Materials<\/p>\n
Chemicals were purchased from commercial suppliers (Thermo Scientific, Merck KGaA, VWR and Honeywell). Solvents and reagents used for peptide synthesis were obtained in peptide grade. Solvents used for peptide purification were of high-performance liquid chromatography (HPLC) grade; dimethyl sulfoxide (DMSO) was BioUltra grade for molecular biology (\u226599.5%, Sigma Aldrich); and water was obtained from a Millipore purification system. Dulbecco\u2019s phosphate buffered saline (DPBS; 1x, without CaCl2 and MgCl2, composition: KCl 0.2\u2009g\u2009l \u22121, KH2PO4 0.2\u2009g\u2009l \u22121, NaCl 8.0\u2009g\u2009l \u22121, Na2HPO4 (anhydrous) 1.15\u2009g\u2009l \u22121) was purchased from Sigma Aldrich (DPBS) or from Thermo Fisher Scientific (Dulbecco\u2019s balanced salt solution) with minor variations in inorganic salt water content. DILT1 was in part purchased from GenScript Biotech with a purity of \u226598%. DILT2-5 and peptides P1 and P3\u2013P8 were purchased from GenScript Biotech with a purity of \u226598%.<\/p>\n
Solid-phase peptide synthesis of DILT1 (KVKVSQINM) and P2 (KLKLSQINM)<\/p>\n
For peptide synthesis, the fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis strategy was applied from the C-terminus to the N-terminus using N,N\u2032-diisopropylcarbodiimide (DIC) and ethyl cyano(hydroxyimino)acetate (Oxyma) on an automated microwave-assisted peptide synthesizer (CEM, Liberty Blue). The peptides were synthesized onto pre-swollen Fmoc-Met-Wang resin (0.1\u2009mmol, 0.147\u2009g, substitution 0.68\u2009mmol\u2009g\u22121, 100\u2013200 mesh, pre-swollen in dimethylformamide (DMF; 2\u20133\u2009ml) on a shaker at room temperature) by sequential coupling of activated N\u03b1-Fmoc-amino acid in DMF (2.5\u2009ml, 0.2\u2009M) in the presence of DIC (1\u2009ml, 0.5\u2009M) and Oxyma (0.5\u2009ml, 1.0\u2009M) in DMF via microwave-assisted reaction at 75\u2009\u00b0C (170\u2009W) for 15\u2009s and 90\u2009\u00b0C (30\u2009W) for 110\u2009s followed by multiple washing of the resin (DMF, 2\u2009\u00d7\u20093\u2009ml, 1\u2009\u00d7\u20094\u2009ml). Sequentially, for DILT1 Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH and Fmoc-Lys(Boc)-OH were coupled to the resin via cycling coupling with each amino acid introduced in one sequential coupling cycle, washing, and N\u03b1-Fmoc-deprotection steps. For P2, Fmoc-Leu-OH was used instead of Fmoc-Val-OH. N\u03b1-Fmoc deprotection was conducted by application of piperidine (3\u2009ml, 20% v\/v in DMF) under heating (75\u2009\u00b0C, 155\u2009W, 15\u2009s, followed by 90\u2009\u00b0C, 30\u2009W, 50\u2009s), followed by washing with DMF (2\u2009\u00d7\u20092\u2009ml, 1\u2009\u00d7\u20093\u2009ml). The synthesized peptides on resin were kept in DMF at 4\u2009\u00b0C until further use. Subsequently, the peptides were cleaved from the resin using a cleavage cocktail (10\u2009ml) comprising trifluoroacetic acid (TFA) (95% v\/v), Milli-Q water (2.5% v\/v) and triisopropylsilane (TIPS) (2.5% v\/v). After 2\u2009h incubation on a shaker at room temperature, the peptides were precipitated with diethyl ether pre-cooled to 4\u2009\u00b0C (40\u2009ml) and centrifuged at 4,000\u2009rpm for 16\u2009min (\u00d72, 0\u2009\u00b0C). The resultant crude products were dried overnight at room temperature. The crude products were purified by preparative reverse-phase HPLC using a 0.1% TFA water\u2013acetonitrile mixture as eluant and lyophilized to yield the peptide as a white amorphous powder. The purified peptides were characterized by matrix-assisted laser desorption\/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The lyophilized solids were stored at \u221220\u2009\u00b0C until use.<\/p>\n
MALDI-TOF MS (DILT1): m\/zcalc\u2009=\u20091,045.60, m\/zfound: 1,046 [M\u2009+\u2009H]+, 1,062 [M\u2009+\u2009O\u2009+\u2009H]+, 1,068 [M+Na]+, 1,084 [M\u2009+\u2009K]+, 1,100, 1,106.<\/p>\n
MALDI-TOF MS (P2): m\/zcalc\u2009=\u20091,073.63, m\/zfound: 1,074 [M\u2009+\u2009H]+, 1,090 [M\u2009+\u2009O\u2009+\u2009H]+, 1,096 [M+Na]+, 1,112 [M\u2009+\u2009K]+, 1,128, 1,134, 1,150, 568.<\/p>\n
High-performance liquid chromatography<\/p>\n
The synthesized crude peptide was purified via preparative HPLC of the dissolved and filtered (0.22\u2009\u00b5m) precipitate (Shimadzu system). Multiple manual injections (2\u00d7 or 3\u2009\u00d7\u20098\u2009mL;) of the peptide crude solution (c\u2009=\u20091.3\u2009mg\u2009ml\u22121, water with 0.1% TFA (v\/v) and 5% acetonitrile (ACN) (v\/v)) were performed. Separation was performed by a preparative reverse-phase HPLC column (Phenomenex 5\u2009\u00b5m C18, 150\u2009\u00d7\u200930\u2009mm) using a 0.1% TFA water\u2013ACN mixture as eluant (flow 25\u2009ml\u2009min\u22121), the applied gradient was as follows: (1) 0.01\u2009min: 5% ACN, 0.1% TFA (A) with 95% 0.1% TFA in water (B); (2) 5\u2009min: 5% A with 95% B; (3) 17\u2009min: 70% A with 30% B; (4) 19\u2009min: 100% A with 0% B; (5) 24\u2009min: 100% A with 0% B; (6) 28\u2009min: 5% A with 95% B; (7) 30\u2009min: 5% A with 95% B. Chromatography was monitored with an ultraviolet absorption detector at 214\u2009nm. Alternatively, the gradient can be applied as follows (for example, P2); (1) 0.01\u2009min: 0% ACN, 0.1% TFA (A) with 100% 0.1% TFA in water (B); (2) 5\u2009min: 0% A with 100% B; (3) 15\u2009min: 40% A with 60% B; (4) 17\u2009min: 100% A with 0% B; (5) 19\u2009min: 100% A with 0% B; (6) 21\u2009min: 5% A with 95% B; (7) 24\u2009min: 5% A with 95% B. Data were evaluated in LabSolutions software.<\/p>\n
