm3 colloidal particles and decolorized by incubating with 1 ml 50% ACN-50% 50 mmol/L NH4HCO3 solution for 30 min. This operation was repeated until the colloidal particles were colorless. Then, 1 mL 100% ACN 1000 μL was incubated for 30 min, dehydrated, and left to dry at room temperature.

Dried gel slices were reduced with 10 mM dithiothreitol (Sigma-Aldrich), in a water bath at 56°C for 1 h and subsequently alkylated with 55 mM iodoacetamide (Sigma-Aldrich) for 1 h at 37°C.

The gel slices were placed in six different protease solutions (0.025 μg/μl trypsin in 50 mM NH4HCO3; 1 μg/μl pepsin in HCl, pH 5.5; 0.05 μg/μl chymotrypsin in 1 mM HCl, 2 μM CaCl2; 0.1 μg/μl Elastase protease in 100 mM Tris-HCl buffer, pH 8.0; Protease K with a substrate mass ratio of 1:40; 0.025 μg/μl Trypsin in 50 mM NH4HCO3). Each protease was digested overnight at 37°C.

The resulting peptide is released from the gel section in two steps: the first step is to add 200 μl of a solution containing 5% TFA-50% ACN-45% water (Elastase protease add 100 μl); The second step is to repeat extraction after 1 h in 37°C water bath, ultrasound for 5 min, centrifugation for 5 min, combined extraction solution, vacuum centrifugation drying, and vacuum drying at 45°C after desalting.

Samples were separated by liquid chromatography using a Vanquish Core HPLC system (Thermo Fisher Scientific) with an Acclaim PepMap analytical column (150 μm × 150 mm, 3 μm, 100-A aperture, Thermo Fisher Scientific) in combination with a C18 μ-pre-column (PepMap, Thermo Fisher Scientific). A gradient of solvent A [0.1% formic acid] and solvent B [80% ACN, 0.1% formic acid] was used for separation at a flow rate of 600 nL/min. The main column is initially balanced in A mixture containing 5% solvent B and 95% solvent A. For elution, the percentage of solvent B was increased from 5 to 15% in 5 min and followed by an increase from 15 to 40% over 65 min. The fractions of the main column are eluted directly into the ionization module and further analyzed by MS.

Samples were measured using the Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Fisher Scientific). Primary mass spectrometry obtained a full-scan mass charge ratio (m/z) of 150–2000 in Orbitrap with a resolution of 120,000, automatic gain control set to 400,000 ions, and a maximum fill time of 50 ms. Collision-induced dissociation as a crushing method for a single sample set. In a linear ion trap, automatic gain control is set to 5000 ions and a maximum fill time of 118 ms. For MS/MS fragments, fragmentation spectra were detected on an Orbitrap mass spectrometer with a resolution of 60,000.

The secondary mass spectrum was generated by the PEAKS Studio software. The mass spectrometry raw data have been deposited to the ProteomeXchange Consortium via the iProX partner repository under the dataset identifier PXD06658740,41.

Negative-staining electron microscopy
Five microliters of an aliquot of fibril sample were applied to a glow-discharged 200 mesh carbon support film (Beijing Zhongjingkeyi Technology Co., Ltd.) for 45 s. Then the grid was washed with 5 μl double-distilled water and followed by another wash of 5 μl 3% w/v uranyl acetate. Another 5 μl 3% w/v uranyl acetate was added to the grid to stain the sample for 45 s. After removing the excess buffer by filter paper, the grid was dried by an infrared lamp. The sample imaging was applied by a Talos L120C microscope (Thermo Fisher) operated at 120 KV.

Cryo-electron microscopy
An aliquot of 4 μl extracted fibril sample was applied to a glow-discharged holey carbon grid (Quantifoil R1.2/1.3, 300 mesh), blotted for 6.5 s, and plunge-frozen in liquid ethane using Thermo Fisher Vitrobot Mark IV with 95% humidity at 16°C. The grids were loaded onto a Thermo Fisher Titan Krios microscope operated at 300 kV equipped with a Falcon 4 direct electron detector. EPU software was used for automated data collection according to standard procedures. Magnification at 130,000× was used for imaging, yielding a pixel size of 0.93Å for the images. The defocus range was set from −0.7 to −1.5 μm. Each micrograph was collected under a dose rate of about 13.24 electrons per Å2 per second, with a total exposure time of 3.92 s, resulting in a total dose of about 52 electrons per Å2. A total of 1626 movies were collected.

Helical reconstruction
Image stacks were corrected for beam-induced sample motion and implemented dose weighting using MotionCor242. Contrast transfer function estimation of aligned, dose-weighted micrographs was performed by CTFFIND4.1.843. Subsequent image-processing steps, including manual picking, particle extraction, 2D classification, 3D classification, 3D refinement, and postprocessing, were performed using helical reconstruction methods in RELION3.044. In total, 5786 fibrils were picked manually from 1626 micrographs, and 400 pixel boxes were used to extract particles by a 90% overlap scheme. 2D classification of 400-box-size particles was used to calculate the initial twist angle. In regard to helical rise, 4.8 Å was used as the initial value. After several iterations of 2D and 3D classification, particles with the same morphology were picked out. Local searches of symmetry in 3D classification were used to determine the final twist angle and rise value. A featureless cylinder model created by the relion_helix_toolbox program was used as the initial model; 3D classification was performed several times to generate a proper reference map for 3D refinement. 3D refinement of the selected 3D classes with appropriate reference was performed to obtain the final reconstruction.

The final map of the Polymorph A was convergence with the rise of 2.46Å and the twist angle of −179.58°. Postprocessing was performed to sharpen the map with a B-factor of −46.07 Å2. Based on the gold-standard FSC = 0.143 criteria, the overall resolution was reported as 2.93Å. The final map of Polymorph B was convergence with the rise of 4.92Å and the twist angle of 0.87°. Postprocessing was performed to sharpen the map with a B-factor of −35.29 Å2. The overall resolution was reported as 3.00Å.

Atomic model building and refinement
Atomic models were subsequently built into the refined maps with COOT45,46. The model of the Polymorph A was built based on the model of the cardiac IGLV1-44 fibril (PDB ID: 6IC3). To build the polymorph B model, a single chain from the polymorph A structure was extracted and fitted into the density map of the twist-trimer. For both fibrils, a three-layer model was generated for structure refinement. Structure models were refined by using the real-space refinement program in PHENIX47.

