1. ABOUT THE DATASET -------------------- Title: An ALS-Associated Mutation in the C-terminal α-helix of TDP-43 Uncouples Condensate Formation and Amyloid Assembly Creator(s): Emily J. Byrd[1], Joel A. Crossley[1], Chalmers C. C. Chau[2,3], Paolo Actis[2,3], Antonio N. Calabrese[1] Organisation(s): [1] Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK. [2] School of Electronic and Electrical Engineering, University of Leeds, LS2 9JT, UK. [3] Bragg Centre for Materials Research, University of Leeds, LS2 9JT, UK Rights-holder(s): Unless otherwise stated, Copyright 2026 University of Leeds Publication Year: 2026 Description: This data set includes native mass spectrometry and ion mobility mass spectrometry data collected on a Synapt G2 Si HDMS instrument (Waters Corporation at the University of Leeds, processed using MassLynx 4.1. Thioflavin T fluorescence data collected on a FLUOstar omega plate reader (BMG Labtech, Ortenburg, Germany). Nephelometry measurements collected on a Nephelostar plate reader (BMG Labtech, Ortenburg, Germany). SLAB simulations generated using the CALVADOS python package and the CALVADOS2 force field. Cite as: Emily J. Byrd, Joel A. Crossley, Chalmers C. C. Chau, Paolo Actis, Antonio N. Calabrese (2026): An ALS-Associated Mutation in the C-terminal α-helix of TDP-43 Uncouples Condensate Formation and Amyloid Assembly. University of Leeds. [Dataset] https://doi.org/10.5518/1785 2. TERMS OF USE --------------- Copyright 2026 University of Leeds. Unless otherwise stated, this dataset is licensed under a Creative Commons Attribution 4.0 International Licence: https://creativecommons.org/licenses/by/4.0/. 3. PROJECT AND FUNDING INFORMATION ---------------------------------- Title: Molecular basis of phase separation in viral replication and neurodegenerative disease Dates: 01/09/2020-01/09/2026 Funding organisation: Wellcome Trust Grant no.:RG.IMCB.118846 4. CONTENTS ----------- File listing IM-MS data: RAW files acquired on a Synapt G2 Si HDMS instrument (Waters Corporation), analysed using MassLynx V4.1. Nephelometry_CTD 20,170,nacl: Excel spreadsheet of raw TDP-43_LCD_SLAB_DOI: simulations data ThT_raw: Excel spreadsheet of raw ThT fluorescence data collected on a FLUOstar omega plate reader (BMG Labtech, Ortenburg, Germany) 5. METHODS ---------- Recombinant TDP-43 CTD expression and purification A plasmid containing the DNA sequence for the C-terminal domain of TDP-43 (residues 267- 414) was kindly gifted from Nicolas Fawzi (Addgene plasmid #98669) and mutations Q331K and R361S were introduced by site-directed mutagenesis (Q5® site-directed mutagenesis kit, New England Biolabs). For protein expression, chemically competent BL21 DE3 E. coli cells were transformed with plasmids containing the TDP-43 CTD gene. Cells were grown in LB media with kanamycin (50 µg/mL) at 37 ˚C with shaking (200 rpm) to an OD600 of 0.6, and protein expression was induced by the addition of 0.01 mg/mL Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 4 hr at 37°C, 200 rpm. Cells were harvested by centrifugation, resuspended in lysis buffer (20 mM Tris-Cl, 300 mM NaCl, 10 mM Imidazole, 1 mM DTT, pH 8.0) supplemented with EDTA-free protease inhibitor cocktail (Roche) and lysed using a cell disruptor (Constant cell disruption systems). The lysate was centrifuged (20,000 xg, 60 min) and the insoluble material, including inclusion bodies (IBs) containing the expressed TDP-43 CTD, was resuspended in denaturing binding buffer (20 mM Tris-Cl, 300 mM NaCl, 10 mM Imidazole, 1 mM DTT, 8 M Urea, pH 8.0). The solubilised IBs were centrifuged (20,000 xg, 60 min) to remove remaining insoluble debris and then applied to a 5 mL Histrap HP column (Cytiva). The column was washed with 5 column volumes of binding buffer, before elution with a linear gradient (0-100% B) of buffer B (20 mM Tris-Cl, 300 mM NaCl, 500 mM Imidazole, 1 mM DTT, 8M Urea, pH 8.0) over 20 column volumes. For His-tag cleavage, the protein was incubated with Tobacco Etch Virus (TEV) (1:25 w/w) protease at 4°C overnight whilst dialysing into 20 mM MES buffer, pH 5.5. Cleaved protein was resuspended in denaturing binding buffer and reapplied to a 5 mL Histrap HP column (Cytiva). The flow through was collected and concentrated to ~ 1 mM using Amicon® 10 kDa ultra centrifugal filter units (Merck Millipore, Darmstadt, Germany) before being snap-frozen and stored at – 80 °C. Nanopipette nanoESI Emitter fabrication The nanopipette nanoESI emitter tips were fabricated using 1.0 mm outer diameter and 0.5 mm inner diameter quartz capillaries (QF100-50-7.5; Sutter Instrument) with the Sutter Instrument P2000 laser puller (World Precision Instruments). A two-line protocol was used: line 1 with HEAT 750/FIL 4/VEL 30/DEL 150/PUL 80, followed by line 2 with HEAT 850/FIL 3/VEL 40/DEL 135/PUL 225. This protocol generates nanopipettes with openings of ~40nm in diameter58. The pulling protocol is specific to the instrument and can vary between different pullers, so individual optimisation of the protocol is needed in each laboratory. For native ion mobility measurements, the emitters were back-filled with analyte solution and fitted with a platinum wire (PT00-WR-000117; Goodfellow) prior use. Native ion mobility mass spectrometry Native IM-MS experiments were performed on a Synapt G2-Si HDMS