MALDI mass spectrometry<\/p>\n
Peptide mass spectra were recorded in positive ion reflector mode on a Bruker rapifleX MALDI-TOF\/TOF mass spectrometer (Bruker Daltonik, scanning smartbeam 3D 10-kHz neodymium-doped yttrium aluminium garnet (Nd:YAG) laser, wavelength of 355\u2009nm, and 10-bit 5-GHz digitizer, acceleration voltage 20\u2009kV) using \u03b1-cyano-4-hydroxycinnamic acid (HCCA) as the matrix. Samples were measured with random walk ionization across the sample spot, and usually 8,000 shots were averaged per spectrum. The sample was prepared by mixing an acidic aqueous analyte solution (0.1% TFA) in equal amounts with the supernatant of a saturated HCCA solution acetonitrile\/water (30:70\u2009+\u20090.1% TFA). Subsequently, this mixture (1\u2009\u00b5l) was applied to a stainless steel target and allowed to dry before measurement. Calibration was conducted with sodium adducts of polyethylene glycol in a mass range of 500\u20133,000\u2009Da. Data were evaluated with mMass software.<\/p>\n
MSMS sequencing<\/p>\n
The sequence analysis was carried out on a rapifleX MALDI-TOF\/TOF mass spectrometer from Bruker Daltonik. The instrument is equipped with a scanning smartbeam 3D 10-kHz Nd:YAG laser at a wavelength of 355\u2009nm and a 10-bit 5-GHz digitizer. The acceleration voltage was set to 20\u2009kV, and the mass spectra were recorded in positive ion MSMS mode. The protonated molecular ion of the peptide at 1,046\u2009Da was selected as precursor with an isolation window of +6\u2009Da and \u22126\u2009Da. Basic instrument calibration was done with the Bruker peptide mix and the Bruker protein calibration standard I and II in a mass range up to 70\u2009kDa. Before fragment ion analysis, the instrument was manually recalibrated with the exact mass of the precursor ion at 1,046.5418\u2009m\/z. Samples were measured with random walk ionization across the sample spot. The laser power was adjusted to provide significant signal intensity for b- and y-series fragments, and 2,000 shots were averaged per spectrum. Sample preparation was done by mixing a 50\u2009mg\u2009ml\u22121 solution of super-DHB matrix in acetonitrile\/water (1:1\u2009+\u20090.1% TFA) with equal amounts of the peptide solution (10\u2009mg\u2009ml\u22121) in acetonitrile\/water (1:1\u2009+\u20090.1% TFA). Finally, 1\u2009\u00b5l of the mixture was applied to a stainless steel target and allowed to dry before measurement. The mass spectrometer was controlled by the software Bruker flexControl (Version 4.0) and data analysis was done by Bruker flexAnalysis (Version 4.0).<\/p>\n
Peptide nanofibril formation<\/p>\n
Nanofibril formation was induced by introducing the pre-dissolved peptide stock solution (10\u2009mg\u2009ml\u22121, DMSO) into DPBS (pH 7.4), yielding a 1\u2009mg\u2009ml\u22121 solution. After brief vortexing, the samples were incubated on a shaker (500\u2009rpm, room temperature) overnight. Further incubation was conducted as indicated without shaking at room temperature or at 37\u2009\u00b0C for the indicated periods.<\/p>\n
Peptide nanofibril electron microscopy screeningCryo-EM<\/p>\n
For cryo-EM micrograph screening of DILT2\u2013DILT5 and peptides P1 and P3\u2013P8, samples were incubated as described above (1\u2009mg\u2009ml\u22121) overnight (500\u2009rpm, 25\u2009\u00b0C), subsequently kept at room temperature without shaking and plunge-frozen after 4\u2009days. Holey-carbon-coated grids (400 mesh C-flat 1.2\/1.3) were glow-discharged (PELCO easiGlow glow discharge cleaning system; TED PELLA), 3\u2009\u00b5l sample was applied, blotted for 8\u2009s and plunge-frozen in liquid ethane using an Automatic Plunge Freezer EM GP2 (Leica Microsystems) for each sample. Grids were screened on a JEM-2100 transmission electron microscope (Jeol) at 200\u2009kV using a TVIPS F416 camera.<\/p>\n
Transmission electron microscopy<\/p>\n
P2 (L2\/L4) was imaged after overnight incubation via TEM as described previously42<\/a>,43<\/a>. The pre-incubated sample (5\u2009\u03bcl, 1\u2009mg\u2009ml\u22121) was placed on a copper grid coated with formvar layer (etched with oxygen plasma). Following a short incubation, excess liquid was removed with filter paper, and the grids were stained with uranyl acetate solution (4% w\/v, 2\u2009min) and washed with water. Measurements were performed on a Jeol 1400 electron microscope with 120\u2009kV acceleration voltage.<\/p>\n Cryo-EM sample preparation and data acquisition<\/p>\n For DILT1 analyses, samples were incubated overnight (500\u2009rpm, 22\u2009\u00b0C), and further sample incubation was conducted without shaking at 37\u2009\u00b0C for the indicated periods. For DILT2 (2-2-2) and DILT3 (2-2-2) cryo-EM reconstruction analyses, samples were incubated overnight (500\u2009rpm, 25\u2009\u00b0C), subsequently kept at 37\u2009\u00b0C without shaking, and plunge-frozen after 7\u2009days and 1\u2009day, respectively. In case of DILT1 and DILT3, a 4\u2009\u00b5l aliquot of the DILT1 fibril sample (from the indicated incubation time points) was applied on a Quantifoil grid (Q R2\/2, 400 mesh) that was glow-discharged in a 6:1 oxygen\/hydrogen plasma (Diener Nano, Diener Electronic) for 30\u2009s shortly before. The excess solution was blotted for 4\u2009s at 4\u2009\u00b0C and >80 % humidity. The specimen was cryo-plunged in liquid ethane using a Vitrobot Mark V (Thermo Fisher Scientific). Subsequent imaging was done using a Titan Krios G4 transmission electron microscope (Thermo Fisher Scientific) at 300\u2009kV, equipped with a Gatan GIF continuum spectrometer. Micrographs and videos were acquired on a 6,912\u2009\u00d7\u20096,912-pixel K3 Gatan direct electron-detection camera with the spectrometer operated in imaging mode using a slit width of 20\u2009eV. For data acquisition, the K3 camera was operated in super resolution and correlated double sampling mode. Extended Data Table 1<\/a> lists the data acquisition parameters.