Ethical statement
This study was approved by the Ethical Committee of the First Affiliated Hospital, Zhejiang University School of Medicine ([2025B] IIT Ethics Approval No. 0278) and was performed in accordance with the Declaration of Helsinki. We have written informed consent from the patient to publish clinical information potentially identifying the individual.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Yu et al. provides a concise and clear structural characterization of amyloid fibrils from renal AL amyloidosis derived from the IGLV1-44 light chain. The authors successfully determine high-resolution cryo-EM structures of two fibril polymorphs and compare them with the previously reported heart-derived IGLV1-44 amyloid structure to extract conclusions about conformational differences.\nWhile the core findings are solid and of interest to the field, I believe the manuscript can be significantly improved in both presentation and clarity. Below, I outline two major concerns and several minor points that should be addressed prior to publication.\nMajor Comments\n1. Symmetry of Polymorph A\nThe authors state (P4LN145–148) that \"Polymorph A displays an approximate two-fold helical symmetry with protofilaments staggered along the helical axis.\" This is not accurate. The symmetry appears more consistent with a 2₁ helical axis. Furthermore, the sentence “If not considering the pseudo twofold symmetry, the helical twist of the polymorph A fibril would be 0.84° and the helical rise would be 4.92 Å” again mischaracterizes the symmetry. This is not a \"pseudo twofold\" but rather a (pseudo) 2₁ screw symmetry. Most concerningly, the density map of Polymorph A visually supports a 2₁ symmetry.\nTherefore, the authors should:\n◦ Reprocess the data with 2₁ symmetry and redeposit the maps, or\n◦ Justify in detail why this symmetry was not applied, and convincingly explain their symmetry assignment in the manuscript.\n2. Organ-specific conclusions not supported\nIn several parts of the text (e.g., P1LN34–35 and P7LN240–242), the authors conclude that their results reveal “organ-specific structural variations.” This is not supported by the data. The structural differences highlighted in the manuscript derive from sequence variations, not from the tissue of origin. The authors provide no evidence that the organ environment drives differential assembly.\nPlease revise the abstract and discussion accordingly to reflect that the observed structural differences are sequence-dependent, not organ-specific.\nMinor Comments\n• Title and Section Headings\nThe current title (\"Cryo-EM structure of amyloid fibrils from renal AL amyloidosis\") is too generic and reads more like a review article title. Please revise it to reflect the specific fibril type studied and the key findings. Similarly, the result section titles are generic and should be updated for better scientific clarity, though this is left to the authors’ discretion.\n• Terminology and Acronyms\n◦ P2LN43: Define \"LC\" (light chain) here, and remove the definition from P2LN59. Use “LC” throughout afterward.\n◦ P3LN98, 106: \"Light chain (LC)\" is already defined. Use \"LC\" only.\n◦ P3LN105, P4LN137–139: \"Cryo-electron microscopy (cryo-EM)\" was defined in P3LN91. Please omit redundant definitions.\n◦ P4LN127: Use the correct acronym “IGLV,” not “IGVL.”\n• Negative Staining Description\n◦ P4LN137–139: The logical flow is confusing. Authors jump from cryo-EM to negative stain (NS) and back. Please rewrite for clarity: first describe the NS data, then describe cryo-EM.\n◦ P4LN138–139: The NS data are low-resolution and insufficient to distinguish polymorphs, particularly with measurements taken from single fibrils. Please either:\n1. Provide statistically robust measurements supporting polymorph discrimination in NS micrographs, or\n2. Revise the text to state only that NS shows fibrils with helical symmetry consistent with amyloid.\n• Helical Processing and Figures\n◦ P4LN140–142: Authors incorrectly state they processed “5,786 fibril segments.” This number refers to filaments. Please correct.\n◦ The claim that polymorphs are distinguishable in 2D class averages is not supported by the figure. According to Sup Table 1, the separation occurs during 3D classification. Please revise accordingly.\n◦ P4LN142: Update SI Fig. 3 to include a standard data processing workflow figure, with a representative micrograph, 2D classes, initial model, and final volume. Include all relevant particle numbers and classification steps.\n◦ P4LN144: In SI Fig. 4, add a horizontal FSC = 0 line for clarity.\n• Structural Description and Labeling\n◦ P4LN145: In Fig. 1, the twist angle is incorrectly described for polymorph B (0.87°). Please correct.\n◦ P4LN152–153: Fig. 2a should include a magnified inset of the disulfide bond (Cys22–Cys89). Label Gln16 and Ser95 rather than just “N-” and “C-terminus.”\n◦ P5LN164–165: Call out Suppl. Fig. S3b,c to better support the statement about structural disorder.\n◦ P5LN167: Secondary structure annotation (β1–β9) is better represented in panel 2c. Please refer to it explicitly.\n• Molecular Interactions\n◦ P5LN172–173 and P6LN202: Proline cannot participate in π–π interactions. Likely interactions are CH–π or hydrophobic contacts. Please revise.\n◦ P5LN176: The similarity in β-sheet layering is described qualitatively. Please calculate and report the RMSD of Cα atoms for both protofilament chains.\n◦ P5LN180–181 & P7LN224: Replace “domain” with “region” since these are not independently folded units.\n◦ P6LN205–207: Include a figure (or additional panels in an existing one) illustrating mutant residues (e.g., Tyr37Phe, Asp86His) and their local environments.\n◦ P6LN211: The phrase “providing a rationale for clinical.” is incomplete. Clarify whether this refers to treatment, diagnosis, etc.\n• Topology and Comparative Analysis\n◦ P7LN224–226: The claim about the N→C parallel-to-antiparallel transition requires visual support. Please show this clearly in Fig. 3c/d or a supplementary figure.\n◦ P7LN234–236: The explanation of the cavity formation by Gln39His and Tyr37Phe is not entirely correct. These residues contribute differently: H39 interacts across protofilaments; F37 flips into the core. Please revise for accuracy and include a structural overlay of the renal and heart fibrils to illustrate the folding difference.\n◦ P7LN238: Remove “network”—a single salt bridge is not a network.\n◦ P7LN249: Clarify “Ser25Arg” — is this Ser25 in heart and Arg25 in renal? Please explain.\n◦ P9LN267–268: The statement that “fibril cores are universally formed by the VL domain” is outdated. See:\nSchulte T, et al. Nat Commun. 2024 Jul 28;15(1):6359.\nUpdate the discussion accordingly to reflect this more nuanced understanding.\n• Figures\n◦ Fig. 4A: Label clearly which fibril is from kidney and which from heart.\n◦ Include a structural overlap between the renal and heart IGLV1-44 fibrils to illustrate folding differences.\n• Methods Clarity\n◦ In “Helical reconstruction,” the initial model generation is unclear. Please specify the software and protocol used to create the initial volume.\nWhile I find the manuscript scientifically sound and the structures relevant to the field, several issues must be addressed—especially regarding the interpretation of symmetry and the overstated conclusion about organ specificity. Once these points are corrected, I believe the manuscript will be suitable for publication in Nature Communications.\nSincerely,\nAntonio Chaves-Sanjuan", "reviewer_2": "The manuscript “Cryo-EM structure of amyloid fibrils from renal AL amyloidosis” by Yu et.al present a structural analysis of fibrils from mutated IGLV 1-44 protein by cryo electron microscopy. Similar studies published previously report high-resolution Cryo-EM structures of immunoglobulin light chain fibrils associated with cardiac amyloidosis, originating from various lambda germline gene segments (Swuec et al., 2019; Radamaker et al., 2019; Radamaker et al., 2021 [two articles]; Schulte et al., 2024).