mass spectrometer (Waters Corporation, Wilmslow, UK) with travelling (T-wave) ion mobility and a nano-ESI source. TDP-43 CTD variants (WT, Q331K, R361S) were analysed at a concentration of 10 µM in 20 mM ammonium acetate, pH 5.5. Instrument parameters were as follows: capillary voltage 1-1.4 kV, source temperature 30 °C, sampling cone 18 V, trap collision energy 5 V, transfer collision energy 2.0 V, trap DC bias 30 V, IM wave velocity 550 m/s, IM wave height 10 V. The trap gas flow was 2.0 mL/min, IMS gas flow was 6.0 mL/min (using N2 gas) and the helium gas flow was 80.0 mL/min. The IM spectra were calibrated65 using denatured cytochrome c (charge states 13+ to 19+), myoglobin (charge states 15+ to 24+) and ubiquitin (charge states 7+ to 13+) (10 µM solutions of each calibrant in 50 % (v/v) acetonitrile, 0.1 % (v/v) formic acid were used for calibration) to obtain CCS distributions for each TDP-43 CTD charge state detected. Here we term these values TWCCSN2, consistent with community standards66, signifying that the CCS values were calculated using traveling wave ion mobility in N2 buffer gas using calibrants acquired in N2 buffer gas. MassLynx v4.1 (Waters Corporation) was used for data processing. For collision induced unfolding (CIU), instrument parameters were identical except for the trap collision energy which was systematically increased from 5 V to 60 V in 5 V increments. Arrival time distributions for the 7+ charge state were selected to generate CIU plots. Drift time profiles were extracted at each collision voltage from the spectral peak using the full width of the peak at half maximum (FWHM) intensity. ThT amyloid assembly kinetics Kinetics of TDP-43 CTD amyloid formation were monitored in a 96-well, non-binding, flat-bottom microplate (Corning; 10438082). Samples (100 µL) containing 50 µM protein with 20 µM Thioflavin T (ThT) in 20 mM ammonium acetate, pH 5.5 with 0 mM NaCl and 150 mM NaCl were incubated at room temperature, quiescently in a FLUOstar Omega plate reader (BMG Labtech). Fluorescence intensity was measured by exciting at 440 ± 10 nm and collecting emission at 482 ± 12 nm using a bandpass filter. Four replicate measurements were conducted, and results were blank corrected and normalised to the maximum fluorescence value of each curve except for when amyloid formation did not occur (in the case of Q331K TDP-43 CTD variant in the absence of added NaCl) the curve was normalised to WT no salt as a control. Negative Stain Transmission Electron Microscopy A sample of 5 μL was taken from the ThT plate at the endpoint of each reaction, loaded onto a glow discharged (30s, Pelco Easi-glow), 400 mesh continuous carbon grid, and incubated for 2 min. The sample was blotted and washed twice with H2O before being blotted and stained twice with 2% (w/v) uranyl acetate. Grids were imaged on FEI Tecnai T12 electron microscope using a nominal magnification of 30,000×. DIC microscopy 100 µL of the TDP-43 CTD (WT, Q331K or R361S) protein was added at a concentration of 50 µM, in 20 mM ammonium acetate, pH 5.5 containing 0 mM or 150 mM NaCl to individual wells of an 18-well high glass bottom chamber slide (Ibidi). To make phase diagrams, 10 µM, 50 µM, 100µM and 200 µM was imaged at 0 mM, 50 mM and 150 mM NaCl in 20 mM ammonium acetate, pH 5.5. Condensates were imaged using a LSM700 Airyscan confocal microscope (Zeiss) using a DIC20x 0.3 objective. Nephelometry WT, Q331K and R361S CTD variants were added to a 96-well, non-binding, flat-bottom, half-area microplate (Corning, USA; 10629151) at a concentration of 50 µM in 20 mM ammonium acetate, pH 5.5 with 0 mM NaCl or 150 mM NaCl added. Light scattering of 40 μL of each solution was then monitored using a Nephelostar plate reader (BMG Labtech, Ortenburg, Germany) using an excitation wavelength of 635 ± 10 nm, over 6 h at 25 °C. Data were blank corrected and four replicate measurements were conducted. Coarse-grained molecular dynamics simulations Coarse-grained implicit-solvent simulations of αS were performed in the in the OpenMM framework (version 8.1.1)67, 68 using the CALVADOS python package and the CALVADOS2 force field49. Simulations were performed for TDP-43 CTD WT, Q331K and R361S at 0 and 125 mM ionic strength. Unless stated otherwise simulation parameters used were kept as their default values for slab simulations in the software. Simulations were performed in a 15 x 15 x 150 nm box with 100 TDP-43 CTD molecules at pH 7.4. Following energy minimization, simulations were run at a temperature of 21 °C for a total of 10 μs each with coordinates being saved every 0.1 ns. The simulation had a time step of 10 fs and a friction coefficient of 10 fs−1. The first 0.1 μs of each simulation was considered to still be part of the equilibration time and therefore not included in the analysis. To reduce the total number of frames in the analysis, every 10th frame was included such that the analysis was performed on frame each 1 ns. The Csat was calculated with inbuilt functions in the CALVADOS software, where the concentration is calculated for frames with z values greater than= ~50 nm and less than ~-50 nm (i.e., outside the dense phase). Contact maps were calculated between the chain closest to the mid-plane (z = 0 nm) of the protein-dense slab and all the surrounding chains.]