<\/p>\n For DILT2 (4-4-4) and DILT3 (3-3-3) cryo-EM reconstruction analyses, samples were incubated overnight (500\u2009rpm, 25\u2009\u00b0C), subsequently kept at room temperature without shaking, and plunge-frozen after 15\u2009days for DILT2 and after 8\u2009days for DILT3. For each, 3\u2009\u00b5l of sample was applied to a glow-discharged (PELCO easiGlow glow discharge cleaning system; TED PELLA) holey-carbon-coated grid (400 mesh C-flat 1.2\/1.3). After blotting with filter paper for 8\u2009s and plunge-freezing in liquid ethane using an Automatic Plunge Freezer EM GP2 (Leica Microsystems), the grids were screened on a JEM-2100 transmission electron microscope (Jeol) at 200\u2009kV, equipped with a TVIPS F416 camera. The dataset for reconstruction was recorded on a Krios G4 transmission electron microscope (Thermo Fisher Scientific) with a Falcon4i (Thermo Fisher Scientific) direct electron detector and a Selectris X energy filter (Thermo Fisher Scientific) operated with a 10-eV slit width. Extended Data Table 1<\/a> lists the data acquisition parameters.<\/p>\n Helical reconstruction<\/p>\n In the case of DILT1 fibrils, MotionCor244<\/a> was used for movie correction. Contrast transfer function (CTF) for all micrographs was performed using CTFFIND45<\/a>. Further image processing and 3D helical reconstructions were performed with RELION-4.046<\/a>,47<\/a> on the 1-1-1 (hexagon fibril) and 3-3-3 (honeycomb fibril) morphologies. For the 3-3-3 morphology, micrographs containing ice or lacking the 4.8-\u00c5-resolution information were excluded. The 3-3-3 morphology filaments were manually picked. As an initial model for the refinement, a featureless cylinder was used with a 60-\u00c5 low-pass filter and C1 symmetry. From the 3D auto-refinement, a C6 symmetry was observed and imposed, reaching a 4.3-\u00c5-resolution volume with clearly visible \u03b2-sheets. To further improve resolution, the full dataset was re-processed via 3D classification and further 3D auto-refine to reach the final 3\u2009\u00c5 volume. The volume was then post-processed with a soft-edge mask and sharpened with a B-factor of \u221250. All 3D classifications were carried out on the central 10% of the reconstruction. For the 1-1-1 morphology, a similar approach was performed. Details of particle extraction and helical reconstruction are reported in Extended Data Table 1<\/a>.<\/p>\n In the case of the 2-2-2 DILT2 fibrils, CTF for all single images, acquired with drift correction in SerialEM, was performed using CTFFIND45<\/a>. Further image processing and 3D helical reconstructions were performed with RELION 5.046<\/a>. The 2-2-2 morphology filaments were manually picked. As an initial model for the refinement, a simulated 2-2-2 hexagonal morphology was used. From the 3D auto-refinement, a C3 symmetry was imposed. The volume was then post-processed with a soft-edge mask and sharpened with a B-factor of \u221250. All 3D analyses were carried out on the central 10% of the reconstruction.<\/p>\n In the case of DILT2 (4-4-4), helical reconstruction was performed in RELION 5.046<\/a>. Raw video frames were aligned and corrected for beam-induced motion using RELION\u2019s own implementation of MotionCorr244<\/a>, followed by CTF estimation using CTFFIND 4.145<\/a>. Fibrils displaying the 4-4-4 morphology were manually selected from the micrographs. Binned segments were extracted and subjected to reference-free two-dimensional classification until a homogeneous set of particles was obtained. Selected segments were extracted for 3D refinement without binning for 3D reconstruction. Helical refinement was performed using a low-passed cylinder and helical twist estimated from the raw micrographs by manual measurement in Fiji48<\/a>, and a helical rise of 4.75\u2009\u00c5 without the imposition of rotational symmetry. For the purpose of map improvement, rotational symmetries C2, C3 and C6 were imposed in the subsequent refinement, of which C3 symmetry yielded the best quality map as validated by visual inspection. Furthermore, the local search for helical parameters was turned on to optimize the twist and rise of the fibril. After the initial reconstructions, 3D classification and selection of the refined particle stack with progressive increase of the Tau fudge factor were carried out to remedy residual heterogeneity. The resulting particles were subjected to Bayesian polishing. The polished, final stack underwent two cycles of CTF refinement and reconstruction until no further improvement of the reconstructed map was noticeable. The final map was masked with a soft-edged mask and post-processed, yielding a map with a nominal gold-standard resolution of 1.9\u2009\u00c5.