\nIn the current manuscript, the authors identify and characterize two structurally distinct fibril polymorphs from renal tissue samples, resolved at up to 3.0 Å resolution. They describe the chemical interactions that stabilize the fibril cores and analyze the surface charge distributions. Additionally, they highlight key structural differences between IGLV 1-44 derived fibrils in renal versus cardiac amyloidosis. Based on these differences, the authors propose potential mechanisms underlying organ tropism and tissue-specific pathogenic pathways of fibril deposition.\nWhile the study contributes valuable structural insights, certain aspects of the manuscript could be improved to better distinguish it from existing literature and ensure it does not appear incremental relative to prior Cryo-EM studies on fibrils from systemic amyloidosis.\n1. The manuscript appropriately focuses on the structural analysis of fibrils using Cryo-EM, the study also touches on organ-specific deposition patterns and pathogenic mechanisms. These aspects, however, are addressed only superficially and lack clear integration with the structural findings. The authors should explicitly state the primary goals of the study at the outset and clarify how the structural data are intended to support these conclusions.\n2. Line 203-205 – It is unclear what the aggregation kinetics are being compared against. Are the authors referring to aggregation kinetics derived from cardiac derived IGLV 1-44 fibrils?\n3. Line 210-211 – Detecting mutation profiles………: this statement needs further elaboration. How could specific mutations influence therapeutic choices?\n4. Given the significance of in vitro systems in studying aggregation kinetics and therapeutic screening, it would be valuable to compare the structure of in vitro fibrils (seeded with renal tissue-derived fibrils) to the ex vivo structures presented. Such a comparison could further enhance the understanding of in vitro models in replicating disease-relevant fibril structures.\n5. Lines 237-242 – The discussion on changes in residues involved in the formation of intermolecular salt bridges leading to organ-specific deposition patterns is intriguing but are these changes sufficient to explain the observed tropism?\n6. The role of surface charge and hydrophobicity in fibril formation and stability must be elaborated. How do these physicochemical properties in renal IGLV 1-44 fibrils compare with those found in cardiac-derived fibrils?\n7. What is structure shown in figure 3d? The PDB:6IC3 mentioned belongs to cardiac IGLV 1-44 fibrils and not natively folded LC variable domain. It is difficult to ascertain the N , C -terminus orientation of the native LC from fig 3d. The figure should be revised for accuracy, and the legend should be corrected accordingly.\n8. What is the key step in the misfolding of the natively folded light chain proteins that initiate fibril formation and arrangements into polymorph A and B?\nMinor points: Check the manuscript for typographical errors – especially in the references."}, "author_response": "Point-by-point response to the reviewers’ comments:\n* The reviewer’s comments are in Italic. Author’s responses are in blue. All revisions\nare highlighted in yellow in the revised manuscripts.\nReviewers' comments:\nReviewer #1:\nThe manuscript by Yu et al. provides a concise and clear structural characterization\nof amyloid fibrils from renal AL amyloidosis derived from the IGLV1-44 light chain.\nThe authors successfully determine high-resolution cryo-EM structures of two fibril\npolymorphs and compare them with the previously reported heart-derived IGLV1-44\namyloid structure to extract conclusions about conformational differences.\nWhile the core findings are solid and of interest to the field, I believe the manuscript\ncan be significantly improved in both presentation and clarity. Below, I outline two\nmajor concerns and several minor points that should be addressed prior to\npublication.\nREPLY: We sincerely thank this reviewer for the positive assessment of this work. We\nespecially thank the reviewer for his/her insightful suggestions provided below, which\nare of great help to improve this manuscript.\nMajor Comments:\n1. Symmetry of Polymorph A\nThe authors state (P4LN145–148) that \"Polymorph A displays an approximate two-\nfold helical symmetry with protofilaments staggered along the helical axis.\" This is\nnot accurate. The symmetry appears more consistent with a 2₁ helical axis.\nFurthermore, the sentence “If not considering the pseudo twofold symmetry, the\nhelical twist of the polymorph A fibril would be 0.84° and the helical rise would be\n4.92 Å” again mischaracterizes the symmetry. This is not a \"pseudo twofold\" but\nrather a (pseudo) 2₁ screw symmetry. Most concerningly, the density map of\nPolymorph A visually supports a 2₁ symmetry.\nTherefore, the authors should:\n◦ Reprocess the data with 2₁ symmetry and redeposit the maps, or\n◦ Justify in detail why this symmetry was not applied, and convincingly explain their\nsymmetry assignment in the manuscript.\nREPLY: We thank the reviewer for this insightful and critical comment regarding the\nsymmetry of the Polymorph A. The reviewer is absolutely correct. We apologize for\nthe inaccuracies in our initial symmetry description.\nFollowing the reviewer's recommendation, we have taken the following actions:\n1. Data Reprocessing: We have accordingly reprocessed the cryo-EM data for\nPolymorph A by imposing a 2₁ helical symmetry during the final 3D reconstruction.\nThis has resulted in a significantly improved density map with better-defined\nfeatures.\n2. Map and Model Redeposition: The newly reconstructed map and the refined atomic\nmodel, which now strictly adheres to the 2₁ symmetry, have been redeposited in the\nrespective public databases. The updated accession codes (or PDB/EMDB IDs) are\nnow provided in the revised manuscript.\n3. Manuscript Revision: The text has been revised throughout to correctly describe the\n2₁ screw symmetry, and all relevant figures and parameters have been updated.\nWe believe that the application of the correct symmetry, as suggested by the\nreviewer, has strengthened the quality and accuracy of our structural model and the\noverall manuscript. We are grateful for the reviewer's expertise, which has been\ninvaluable in correcting this important aspect of our work.\n“……Consistent with the density map, polymorph A fibril displays a (pseudo) 2₁ screw\nsymmetry, characterized by a helical twist of -179.58° and a rise of 2.46 Å……”\n“……we resolved contain two protofilaments: polymorph A fibril displays a (pseudo) 2₁\nscrew symmetry, while polymorph B displays C1 helical symmetry……”\n2. Organ-specific conclusions not supported\nIn several parts of the text (e.g., P1LN34–35 and P7LN240–242), the authors\nconclude that their results reveal “organ-specific structural variations.” This is not\nsupported by the data. The structural differences highlighted in the manuscript derive\nfrom sequence variations, not from the tissue of origin. The authors provide no\nevidence that the organ environment drives differential assembly.\nPlease revise the abstract and discussion accordingly to reflect that the observed\nstructural differences are sequence-dependent, not organ-specific.\nREPLY: We thank the reviewer for this critical comment. We agree that the term\n“organ-specific structural variations” was inaccurate and could be misleading, as it\nmight imply that the organ environment directly drives the assembly of different fibril\nstructures. This was not our intention.\nAs the reviewer correctly pointed out, our data demonstrate that the structural\ndifferences originate from sequence variations (i.e., the mutations). Our intended\nmeaning was that these sequence-dependent structural variations could, in turn,\ninfluence the organ tropism (i.e., the propensity to deposit in specific organs) of the\namyloid fibrils.