<\/p>\n In the case of DILT3 (3-3-3), image processing was performed in cryoSPARC 4.7.149<\/a>. Raw video frames were aligned and corrected for beam-induced motion using cryoSPARC\u2019s internal patch motion correction. CTF parameter estimation was performed by patch CTF estimation. Exposures were manually curated to exclude micrographs with thick ice and fibril overcrowding. Filament segments were automatically picked using the filament tracer, and segments were extracted with binning for initial classification. Reference-free two-dimensional classification was used to separate segments belonging to different fibril morphologies. Particles corresponding to the 3-3-3 morphology were re-extracted without binning. The resulting stack was subjected to multiple rounds of two-dimensional classification to remove low-quality particles. An initial 3D helical reconstruction was performed using a featureless cylinder without the imposition of helical symmetry. The obtained map was used to bootstrap a 3D helical refinement with enforcement of helical symmetry parameters as obtained by measurement in Fiji48<\/a>. Three-dimensional classification without particle alignment was used to resolve structural heterogeneity. Classes displaying clear separation of fibril layers along the helical axis were selected, and helical symmetry search was enabled during subsequent reconstructions. The homogeneous particle stack was subjected to reference beam motion correction in cryoSPARC and subsequent local and global CTF refinements. Several rounds of local CTF refinement and helical refinement were performed until no further improvement of the 3D map was observable. The final particle stack was symmetry expanded, and a final local refinement with recentring was performed. The resulting half-maps and reconstruction mask were imported into RELION 5.046<\/a> where masking and post-processing were performed, yielding a final map with a gold-standard resolution of 1.78\u2009\u00c5. Details of particle extraction and helical reconstruction are reported in Extended Data Table 1<\/a>.<\/p>\n Atomic model building and refinement<\/p>\n In the case of DILT1, the atomic model for morphology 3-3-3 was built de novo using the program Coot50<\/a>. Given the large number of chains present in the structure, model building was performed stepwise. The fundamental chains of the fibril are termed I.II.III\u2013IV.V.VI\u2013VII.VIII\u2013IX.X (Extended Data Fig. 2g<\/a>). Initially three chains (I-II-III) were constructed in Chimera51<\/a> by applying the standard \u03b2-parallel parameters ((\u03a6,\u2009\u03a8)\u2009=\u2009(\u2013135\u00b0,\u2009135\u00b0)). These were manually aligned to the junction motif in the 3D volume. Next, to limit atomic clashes in the fibril axis, three layers of such chains were constructed. The three-layer system was refined further in Coot50<\/a> and Chimera51<\/a>. An iterative optimization process was performed to achieve optimal fitting. Next, the structure was further extended to take into consideration the cross-\u03b2-motif. Once the I-II-III chains were optimized, they were imposed on the remaining outer electron density. MolProbity52<\/a>, the comprehensive validation tool in Phenix53<\/a>, was used to produce a validation of the atomic model to assess atomic clashes, rotamer and Ramachandran outliers, and model geometry. The structural statistics for refinement and model building are listed in Extended Data Table 1<\/a> and refer to the deposited atomic model (18 chains and 3 layers).<\/p>\n In the case of 2-2-2 DILT2 fibrils, the starting model for refinement was the atomic model of DILT1, and the applied rotational symmetry was C3. The structural statistics for refinement and model building are listed in Extended Data Table 1<\/a> and refer to the deposited atomic models.<\/p>\n In the case of DILT3 fibrils, an initial atomic model was generated de novo using ModelAngelo54<\/a>. An iterative model refinement process was used in which an asymmetric unit of a single layer of the C6-symmetrical DILT3 fibril cross-sections was refined in WinCoot 1.1.1849<\/a>. Phenix 2.0.593653<\/a> was used to evaluate model geometry and clash score. ChimeraX 1.1055<\/a> was used to obtain a helical assembly of three layers, which was again validated. The three-layer model was further manually refined in WinCoot 1.1.1849<\/a> using regularization with Ramachandran, torsion and planar peptide restraints, followed by a subsequent real-space refinement. ISOLDE56<\/a> was used to improve the clash score. After each model refinement cycle, the contact sites between asymmetric units were checked for steric clashes by symmetry expansion to the original fibril symmetry and renewed validation in Phenix. Final images were produced using Fiji48<\/a> and ChimeraX55<\/a>.<\/p>\n In the case of DILT2 fibrils, the same procedure was used as for DILT3 fibrils, with the exception of the starting model and the applied symmetry. The starting model for refinement of DILT2 was the atomic model of DILT3, and the applied rotational symmetry was C3. The structural statistics for refinement and model building are listed in Extended Data Table 1<\/a> and refer to the deposited atomic models.<\/p>\n Polymorphism analysis<\/p>\n The CTF-corrected micrographs were used for the polymorphism analysis. A subset of 100 micrographs was randomly generated with the RELION \u2018Subset Selection\u2019 function. Each of these micrographs was analysed, and the different morphologies were manually picked and extracted using a box size of 350\u2009pixels, nanochannel diameter of 220\u2009\u00c5, number of asymmetric units 7 and rise of 4.7\u2009\u00c5. The number of extracted particles was used to quantify the different morphologies. The polymorphism prediction was performed using Chimera51<\/a>, RELION-4.047<\/a> and Fiji48<\/a>. Three layers of the atomic model of the central hexagon obtained from the 3-3-3 morphology were copied and shifted manually to create plausible hexagonal patterns. In Chimera51<\/a>, the atomic model was converted into a volume with a 3-\u00c5 resolution (molmap #0 3) and saved as an\u00a0.mrc file. The file was then processed with RELION-4.047<\/a> to create a prolonged volume. Specifically, the file was converted into a file with a box size of 1,750\u2009pixels, a nanochannel diameter of 250\u2009\u00c5, a rise of 4.77\u2009\u00c5 and a twist of \u22120.64\u00b0. The following commands were used:<\/p>\n \n relion_image_handler\u2013i\u00a02Hex_221.mrc\u2013new_box 1750\u2013o 2Hex_221_BS1750.mrc The volume was then processed in Fiji48<\/a>. The central 1,511 slices of the volume (which correspond to the number of slices that generate a single crossover distance) were analysed to visualize the projection. To visualize the full 360\u00b0 rotation pattern, the images were rotated by 180\u00b0, and the 2 projection images were combined.<\/p>\n Simulation details<\/p>\n Simulations were performed using the GROMACS 2021.757<\/a> molecular dynamics package in the isothermal\u2013isobaric\u00a0NPT ensemble. The temperature was imposed using a velocity rescale thermostat58<\/a> at 298.15\u2009K with a time constant of 1.0\u2009ps\u22121. The pressure was controlled at 1.0\u2009bar using the Parrinello\u2013Rahman barostat59<\/a> with a time constant of 2.0\u2009ps\u22121. The equations of motion were integrated using the leap-frog algorithm with a time step of 1.0\u2009fs. Peptide chains and sodium, potassium and chloride ions were modelled using CHARMM36m force-field parameters60<\/a>, and water was described by the TIP3P model61<\/a>,62<\/a>. The H2PO4\u2212 and HPO42\u2212 ions parameters were taken from CHARMM FF on CHARMM-GUI63<\/a>,64<\/a>. Electrostatic interactions were treated using the smooth particle mesh Ewald method65<\/a>. The solvent molecules and ions were equilibrated by the steepest descent energy minimization with a tolerance of 100.0\u2009kJ\u2009mol\u22121, with position restraints in all peptide atoms, followed by 25\u2009ns of molecular dynamics simulation with position restraints only in the backbone atoms of the peptides. After the equilibration of the solvent, the systems were simulated for 100\u2009ns without restraints. All simulations were performed on RAVEN HPC at Max Planck Computing and Data Facility, Garching, Germany.<\/p>\n Molecular dynamics analysis<\/p>\n All analyses were performed with the GROMACS 2021.7 molecular dynamics package53<\/a>. The RMSF was calculated by residue and averaged over time and layer. The size-independent comparison of the 3D structures was done using the scaled dissimilarity index proposed by Maiorov and Crippen66<\/a>. The trajectories were least-squared fitted to the first snapshot of the simulation, using its backbone atoms as reference. The visualization and simulation snapshots were generated with visual molecular dynamics67<\/a>.<\/p>\n DILT1 fibril integrity and solvent interaction<\/p>\n A DILT1 fibril in explicit solvent, consisting of 94 layers with each layer rotated by \u22120.64\u00b0 (total twist approximately 60\u00b0) was simulated to investigate its integrity. The fibril extends across the 22.0\u2009\u00d7\u200922.0\u2009\u00d7\u200944.8\u2009nm3 simulation box along the z direction, forming an infinitely periodic fibril owing to periodic boundary conditions. The system composition was determined using the systematic equilibrium treatment described previously. The final composition includes: 1,692 peptide chains, 4,739 Cl\u2212, 18 H2PO4\u2212, 96 HPO42\u2212, 44\u2009K+, 1,521 Na+, 14,637 DMSO and 509,721 H2O.<\/p>\n Composition of the system for molecular dynamics<\/p>\n The integrity of the DILT1 junction and cross-\u03b2-motif was studied by simulating 1-, 3-, 5-, 10- and 15-layer models for each motif. The same number of layers was simulated to verify the conformational persistence of the hexagon fibril. The layer structures were generated by rotating and translating the fundamental junction unit along the z direction. The geometric centre of the peptides was placed in a 22.0\u2009\u00d7\u200922.0\u2009\u00d7\u200922.0\u2009nm3 simulation box and solvated with a 9:1 (% v\/v) mixture of PBS and DMSO.<\/p>\n The system composition was determined from equilibrium calculations at 298.15\u2009K based on the procedure described in ref. 68<\/a>. The dissociation constants of ionizable amino acids (for example, lysine, pKa\u2009=\u200910.82) were assumed to be independent of each other. Activity coefficients were computed using the temperature-dependent extended Debye\u2013H\u00fcckel equation68<\/a>. The experimental value of the dielectric constant of the water\/DMSO mixture, needed to obtain the system composition, was obtained from ref. 69<\/a>, corresponding to 78.08 at 298.15\u2009K.<\/p>\n The equilibrium composition of each system was computed numerically using the Newton\u2013Raphson method, with the change in pH, \u0394pH <10\u22127, as the convergence criterion.<\/p>\n Structure\u2013property relationship simulations<\/p>\n As we have observed that 15 layers are a persistent fibril fragment for DILT1, we have generated samples for the other sequences, DILT2\u2013DILT5, and P1 and P4, with this number of layers. Once the peptide sequence has been mutated in PyMOL, systems were built as before, starting from a junction and applying the corresponding symmetry operations, and simulated following the protocol described above.