\nTo accurately reflect this, we have revised the text to use “organ-tropism” and\nrelated phrases in the abstract, introduction and discussion.\n“……Despite significant advances in understanding amyloid fibril structure in AL\namyloidosis, the molecular mechanism of organ-tropism in fibril deposition remains\nunclear……”\n“……These clinical challenges underscore the urgent need for advances in understanding\nthe molecular pathogenesis of the disease and the mechanisms underlying organ-tropism\namyloid deposition.”\n“……These findings reveal pronounced molecular assembly differences between AL\namyloid fibrils derived from distinct tissues (Fig. 4c), establishing a structural basis for how\nmutation-driven fibril polymorphism contributes to organ tropism.”\nMinor Comments：\n1. Title and Section Headings\nThe current title (\"Cryo-EM structure of amyloid fibrils from renal AL amyloidosis\") is\ntoo generic and reads more like a review article title. Please revise it to reflect the\nspecific fibril type studied and the key findings. Similarly, the result section titles are\ngeneric and should be updated for better scientific clarity, though this is left to the\nauthors’ discretion.\nREPLY: We are grateful to the reviewer for their constructive feedback, which has\nhelped us improve the clarity and impact of our manuscript.\nRegarding the manuscript title:\nWe completely agree that the previous title was too generic. As suggested, we have\nrevised it to specifically highlight the key fibril type and our major finding. The new\ntitle is:\n“Cryo-EM structure of renal AL amyloid fibrils from a patient with λ1 light chain\namyloidosis”\nWe believe this new title is more precise, informative, and better reflects the\nspecific structural insights presented in our study.\nRegarding the result section titles:\nWe are grateful to the reviewer for this valuable feedback. We agree that the\noriginal section titles were too generic. Accordingly, we have updated them to precisely\narticulate the scientific advances in each section. We are confident that the new titles\nprovide a much clearer and more compelling guide for the reader.\n“1、Extraction and sequence analysis of amyloid fibrils from a patient renal biopsy\n2、Cryo-EM structural determination of the renal AL fibril\n3、Salt bridges and hydrophobic interactions mediate stability in the fibril protein fold\n4、Implication of patient-specific mutations for fibril morphology\n5、Comparative analysis with cardiac fibrils and native light chain structures”\nWe hope that our revised title, combined with the enhanced clarity in the result\nnarratives, fully addresses the reviewer's concern and improves the overall scientific\nclarity of the manuscript.\n2. Terminology and Acronyms\n1) P2LN43: Define \"LC\" (light chain) here, and remove the definition from P2LN59.\nUse “LC” throughout afterward.\n2) P3LN98, 106: \"Light chain (LC)\" is already defined. Use \"LC\" only.\n3) P3LN105, P4LN137–139: \"Cryo-electron microscopy (cryo-EM)\" was defined in\nP3LN91. Please omit redundant definitions.\n4) P4LN127: Use the correct acronym “IGLV,” not “IGVL.”\nREPLY: We apologize for the mistake. They have been corrected in the revised\nmanuscript. Proofreading has also been applied.\n1)We defined \"LC\" (light chain) here at the very beginning, and \"LC\" was directly used\nin the subsequent content.\n“Systemic light-chain (AL) amyloidosis is a disorder characterized by the abnormal\naggregation of immunoglobulin light chains (LCs) into oligomers and amyloid fibrils……”\n“……As a result, the LC sequences derived from either the λ or κ loci are nearly unique\nto each individual patient……”\n2)We modified the original text and directly used LC.\n“……highlighting the immunoglobulin LC sequence as a key determinant of fibril\nstructure……”\n“……we employed cryo-EM to determine the high-resolution three-dimensional structure\nof LC amyloid fibrils extracted from the kidney tissue of a patient with AL amyloidosis……”\n3)We have corrected the errors in the original manuscript.\n“……we employed cryo-EM to determine the high-resolution three-dimensional structure\nof LC amyloid fibrils extracted from the kidney tissue of a patient with AL amyloidosis……”\n“……To determine the atomic structures, we subsequently employed cryo-EM……”\n4)We have corrected the errors in the original manuscript.\n“……Previous studies have demonstrated that the immunoglobulin light chain variable\nregion (IGLV) is associated with organ tropism……”\n3. Negative Staining Description\n1) P4LN137–139: The logical flow is confusing. Authors jump from cryo-EM to\nnegative stain (NS) and back. Please rewrite for clarity: first describe the NS data, then\ndescribe cryo-EM.\nREPLY: We thank the reviewer for pointing out the issue with the logical flow. We\nhave rewritten the section as suggested, now presenting the NS data first to establish\nthe initial morphological characterization, followed by the high-resolution structural\ninsights from cryo-EM. This restructuring creates a more coherent narrative from initial\nscreening to high-resolution structure determination. We have revised the manuscript\naccordingly.\n“To investigate the structure of the extracted fibrils, we first performed negative-staining\nTEM. This initial analysis revealed the presence of fibrils with the characteristic helical\nsymmetry of amyloid but was insufficient to resolve high-resolution details (SI Fig. 2). To\ndetermine the atomic structures, we subsequently employed cryo-EM. Following cryo-EM data\ncollection, we used RELION for helical reconstruction of this fibril species. Three-dimensional\n(3D) classification from helical image processing of 5,786 filaments revealed two major\npolymorphs of twisted fibrils, herein designated as polymorphs A and B (SI Fig. 3)”\n2) P4LN138–139: The NS data are low-resolution and insufficient to distinguish\npolymorphs, particularly with measurements taken from single fibrils.\nPlease either:\n1. Provide statistically robust measurements supporting polymorph discrimination in\nNS micrographs, or\n2. Revise the text to state only that NS shows fibrils with helical symmetry consistent\nwith amyloid.\nREPLY: We sincerely thank the reviewer for this critical observation. We agree that\nthe previous narrative flow was suboptimal and could cause confusion. As suggested,\nwe have entirely restructured the Results section to present a linear and logical\nprogression of our experimental workflow. We now first describe the NS-TEM data\n(which served as an initial quality check and morphological assessment), followed by\nthe detailed presentation of the cryo-EM data (which provided the high-resolution\ninformation for structural analysis and polymorph distinction). Meanwhile, we have\nalso made corresponding modifications to SI fig2. We believe this revision significantly\nimproves the clarity and readability of the manuscript.\n“To investigate the structure of the extracted fibrils, we first performed negative-staining\nTEM. This initial analysis revealed the presence of fibrils with the characteristic helical\nsymmetry of amyloid but was insufficient to resolve high-resolution details (SI Fig. 2). To\ndetermine the atomic structures, we subsequently employed cryo-EM. Following cryo-EM data\ncollection, we used RELION for helical reconstruction of this fibril species. Three-dimensional\n(3D) classification from helical image processing of 5,786 filaments revealed two major\npolymorphs of twisted fibrils, herein designated as polymorphs A and B (SI Fig. 3).”\nFigure S2 Negative-staining TEM images reveal the characteristic helical symmetry of\nIGLV1-44 fibrils.\n4. Helical Processing and Figures\n1) P4LN140–142: Authors incorrectly state they processed “5,786 fibril segments.”\nThis number refers to filaments. Please correct.\nREPLY: We apologize for the mistake. It has been corrected in the revised manuscript.\nProofreading has also been applied.