<\/p>\n DILT 1 nanofibril characterization<\/p>\n The incubation times and temperatures were varied as indicated below for\u00a0kinetics and stability (solvent, temperature, sonication)\u00a0characterizations\u00a0as follows. (1) Preformed fibrils (1\u2009mg\u2009ml\u22121, 10% v\/v DMSO, 90% v\/v PBS) were obtained by incubating at 22\u2009\u00b0C for 24\u2009h with 500\u2009rpm shaking (Extended Data Fig. 7a<\/a>). (2) Nanofibril formation without pre-dissolved DMSO peptide stock was determined by dissolving the lyophilized peptide directly in DPBS (pH 7.4) yielding a 1\u2009mg\u2009ml\u22121 solution (Extended Data Fig. 7b<\/a>). (3) A dilution stability test was performed on the preformed fibrils as described in (1), which were incubated at 37\u2009\u00b0C for 1\u2009week. The fibrils were then diluted in water. Specifically, 10\u2009\u03bcl of the preformed fibrils (1\u2009mg\u2009ml\u22121) was added to 90\u2009\u03bcl Milli-Q H2O (0.1\u2009mg\u2009ml\u22121) with a final composition of 1% v\/v DMSO, 9% v\/v PBS and 90% v\/v Milli-Q H2O. The sample was incubated for 3\u2009days at 37\u2009\u00b0C (Extended Data Fig. 7c<\/a>). This sample was used for the reconstruction of the 1-1-1 morphology (#2; Extended Data Table 1<\/a>). (4) Early kinetics experiments were conducted by inducing nanofibril formation via the general method with incubation (22\u2009\u00b0C, 500\u2009rpm) for either 30\u2009min, 45\u2009min, 1\u2009h, 3\u2009h, 6\u2009h, 9\u2009h, 15\u2009h, 21\u2009h or 24\u2009h (Extended Data Fig. 7k\u2013s<\/a>). (5) Late kinetics experiments were conducted using the preformed fibrils as described in (1) and transferring them into a 37\u2009\u00b0C oven without shaking. The samples were then imaged after 4\u2009days, 1\u2009week, 2\u2009weeks and 53\u2009days (Extended Data Fig. 7t\u2013w<\/a>). The 53-day sample was used for reconstruction of the 3-3-3 morphology and for the polymorphism analysis (Extended Data Fig. 2<\/a>). (6) Temperature stability tests were performed by using the preformed fibrils as described in (1) and transferring them for 15\u2009min or 60\u2009min in an oven at 60\u2009\u00b0C, 85\u2009\u00b0C or 95\u2009\u00b0C (Extended Data Fig. 7d\u2013i<\/a>). (7) Sonication stability tests were performed by exposing preformed fibrils as described in (1) to sonication (15\u2009min, sonication bath) (Extended Data Fig. 7j<\/a>).<\/p>\n To minimize contamination during long-term incubation, peptide assemblies were prepared from fresh stock solutions using sterile-filtered buffer and clean consumables; full-grid cryo-EM mapping of long-incubation samples did not reveal contaminating micrometre-scale objects.<\/p>\n Atomic force microscopy<\/p>\n For atomic force microscopy (AFM) screening of DILT1\u2013DILT5 and P3 samples were prepared by introducing the pre-dissolved peptide stock solution (10\u2009mg\u2009ml \u22121, DMSO) to DPBS (pH 7.4), yielding a 1\u2009mg\u2009ml\u22121 solution, and analysed after overnight incubation (500\u2009rpm, 25\u2009\u00b0C, DPBS, pH 7.4). For imaging the DILT and P3 architectures, a Bruker Dimension FastScan BioTM atomic force microscope was used in the liquid state, which was operated in PeakForce mode. FastScan-D tips from Burker with a nominal spring constant of 0.25\u2009Nm\u22121 were used.<\/p>\n For AFM sample preparation, the pre-incubated peptide solution (100\u2009\u00b5l, 1\u2009mg\u2009ml\u22121) was added to a circular mica substrate (20\u2009mm) and incubated for 15\u2009min. The excess liquid was removed, washed with 200\u2009\u00b5l buffer (DPBS, pH 7.4), excess liquid was removed and 300\u2009\u00b5l buffer was added to the mica to measure in liquid. Images were analysed with NanoScope Analysis 1.9.<\/p>\n In situ humidity-controlled FTIR experiments<\/p>\n Nanofibril formation was induced either by dissolving the DILT1 solid powder directly in the DPBS to yield a 1\u2009mg\u2009ml\u22121 solution (DILT1, Fig. 6e<\/a> and Extended Data Fig. 9b,e\u2013h<\/a>) or, alternatively, peptide nanofibril formation was induced as described above by introducing the pre-dissolved peptide stock solution (10\u2009mg\u2009ml\u22121, DMSO) to DPBS (pH 7.4), yielding a 1\u2009mg\u2009ml\u22121 solution (DILT 5, Fig. 6e<\/a>; DILT 2, Extended Data Fig. 9c<\/a>; DILT 4, Extended Data Fig. 9d<\/a>). The same protocol was applied for the control-peptide CKFKFQF. After overnight incubation (500\u2009rpm, 25\u2009\u00b0C), DILT1, DILT2, DILT4, DILT5 and CKFKFQF samples, were submitted to in situ humidity-controlled FTIR experiments, respectively. FTIR spectra were recorded in transmission mode using a Bruker VERTEX 70 spectrometer. The spectrometer was purged with nitrogen and all measurements were conducted at room temperature. The pre-incubated peptide samples were loaded into a Teflon flow cell fitted with CaF2 windows (1\u2009mm thickness) and a 2\u2009mm path length; the sample (approximately 8\u2009\u00b5l) was applied with a thickness \u22640.1\u2009mm. The flow cell was mounted in the spectrometer sample compartment and connected to a dry-nitrogen supply to initiate and control dehydration. Spectra were recorded at regular intervals throughout the drying process, which proceeded until no further spectral change indicated complete desiccation.<\/p>\n Estimation of the number of water molecules per peptide<\/p>\n The number of water molecules per peptide was estimated using the known infrared absorption cross-sections of the respective functional groups. Molar extinction coefficients of \u03f5N\u2013H\u2009=\u2009100\u2009M\u22121\u2009cm\u22121 for the peptide N\u2013H (ref. 70<\/a>) and \u03f5O\u2013H\u2009=\u2009400\u2009M\u22121\u2009cm\u22121 for the O\u2013H stretch of water molecules71<\/a> were applied. Considering four N\u2013H groups per peptide and two O\u2013H bonds per water molecule, the integrated intensity ratio satisfies \\(\\frac{{I}_{{\\rm{N}}-{\\rm{H}}}}{{I}_{{\\rm{O}}-{\\rm{H}}}}\\approx \\frac{{N}_{{\\rm{N}}-{\\rm{H}}}\\times {{\\epsilon }}_{{\\rm{N}}-{\\rm{H}}}}{2{N}_{\\mathrm{water}}\\times {{\\epsilon }}_{{\\rm{O}}-{\\rm{H}}}}\\), where NN\u2013H is the number of peptide N\u2013H groups and Nwater is the number of water molecules.<\/p>\n For DILT1, after about 110\u2009min in dry nitrogen, the N\u2013H peak at 3,277\u2009cm\u22121 shows an integrated intensity of 0.15, and the broader water O\u2013H band centred at 3,300\u2009cm\u22121 shows an integrated intensity of 7.5, corresponding to approximately 25 water molecules per peptide, assuming negligible contribution from backbone N\u2013H groups (Extended Data Fig. 9b<\/a>). Considering the channel geometry (0.48\u2009nm peptide-layer height, 6 peptides per layer, 5\u2009nm channel diameter) and a water density of 1.05\u2009g\u2009cm\u22123 from simulations, a fully filled channel would contain about 55 water molecules per peptide. The FTIR-derived estimate is inherently approximate, as it relies on extinction coefficients from related systems and does not account for peptide side-chain O\u2013H or N\u2013H contributions. Nevertheless, the number inferred from the infrared spectral intensities is consistent with fully filled tubes at about 100\u2009min drying time inferred from the convergence of the spectral shape at that time.<\/p>\n In situ dehydration Raman spectroscopy and Cryo-EM experiments<\/p>\n In situ Raman spectra were recorded using a WITec alpha300 Raman microscope equipped with a 532-nm excitation laser. The laser power at the sample was set to 5\u2009mW. The pre-incubated peptide samples (1\u2009mg\u2009ml\u22121, directly dissolved in DPBS, 500\u2009rpm overnight shaking, 25\u2009\u00b0C) were loaded into a Teflon flow cell fitted with CaF2 windows (1\u2009mm thickness) and a 2\u2009mm path length; the sample, approximately 8\u2009\u00b5l, was applied with a thickness \u22640.1\u2009mm. The flow cell was fixed on the microscope sample stage and connected to a dry-nitrogen supply, allowing dehydration to be initiated and controlled during Raman acquisition. Spectra were recorded at regular intervals throughout the drying process until no further spectral changes were observed, indicating complete desiccation.<\/p>\n For cryo-EM analysis after controlled drying, a similarly pre-incubated peptide sample was dehydrated using the same Teflon flow-cell and dry-nitrogen protocol as used for the Raman and FTIR experiments. After approximatey 120\u2009min of drying, the flow cell was opened and a TEM grid was dripped into the residual sample liquid on the CaF2 window and the TEM grid was then immediately plunge-frozen using a Vitrobot Mark V for subsequent cryo-EM examination.<\/p>\n Characterization of concentration-dependent self-assembly of DILT1Sample preparation and incubation<\/p>\n DILT1 solutions of various concentrations were prepared. For this, a DILT1 10\u2009mg\u2009ml\u22121 DMSO stock solution, prepared in filtered DMSO, was diluted to yield DILT1 DMSO stock solutions at varying concentrations. Subsequently, these pre-dissolved DILT1 DMSO stock solutions were introduced to DPBS (pH 7.4) in a 1:9 ratio, respectively, yielding solutions with varied concentrations (1,000\u2009\u00b5g\u2009ml\u22121, 500\u2009\u00b5g\u2009ml\u22121, 100\u2009\u00b5g\u2009ml\u22121, 50\u2009\u00b5g\u2009ml\u22121, 35\u2009\u00b5g\u2009ml\u22121, 17.5\u2009\u00b5g\u2009ml\u22121, 8.8\u2009\u00b5g\u2009ml\u22121, 4.4\u2009\u00b5g\u2009ml\u22121, 2.2\u2009\u00b5g\u2009ml\u22121, 500\u2009\u00b5l, 10% v\/v DMSO in DPBS). The respective samples were mixed by brief vortexing and incubated on a shaker (500\u2009rpm, room temperature) overnight. The given values were accurately rounded up to the full number in Extended Data Fig. 8<\/a>.<\/p>\n Vial flip test<\/p>\n After overnight incubation of the DILT1 samples (2\u20131,000\u2009\u00b5g\u2009ml\u22121, 10% v\/v DMSO in DPBS, 500\u2009\u00b5l), the macroscopic gelation behaviour of DILT1 at varied concentrations was examined by vial flip test.<\/p>\n Proteostat assay<\/p>\n The critical aggregation concentration of the DILT1 peptide was examined via the commercial Proteostat protein aggregation assay kit by Enzo Life Sciences. Following the manufacturer\u2019s recommendation, adapting a previously published protocol72<\/a>, the Proteostat working solution was prepared by diluting Proteostat stock solution (0.50\u2009\u00b5l) and assay buffer (1\u2009\u00b5l 10x assay buffer) in 98.5\u2009\u00b5l Milli-Q water. To conduct the assay, 36\u2009\u03bcl pre-incubated DILT1 peptide solutions at various concentrations (2\u20131,000\u2009\u00b5g\u2009ml\u22121, 10% v\/v DMSO in DPBS) were mixed with 4\u2009\u03bcl Proteostat working solution. The samples were transferred into a Greiner 384 flat black well plate (3 wells per sample (technical replicates n\u2009=\u20093), that is, per peptide concentration, 9\u2009\u00b5l sample per well), incubated in the dark (15\u2009min) while shaking, and the fluorescence intensity of the Proteostat dye was determined \u03bbexcitation\u2009=\u2009550\u2009nm, \u03bbemission\u2009=\u2009600\u2009nm; bandwidths 20\u2009nm, multiple reads per well, respectively. At high DILT1 concentration (956\u2009\u00b5M), the aggregation affected sample loading to a maximum of 2 wells, each containing 9\u2009\u00b5l of the mix (n\u2009=\u20092).