\n“Two-dimensional (2D) and three-dimensional (3D) classification from helical image\nprocessing of 5,786 filaments revealed two major polymorphs of twisted fibrils upon 3D\nanalysis (SI Fig. 3).”\n2) The claim that polymorphs are distinguishable in 2D class averages is not supported\nby the figure. According to Sup Table 1, the separation occurs during 3D classification.\nPlease revise accordingly.\nREPLY: We thank the reviewer for this critical insight and for highlighting the need\nfor greater precision in our data interpretation. We fully agree that the distinction\nbetween the two polymorphs is conclusively demonstrated by the 3D classification\nresults, as correctly noted in Supplementary Table 1, and not by 2D class averages alone.\nAs suggested, we have revised the text on page4 to accurately reflect the source of this\nconclusion. The sentence now clearly states that the two polymorphs were identified\nthrough “upon 3D analysis”. We apologize for the confusion.\n“……Two-dimensional (2D) and three-dimensional (3D) classification from helical image\nprocessing of 5,786 filaments revealed two major polymorphs of twisted fibrils upon 3D\nanalysis (SI Fig. 3).”\n3) P4LN142: Update SI Fig. 3 to include a standard data processing workflow figure,\nwith a representative micrograph, 2D classes, initial model, and final volume. Include\nall relevant particle numbers and classification steps.\nREPLY: We thank the reviewer for this excellent suggestion. We have now updated\nSI Fig. 3 as recommended. The new figure provides a comprehensive workflow,\nincluding: (i) a representative raw micrograph, (ii) representative 2D class averages,\n(iii) the initial model, and (iv) the final reconstructed volume. We have also included a\ndetailed flowchart that outlines the data processing steps, along with the corresponding\nparticle numbers at each classification stage. This revision offers a clearer and more\ncomplete overview of our data processing strategy.\nFigure S3 Work flow of steps of the reconstruction process. A representative micrograph\nand 2D class average are shown at the top left and top right, respectively. Scale bars are shown\nin the pictures. Data statistics, no. of micrographs, and no. of particles used in image\nprocessing are listed. Based on an initial reconstruction, 3D classification separated two\nsubsets exhibiting different density paths. Through several further classification and refinement\nsteps, the final reconstructions were obtained.\n4) P4LN144: In SI Fig. 4, add a horizontal FSC = 0 line for clarity.\nREPLY: Following the reviewer’s suggestion, we added the horizontal FSC = 0 line\nfor clarity as shown in SI Fig. 4.\nFigure S4 FSC curve of the reconstructed density\n5. Structural Description and Labeling\n1) P4LN145: In Fig. 1, the twist angle is incorrectly described for polymorph B (0.87°).\nPlease correct.\nREPLY: We sincerely apologize for the mistake. We have now corrected Figure 1 with\nthe accurate value of 0.87° for polymorph B.\n2) P4LN152–153: Fig. 2a should include a magnified inset of the disulfide bond\n(Cys22–Cys89). Label Gln16 and Ser95 rather than just “N-” and “C-terminus.”\nREPLY: We thank the reviewer for their insightful suggestion, which has significantly\nimproved the clarity and informational value of Figure 2. As suggested, we have\nincluded a new inset panel that provides a clear, close-up view of the disulfide bond\nbetween Cys22 and Cys89. Additionally, the generic “N-terminus” and “C-terminus”\nlabels have been replaced with the specific residue names “Gln16” and “Ser95”,\nrespectively, to provide more precise structural annotation.\n3) P5LN164–165: Call out Suppl. Fig. S3b,c to better support the statement about\nstructural disorder.\nREPLY: We are grateful to the reviewer for this comment. In response, and following\nour update to the workflow in Figure S3, we have revised the text to cite this updated\nfigure. The revised figure provides direct visual support for the described structural\ndisorder.\n“……The N- and C-termini of the protein were surrounded by diffuse density (SI Fig.\n3) ……”\n4) P5LN167: Secondary structure annotation (β1–β9) is better represented in panel 2c.\nPlease refer to it explicitly.\nREPLY: We thank the reviewer for this insightful suggestion. We have now explicitly\nreferenced Panel 2c in the relevant section of the Results part to ensure clarity and direct\nreaders to this improved visualization.\n“……we designated as β1 through β9, respectively (Fig. 2c, d) ……”\n6. Molecular Interactions\n1) P5LN172–173 and P6LN202: Proline cannot participate in π–π interactions. Likely\ninteractions are CH–π or hydrophobic contacts. Please revise.\nREPLY: We sincerely thank the reviewer for catching this important inaccuracy and\nfor providing the correct explanation. We fully agree with the reviewer that proline is\nnot capable of engaging in π-π stacking interactions. We have revised the manuscript\naccordingly to remove all incorrect references to π-π stacking involving Pro41.\n“……Polymorph A’s interface is stabilized by two pairs of inter-chain aromatic residue\ninteractions, specifically a symmetric CH-π stacking between His39-Pro41 and Pro41-His39,\nmarkedly enhancing interface stability (Fig. 2a). In contrast, polymorph B maintains\nprotofilament interactions through a single CH-π interaction between His39 and Pro41, with\nan interface topology exhibiting asymmetric features……”\n“……enabling a cross-chain CH-π stacking interaction with the adjacent Pro41 residue\nof a neighboring protofilament subunit……”\n2) P5LN176: The similarity in β-sheet layering is described qualitatively. Please\ncalculate and report the RMSD of Cα atoms for both protofilament chains.\nREPLY: We thank the reviewer for this excellent suggestion. We have now calculated\nthe Cα RMSD between the two protofilaments to quantitatively assess their structural\nsimilarity. Following structural superposition, the RMSD for Cα atoms between the two\nprotofilaments is 0.001 Å. This very low value provides strong quantitative support for\nour original statement that the core structures of the protofilaments are virtually\nidentical. We have revised the manuscript to include this new data.\n“……Notably, both polymorphs display an identical β-sheet layering pattern within the\ncore of individual protofilaments, which is underscored by a very low Cα RMSD of 0.001 Å\nbetween the two protofilaments……”\n3) P5LN180–181 & P7LN224: Replace “domain” with “region” since these are not\nindependently folded units.\nREPLY: We apologize for the confusion. We agree entirely that the term \"region\" is\nmore accurate than \"domain\" for describing these segments, as they are not\nindependently folded structural units. We have carefully gone through the manuscript\nand replaced all instances where \"domain\" was used inaccurately with the more\nappropriate term \"region\".\n“……The N-terminal region is dominated by positive electrostatic potential (blue),\nwhereas the C-terminal region exhibits a negative potential (red) (Fig. 3a) ……”\n“……which covalently links the N- and C-terminal regions of the protein ……”\n4) P6LN205–207: Include a figure (or additional panels in an existing one) illustrating\nmutant residues (e.g., Tyr37Phe, Asp86His) and their local environments.\nREPLY: We thank the reviewer for the comment. The local environments of the\nTyr37Phe and Asp86His mutant residues, including their salt-bridge networks, are\nalready illustrated in Figure 3b. We have now inserted a reference to Figure 3b in the\nrelevant section of the text to more prominently highlight these specific residues and\ntheir interactions, thereby enhancing clarity. Meanwhile, we have also added magnified\nlocal environment maps of the mutant residues (Tyr37Phe, Asp86His) in fig 3b to more\nclearly display various interactions.\n“……The Tyr37Phe mutation (Fig. 3b) may strengthen the van der Waals forces and\nhydrophobic synergism between β-sheet layers of the fibril, further stabilizing the fibril.\nAdditionally, the Asp86His mutation (Fig. 3b) can form a salt bridge with Glu84, reinforcing\nthe internal fibril architecture ……”\n5) P6LN211: The phrase “providing a rationale for clinical.” is incomplete. Clarify\nwhether this refers to treatment, diagnosis, etc.\nREPLY: We apologize for the confusion. We have revised the sentence to complete\nthe thought. The sentence now clearly states that this approach provides a rationale for\nfirst-line treatment options in clinical practice.\n“……Detecting mutation profiles in patient light chains could therefore guide the\nselection of targeted therapies, providing a rationale for first-line treatment options in clinical\npractice. For instance, if a mutation profile closely aligns with that characteristic of cardiac-\nderived fibrils, a cardiotropic treatment strategy would be prioritized. Conversely, the detection\nof a mutation profile associated with renal amyloidosis would justify a renally targeted\napproach. However, it is important to note that the existing maps of organ-specific mutational\nsignatures remain incomplete. This evolving framework for mutational profiling nonetheless\nallows for a more personalized and biologically rational selection of first-line therapies,\nultimately aiming to improve treatment outcomes.”\n7. Topology and Comparative Analysis\n1) P7LN224–226: The claim about the N→C parallel-to-antiparallel transition\nrequires visual support. Please show this clearly in Fig. 3c/d or a supplementary figure.\nREPLY: We thank the reviewer for this important suggestion. We fully agree that\nproviding direct visual support for the key finding of the “N→C parallel-to-antiparallel\ntransition” is crucial for readers to appreciate the extent of the conformational\nrearrangement. As recommended, we have created a new Supplementary Figure (SI Fig.\n6), which provides a side-by-side structural comparison of the natively folded IGLV1-\n44 and the renal IGLV1-44 fibril. We are confident that this new supplementary figure\nprovides direct structural evidence for our claim of a “parallel-to-antiparallel transition,”\nmaking it immediately clear and incontrovertible. We have now referenced this figure\nin the main text and discuss this finding. We thank you again for this valuable comment,\nwhich has significantly improved the clarity and impact of our manuscript.\n2) P7LN234–236: The explanation of the cavity formation by Gln39His and Tyr37Phe\nis not entirely correct. These residues contribute differently: H39 interacts across\nprotofilaments; F37 flips into the core. Please revise for accuracy and include a\nstructural overlay of the renal and heart fibrils to illustrate the folding difference.\nREPLY: We thank the reviewer for this insightful comment and for highlighting the\nneed for a more precise mechanistic description. The reviewer is absolutely correct. We\nhave now revised the text to accurately describe the distinct roles of these two residues:\nthe Gln39His mutation primarily facilitates inter-protofilament interactions, while the\nTyr37Phe mutation allows the residue to flip into the protein core. We now clearly state\nthat the combined effect of these two distinct mechanisms is the formation of the\nenlarged cavity.\n“……Further structural analysis delineates the mechanisms by which the Gln39His and\nTyr37Phe mutations reshape the fibril core: Gln39His mediates inter-protofilament contacts,\nwhile Tyr37Phe causes a inward flipping of the residue. This dual rearrangement culminates\nin the formation of a larger internal cavity specific to the renal fibril.”\nAdditionally, as requested, we have included a new structural overlay comparing\nthe renal and heart fibrils to visually underscore this folding difference. This new panel\nhas been added as Figure 4c. We believe these revisions have significantly improved\nthe accuracy and clarity of our structural description.\n3) P7LN238: Remove “network”—a single salt bridge is not a network.\nREPLY: We thank the reviewer for this precise correction. We agree that the term\n\"network\" is inaccurate for describing a single salt bridge. We have therefore removed\nthe word \"network\" throughout the manuscript to improve scientific accuracy.\n“……forms a stable intermolecular salt bridge with the neighboring Glu84 residue……”\n4) P7LN249: Clarify “Ser25Arg” — is this Ser25 in heart and Arg25 in renal? Please\nexplain.\nREPLY: We thank the reviewer for raising this point, which allows us to clarify an\nimportant detail. The nomenclature \"Ser25Arg\" does not refer to a difference between\ntissues (e.g., heart vs. renal). Instead, it indicates a somatic mutation from serine (Ser)\nto arginine (Arg) at position 25 when comparing the amyloid fibril sequence (derived\nfrom the heart in this case) to the IGLV1-44 germ line sequence.\nTo prevent any confusion for the reader, we have revised the manuscript to\nexplicitly state this.\n“……whereas in the cardiac fibril, structural stabilization is mediated by a salt bridge\ninteraction between Asp86 and Arg25……”\n5) P9LN267–268: The statement that “fibril cores are universally formed by the VL\ndomain” is outdated. See:\nSchulte T, et al. Nat Commun. 2024 Jul 28;15(1):6359.\nUpdate the discussion accordingly to reflect this more nuanced understanding.\nREPLY: We thank the reviewer for this insightful comment. We agree that the\nstatement “fibril cores are universally formed by the VL domain of precursor λ-light\nchains” is overly absolute and does not reflect recent structural advances. As rightly\npointed out by the reviewer, the study by Schulte et al. (2024, Nat Commun) presents\nthe first cryo-EM structure of an AL amyloid fibril (AL59) that incorporates an\nextended fragment of the constant domain (CL) within its core. We thank the reviewer\nfor bringing this important point to our attention, which has helped us improve the\nmanuscript.\nWe have modified the sentence to reflect this more nuanced understanding. The\nrevised text now reads:\n“……fibril cores are typically formed by the VL domain of precursor λ-light chains,\nalthough emerging evidence demonstrates that the constant domain (CL) can also be\nincorporated into the core structure in some cases ……”\n8. Figures\n1) Fig. 4A: Label clearly which fibril is from kidney and which from heart.\nREPLY: Thank you for this important comment. We agree that clearly labeling the\ntissue origin of the fibrils is essential for the reader's understanding of the experimental\nresults. As suggested, we have revised Figure 4a and 4b. The panels now include clear\ntext labels directly on the images to unambiguously indicate the origin of each fibril or\nstructural model. This modification ensures that readers can easily distinguish between\nthe structures of the fibrils from different tissue origins, avoiding any potential\nconfusion. We thank you again for helping us improve the clarity and precision of our\nmanuscript.\n2) Include a structural overlap between the renal and heart IGLV1-44 fibrils to\nillustrate folding differences.\nREPLY: We thank the reviewer for this valuable comment. We agree that a structural\noverlap between the renal and heart IGLV1-44 fibrils is crucial to illustrate their folding\ndifferences, as this provides a direct visual and quantitative comparison of their atomic-\nlevel structures. As suggested, we have modified the manuscript accordingly.\nSpecifically, we have added a new panel (Figure 4c) to Figure 4, which presents a\nstructural alignment of the renal (colored in light pink) and heart (colored in pale yellow)\nIGLV1-44 fibrils. This structural overlap clearly demonstrates that while the two fibrils\nshare some basic architectural features, there are significant conformational differences,\nparticularly in key loop regions.\n9. Methods Clarity\n1) In “Helical reconstruction,” the initial model generation is unclear. Please specify\nthe software and protocol used to create the initial volume.