<\/p>\n Transmission electron microscopy<\/p>\n To analyse the aggregate and assembly morphologies at various concentrations, the incubated DILT1 sample solutions (2\u20131,000\u2009\u00b5g\u2009ml\u22121, 10% v\/v DMSO in DPBS) were submitted to TEM analysis. Grid preparation was carried out as follows. Five microlitres of the sample solution was deposited onto a TEM grid and allowed to stand for 5\u2009min to enable adequate adsorption. Excess solution was then gently removed using filter paper. Subsequently, staining was performed with 5\u2009\u00b5l of 4% w\/v aqueous uranyl acetate for 2\u2009min. After staining, the grids were rinsed three times with Milli-Q water to remove excess stain. Finally, the water was gently removed from the grid with filter paper, and the grids were left to dry overnight before TEM measurement. TEM was measured at 120\u2009kV.<\/p>\n Hydrogel and honeycomb fibril preparation<\/p>\n Hydrogels were prepared by either pre-dissolving the DILT1 peptide in DMSO (10\u2009mg\u2009ml\u22121) and adding this DMSO peptide stock solution into DPBS to yield a 1\u2009mg\u2009ml\u22121 solution (0.1\u2009wt%) or by dissolving solid DILT1 powder directly in the respective solvent to yield 1\u20134\u2009wt% hydrogels. For example, 0.3\u2009mg peptide was dissolved in 30\u2009\u00b5l buffer (for example, DPBS, pH 7) and mixed for approximately 5\u2009s to yield a 1\u2009wt% (10\u2009mg\u2009ml\u22121) hydrogel. Very soft hydrogels were obtained at 0.1\u2009wt%, for example, by the nanofibril-formation procedure described in \u2018Peptide nanofibril formation\u2019.<\/p>\n Rheology<\/p>\n Rheological characterization was conducted using a DHR3 rheometer (TA Instruments) equipped with a temperature controller and a solvent reservoir to prevent hydrogel drying. Experiments were performed using an 8-mm parallel-plate geometry with hydrogels of approximately 30\u2009\u03bcl volume (gap size of about 0.5\u2009mm). Characterization of the hydrogel mechanical properties was conducted at 25\u2009\u00b0C. Gels were prepared directly on the plate by the procedure described in \u2018Hydrogel and honeycomb fibril preparation\u2019. Oscillatory time-sweep measurements monitored the gelation at a fixed strain of 0.1% and a fixed frequency of 1\u2009Hz. Extended Data Fig. 8e<\/a> presents representative sections after complete gelation (approximately last 300\u2009s of gelation curves), which summarizes individual measurements recorded with time stamps at approximately 6-s intervals (time stamps are indicative). Oscillatory strain sweeps (0.01\u2013200% or 0.01\u20131000%, as indicated) were conducted at a fixed frequency of 1\u2009Hz. Oscillatory frequency sweeps (0.05\u2013100\u2009Hz) were performed at a fixed strain of 0.1%.<\/p>\n DILT1 gel stability<\/p>\n Photographs were taken with a Canon EOS 250D with a Canon EF 24\u2013105\u2009mm f\/4L IS lens. Manual mode, exposure of 1\/50 with aperture of f\/8 and focal length of 105\u2009mm. ISO 100. Saved in jpg format with a resolution of 5,284\u2009dpi.<\/p>\n","protected":false},"excerpt":{"rendered":"Materials Chemicals were purchased from commercial suppliers (Thermo Scientific, Merck KGaA, VWR and Honeywell). Solvents and reagents used…\n","protected":false},"author":2,"featured_media":862340,"comment_status":"","ping_status":"","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[32],"tags":[1159,2528,282814,1160,79,31208,78679,363897],"class_list":["post-862339","post","type-post","status-publish","format-standard","has-post-thumbnail","category-science","tag-humanities-and-social-sciences","tag-materials-science","tag-molecular-self-assembly","tag-multidisciplinary","tag-science","tag-self-assembly","tag-structural-biology","tag-supramolecular-chemistry"],"_links":{"self":[{"href":"https:\/\/www.newsbeep.com\/us\/wp-json\/wp\/v2\/posts\/862339","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.newsbeep.com\/us\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.newsbeep.com\/us\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/us\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/us\/wp-json\/wp\/v2\/comments?post=862339"}],"version-history":[{"count":0,"href":"https:\/\/www.newsbeep.com\/us\/wp-json\/wp\/v2\/posts\/862339\/revisions"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.newsbeep.com\/us\/wp-json\/wp\/v2\/media\/862340"}],"wp:attachment":[{"href":"https:\/\/www.newsbeep.com\/us\/wp-json\/wp\/v2\/media?parent=862339"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.newsbeep.com\/us\/wp-json\/wp\/v2\/categories?post=862339"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.newsbeep.com\/us\/wp-json\/wp\/v2\/tags?post=862339"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}
\n relion_helix_toolbox\u00a0\u2013impose\u2013i\u00a02Hex_221_BS1750.mrc\u2013o 2Hex_221_BS1750_4.77_t0.64.mrc\u2013cyl_outer_diameter 230\u2013angpix 0.8875\u2013rise 4.77\u2013twist -0.64\u2013z_percentage\u00a00.1<\/p>\n