\nREPLY: We sincerely thank the reviewer for pointing out this critical omission and\nthe lack of clarity in our original description. The reviewer is absolutely correct. We\napologize for the error in our initial manuscript, which incorrectly stated that \"The 3D\ninitial model was built by selected 2D classes\". This was a mistake in the method\ndescription. To address this comment, we have now corrected the manuscript as follows:\nThe initial model for helical reconstruction was, in fact, a featureless cylinder\ngenerated de novo using the relion_helix_toolbox program (a part of the RELION\nsoftware suite), not from selected 2D classes. This is a standard and robust approach\nfor ab initio helical reconstruction to avoid potential reference bias.\nWe have now revised the Methods section to precisely state: \"An initial featureless\ncylinder model was created using the relion_helix_toolbox program within RELION-\n3.1 and used as the reference for subsequent 3D classification and refinement.\"\nFurthermore, to enhance clarity and transparency, we have also updated the\nsupplementary Figure S3 (the workflow figure) to explicitly include a visualization of\nthis featureless cylinder initial model in the diagram, as suggested by the reviewer's\ncomment. This provides a clear, visual representation of the starting point for our helical\nprocessing pipeline.\nWe believe these corrections have now accurately and clearly specified the\nsoftware and protocol used for initial model generation, and we thank the reviewer\nagain for ensuring the methodological rigor of our work.\n“……A featureless cylinder model created by the relion_helix_toolbox program was used\nas initial model; 3D classification was performed several times to generate a proper reference\nmap for 3D refinement……”\nReviewer #2 (Remarks to the Author):\nThe manuscript “Cryo-EM structure of amyloid fibrils from renal AL amyloidosis”\nby Yu et.al present a structural analysis of fibrils from mutated IGLV 1-44 protein by\ncryo electron microscopy. Similar studies published previously report high-resolution\nCryo-EM structures of immunoglobulin light chain fibrils associated with cardiac\namyloidosis, originating from various lambda germline gene segments (Swuec et al.,\n2019; Radamaker et al., 2019; Radamaker et al., 2021 [two articles]; Schulte et al.,\n2024).\nIn the current manuscript, the authors identify and characterize two structurally\ndistinct fibril polymorphs from renal tissue samples, resolved at up to 3.0 Å resolution.\nThey describe the chemical interactions that stabilize the fibril cores and analyze the\nsurface charge distributions. Additionally, they highlight key structural differences\nbetween IGLV 1-44 derived fibrils in renal versus cardiac amyloidosis. Based on these\ndifferences, the authors propose potential mechanisms underlying organ tropism and\ntissue-specific pathogenic pathways of fibril deposition.\nWhile the study contributes valuable structural insights, certain aspects of the\nmanuscript could be improved to better distinguish it from existing literature and\nensure it does not appear incremental relative to prior Cryo-EM studies on fibrils from\nsystemic amyloidosis.\nREPLY: We are grateful to the reviewer for their thoughtful assessment and\nconstructive comments, which have helped strengthen our manuscript. We also\nappreciate the chance to elaborate on how our study provides novel and distinct\ncontributions to the field.\n1. The manuscript appropriately focuses on the structural analysis of fibrils using Cryo-\nEM, the study also touches on organ-specific deposition patterns and pathogenic\nmechanisms. These aspects, however, are addressed only superficially and lack clear\nintegration with the structural findings. The authors should explicitly state the primary\ngoals of the study at the outset and clarify how the structural data are intended to\nsupport these conclusions.\nREPLY: We thank the reviewer for this insightful comment. We agree that the term\n\"organ-specific\" was inaccurate in this context. Our intention was to convey that the\nmutation alters the LC sequence, which in turn leads to distinct amyloid fibril structures\nin the heart and kidneys. These structural differences ultimately contribute to differing\norgan tropism of the fibrils, resulting in their deposition in different organs. We\nsincerely appreciate the reviewer’s feedback and have revised the terms \"organ-\nspecific\" to \"organ tropism\" in both the Abstract and Discussion sections to better\nreflect our intended meaning.\n“……Despite significant advances in understanding amyloid fibril structure in AL\namyloidosis, the molecular mechanism of organ-tropism in fibril deposition remains\nunclear……”\n“……These findings reveal pronounced molecular assembly differences between AL\namyloid fibrils derived from distinct tissues (Fig. 4c), establishing a structural basis for how\nmutation-driven fibril polymorphism contributes to organ tropism.”\n2. Line 203-205 – It is unclear what the aggregation kinetics are being compared\nagainst. Are the authors referring to aggregation kinetics derived from cardiac derived\nIGLV 1-44 fibrils?\nREPLY: We thank the reviewer for this question, which allows us to clarify an\nimportant point. The comparison of aggregation kinetics is made against the native,\nnon-amyloidogenic state of the protein subunits themselves, not against fibrils derived\nfrom other organs. This benchmark is used to demonstrate how the identified interaction\nenhances the propensity for aggregation relative to the protein's normal state. We\napologize for the lack of clarity in the original manuscript and have revised the text to\nexplicitly state \"…compared to their native state\" to prevent any future\nmisunderstanding.\n“……This interaction facilitates the formation of a unique fibril interface, leading to a\ndimer architecture distinct from other amyloid fibrils. It thereby constitutes a core structural\nbasis for IGLV1-44 fibril assembly and enhances the aggregation kinetics of the protein\nsubunits compared to their native or non-amyloidogenic states ……”\n3. Line 210-211 – Detecting mutation profiles………: this statement needs further\nelaboration. How could specific mutations influence therapeutic choices?\nREPLY: We thank the reviewer for raising this important point. We agree that the\nconnection between mutations and therapy should be made more explicit. We have now\nelaborated on this in the manuscript. The revised text now explains how mutations that\ndictate fibril structure could influence the efficacy of amyloid-depleting therapies and\naid in personalizing treatment strategies.\n“……Detecting mutation profiles in patient light chains could therefore guide the\nselection of targeted therapies, providing a rationale for first-line treatment options in clinical\npractice. For instance, if a mutation profile closely aligns with that characteristic of cardiac-\nderived fibrils, a cardiotropic treatment strategy would be prioritized. Conversely, the detection\nof a mutation profile associated with renal amyloidosis would justify a renally targeted\napproach. However, it is important to note that the existing maps of organ-specific mutational\nsignatures remain incomplete. This evolving framework for mutational profiling nonetheless\nallows for a more personalized and biologically rational selection of first-line therapies,\nultimately aiming to improve treatment outcomes.”\n4. Given the significance of in vitro systems in studying aggregation kinetics and\ntherapeutic screening, it would be valuable to compare the structure of in vitro fibrils\n(seeded with renal tissue-derived fibrils) to the ex vivo structures presented. Such a\ncomparison could further enhance the understanding of in vitro models in replicating\ndisease-relevant fibril structures.\nREPLY: We thank the reviewer for their insightful suggestion. We fully agree that a\ndirect comparison between in vitro-generated fibrils and those derived from renal\ntissues would strengthen the relevance of our in vitro model.\nIn our in vitro experiments, we employed kidney tissue-derived seeds to induce\nfibril formation under various conditions such as different pH levels, salt concentrations,\nand temperatures. Although this approach yielded a diversity of fibrillar morphologies,\nthese structures differed from patient-derived fibrils, suggesting that chemical\nmodifications within the physiological microenvironment play a crucial role in shaping\nfibril architecture.\nAfter multiple attempts, these in vitro fibrils exhibited heterogeneous morphology\nunder negative-stain EM imaging, as shown in the figure below. Our negative-stain EM\ndata revealed that the in vitro fibrils are structurally heterogeneous, with poorly defined\nhelical symmetry, pronounced twisting, and a tendency to aggregate. These properties\nhindered homogeneous particle alignment and prevented high-resolution structure\ndetermination by cryo-EM. This suggests that, unlike their in vivo counterparts which\nbenefit from tissue-specific microenvironments, fibrils formed in vitro may lack the\nstructural homogeneity required for such analysis. We thank the reviewer again for\nemphasizing this valuable point.\n5. Lines 237-242 – The discussion on changes in residues involved in the formation of\nintermolecular salt bridges leading to organ-specific deposition patterns is intriguing\nbut are these changes sufficient to explain the observed tropism?\nREPLY: We agree with the reviewer's insightful comment that organ tropism is\nundoubtedly multifactorial. Our data demonstrate that these changes are a powerful and\npreviously delineated structural feature that can predispose fibrils to deposit in specific\norgans by altering their surface electrostatic potential. While we do not claim this to be\nthe exclusive factor, we posit it as a fundamental one that establishes a structural basis\nfor electrostatic interactions with the tissue microenvironment. Future work will be\nessential to integrate our findings with other potential determinants to build a\ncomprehensive model.\n6. The role of surface charge and hydrophobicity in fibril formation and stability must\nbe elaborated. How do these physicochemical properties in renal IGLV 1-44 fibrils\ncompare with those found in cardiac-derived fibrils?\nREPLY: We thank the reviewer for this excellent and critical point, which allows us\nto deepen the mechanistic discussion of our findings. As suggested, we have now\nextensively elaborated on the roles of surface charge and hydrophobicity in a paragraph\nwithin the results section.\nSpecifically, we have added a new Supplementary Figure 5 (SI Fig. 5)\ncomparing the surface charge distribution and hydrophobicity of a cardiac-derived AL\nfibril with our renal fibril. The analysis reveals that the renal IGLV1-44 fibril exhibits\na richer and more complex surface charge distribution relative to its cardiac counterpart,\na feature we propose as an adaptation to the high-ionicity microenvironment of the\nkidney, potentially influencing fibril stability and assembly.\n“……The fibril surface exhibits a pronounced charge distribution pattern, with a\nmolecular surface highly enriched in charged and polar amino acid residues. Notably,\ncompared to cardiac fibrils (SI Fig. 5a, b), our kidney-derived fibrils display a more abundant\nand complex surface charge pattern, which is likely an adaptation to the charge-rich\nmicroenvironment of the kidney.……”\n7. What is structure shown in figure 3d? The PDB:6IC3 mentioned belongs to cardiac\nIGLV 1-44 fibrils and not natively folded LC variable domain. It is difficult to ascertain\nthe N , C -terminus orientation of the native LC from fig 3d. The figure should be revised\nfor accuracy, and the legend should be corrected accordingly.\nREPLY: We sincerely thank the reviewer for this insightful comment and for pointing\nout the inaccuracy in our original labeling of Figure 3D. We acknowledge the error in\nreferencing PDB:6IC3, which indeed corresponds to cardiac IGLV 1-44 fibrils rather\nthan the natively folded LC variable domain. We have now corrected the figure legend\nto accurately describe the structure shown in Figure 3D as the natively folded IGLV1-\n44.\n“Fig. 3 Surface properties and mutations of the IGLV1-44 fibril. (d) Location of mutations\nin the corresponding, natively folded LC VL domains that are based on the GL segments\nIGLV1-44*01(PDB entry 1BJM). Mutations are colored in green.”\nRegarding the orientation of the N and C termini, we agree with the reviewer that\nthis was not clearly depicted in the original figure. To address this, we have included a\nnew Supplementary Figure (SI Fig. 6) that explicitly illustrates the N→C parallel-to-\nantiparallel transition in the native LC. This addition provides a clearer representation\nof the terminal orientations, enhancing the accuracy and interpretability of our\nstructural data.\nWe have ensured that all revisions align with the reviewer's suggestions and hope\nthat these modifications meet their expectations. The manuscript has been updated\naccordingly, and the changes are highlighted in the revised version for easy reference.\n8. What is the key step in the misfolding of the natively folded light chain proteins that\ninitiate fibril formation and arrangements into polymorph A and B?\nREPLY: We thank the reviewer for this important question. As the reviewer surmises,\na key step is the initial formation of a protofilament from the natively folded protein,\ndriven by a combination of factors including mutations, co-factors, and the tissue\nenvironment. The subsequent arrangement into distinct polymorphs (A and B) depends\non the stability of the intermolecular interactions between these protofilaments. Stable\ninteractions allow them to dimerize (or form higher-order assemblies like trimers), and\nthe specific nature of these interactions dictates the final polymorphic structure.\nWe have added a description of this key step and its implications for\npolymorphism to the discussion section to clarify this point.\n“……the renal AL amyloid fibrils exhibit unique topological features. Native LC can\nform protofilaments under the combined effects of somatic mutations, co-factors, and the local\ntissue microenvironment. When stable intermolecular interactions exist between protofilaments,\ntwo protofilaments can further associate into a dimeric structure, and may even assemble into\ntrimers or higher-order aggregates. Variations in the intermolecular forces can also lead to\nthe formation of distinct polymorphs. The 3D reconstructed fibril is composed of two\nprotofilaments entwined together……”\nMinor points: Check the manuscript for typographical errors – especially in the\nreferences.\nREPLY: We thank the reviewer for this important reminder. We have carefully\nproofread the entire manuscript, including the reference list, and corrected all identified\ntypographical and formatting errors."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done an excellent job addressing all the points raised during the previous review round. The manuscript has improved substantially in clarity, rigor, and overall presentation. The experimental design and data interpretation are now well explained, and the figures and supplementary materials are clear and consistent with the conclusions.\nI particularly appreciate the care and thoroughness with which the revisions were carried out — the responses are detailed and convincing, and the resulting manuscript is stronger and more readable.\nCongratulations on the excellent revision. I have no further concerns and recommend the paper for publication in its current form.\nAntonio Chaves-Sanjuan", "reviewer_2": "I am satisfied with the revisions the authors have made in response to my review comments. The manuscript has been adequately improved, and no further modifications are required from my side."}, "author_response": ""}]

Decision letter