(A) Domain architecture diagrams for IC from Drosophila melanogaster (Dros. IC) and Rattus norvegicus (Rat IC2C) and constructs used in earlier work are provided for comparison. Proteins and …
Alignment of the first 260 residues of IC from Drosophila, human, and Chaetomium thermophilum using the Multiple Alignment using Fast Fourier Transform (MAFFT) alignment program (Katoh and Standley, …
(A) Sedimentation velocity analytical ultracentrifugation profiles for LC8 (red), LC7 (yellow), and Tctex (orange) (top), and, IC1-260 (gray), p150CC1B (blue), and NudECC (green) (bottom). All …
Source files for sedimentation velocity analytical ultracentrifugation, sedimentation equilibrium analytical ultracentrifugation, circular dichroism, and size exclusion chromatography with multi-angle light scattering data.
This Excel workbook contains all the data plotted in Figure 2. The different sheets correspond to different panels within the figure. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.
(A) Agadir prediction for IC1-260 showing the percent helicity by residue (purple). Shown above the plot is a schematic structure for IC1-260 with labels for single α-helix (SAH), H2, and H3 above …
Source files for IC1-260 helicity prediction and CD data.
This Excel workbook contains all the data plotted in Figure 3. The different sheets correspond to different panels within the figure. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.
Residue-level percent helicity predictions generated using the Agadir algorithm for the first 260 amino acids of IC from Homo sapiens (human), Rattus norvegicus (rat), Danio rerio (zebrafish), Callor…
Source file for helix prediction of intermediate chain across species.
This Excel workbook contains the data plotted for the helix predictions shown in Figure 3—figure supplement 1. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.
(A) Domain architecture diagrams of IC1-260, IC1-88, and IC100-260. (B) SV-AUC experiments of IC100-260 (gray), IC100-260 mixed with IC1-88 at a 1:2 molar ratio (black), and IC1-260 (gray dashes). (C…
Source file for sedimentation velocity analytical ultracentrifugation (SV-AUC) and size exclusion chromatography with multi-angle light scattering (SEC-MALS) of binding interactions between IC1-88 and IC100-260.
This Excel workbook contains the data plotted for the SV-AUC and SEC-MALS data shown in Figure 3—figure supplement 2 with different sheets corresponding to different panels within the figure. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.
(A) Plot showing the normalized peak volumes at 10°C in the 1H-15N TROSY spectrum (gray) and in the CLEANEX spectrum (orange) of the amides that could be assigned. Assigned residues are in black in …
Source files for IC1-260 nuclear magnetic resonance (NMR) TROSY and CLEANEX data.
This Excel workbook contains the peak heights and volumes for residues within IC1-260 in the TROSY and CLEANEX NMR experiments collected at 10°C. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.
1H-15N TROSY spectra of IC1-260 in 20 mM NaCl (black) overlaid with IC1-260 in 250 mM NaCl (pink) at both 10°C (left) and at 40°C (right).
(A) Domain architecture diagram for IC1-260 with bars shown below corresponding to the IC100-260, IC1-88, and IC160-240 constructs. (B) 1H-15N TROSY overlays of free 15N-labeled IC1-88 (black) and 15…
Source files for nuclear magnetic resonance binding studies of intermediate chain (IC) fragments.
This Excel workbook contains the peak heights and volumes for residues within the 15N-labeled IC1-88 and 15N-labeled IC216-260 constructs upon binding of IC100-260, IC160-240, and 15N-labeled IC216-260 or 15N-labeled IC1-88. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.
(A) Sedimentation velocity analytical ultracentrifugation profiles for samples containing p150CC1B (blue dashed line), NudECC (green dashed line), IC1-88/p150CC1B complex (blue solid line), and IC1-8…
Source files for sedimentation velocity analytical ultracentrifugation (SV-AUC) and nuclear magnetic resonance (NMR) binding studies of intermediate chain (IC) fragments with p150CC1B and NudECC.
This Excel workbook contains the data plotted for the SV-AUC experiments as well as the NMR peak volume ratios for binding studies between 15N-labeled IC1-88 and 15N-labeled IC37-88 with p150CC1B and NudECC. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.
The estimated mass of p150CC1B from MALS is 49 kDa (light blue) while the addition of IC1-88 increases the mass to 66 kDa (dark blue) indicating a complex containing a p150CC1B dimer and two IC1-88 …
This Excel workbook contains the data plotted for the size exclusion chromatography with multi-angle light scattering data shown in Figure 6—figure supplement 1.
Data were plotted using gnuplot.
ITC thermograms for p150CC1B titrated with IC37-88 (left) and NudECC titrated with IC37-88 (right), collected at 25°C (pH 7.5). IC37-88 contains the H2 region of intermediate chain (IC) and binds …
(A) Sedimentation velocity analytical ultracentrifugation of IC1-260/LC8, IC1-260/Tctex, IC1-260/LC7, IC1-260/p150CC1B, and IC1-260/NudECC. Data for the binary complexes is overlayed with data for …
Source files for sedimentation velocity analytical ultracentrifugation (SV-AUC) binding studies of IC1-260.
This Excel workbook contains the data plotted for the SV-AUC experiments shown in Figure 7. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.
(A) Size exclusion chromatography (SEC) traces of the dynein subcomplex (IC/light chains) (purple) and the dynein subcomplex with the addition of either p150CC1B (blue) or NudECC (green). (B) Sodium …
Source files for size exclusion chromatography (SEC) and sedimentation velocity analytical ultracentrifugation (SV-AUC) of reconstituted IC1-260 subcomplexes.
This Excel workbook contains the data plotted for the SEC and SV-AUC experiments shown in Figure 8 with individual sheets corresponding to panels within the figure. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.
Original sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel images.
This zipped folder contains the original files of the full raw unedited gel images for the size exclusion chromatography purification of each IC1-260 subcomplex. There is also a combined image with the uncropped gels with the relevant bands clearly labeled.
(A) The estimated mass of ICFL from multi-angle light scattering is 75.4 kDa, which indicates that ICFL is a monomer in the absence of binding partners. (B) Sedimentation velocity analytical …
Source files for size exclusion chromatography with multi-angle light scattering (SEC-MALS) and sedimentation velocity analytical ultracentrifugation (SV-AUC) of ICFL.
This Excel workbook contains the data plotted for the SEC-MALS experiment of ICFL and the SV-AUC binding experiments shown in Figure 9 with individual sheets corresponding to panels within the figure. Additional information regarding data collection can be found in the corresponding Methods section. Data were plotted using gnuplot.
Original sodium dodecyl sulphate–polyacrylamide gel electrophoresis gel image.
This zipped folder contains the original file of the full raw unedited gel image for the IMAC fractions of ICFL. There is also an image with the uncropped gel with the relevant bands clearly labeled. Notice, two batches of ICFL were purified in tandem.
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Chaetomium thermophilum) | Ct IC | NA | UniProt: G0SCF1-1 | |
Strain, strain background (Escherichia coli) | Rosetta (DE3) | Sigma-Aldrich | Catalog number: 70954 | |
Cell line (Insect) | Sf9 | ThermoFisher | Catalog number: 11496015 | Used within 1 yr of purchase |
Strain, strain background (E. coli) | DH10EMBacY | Geneva Biotech | DH10EMBacY | Used within 1 yr of purchase |
Peptide, recombinant protein | Ct IC full length protein | GenScript | pFastbac1 vector | |
Peptide, recombinant protein | IC1-88 | Ref. 32 | ||
Peptide, recombinant protein | IC37-88 | GenScript | ||
Peptide, recombinant protein | IC160-240 | GenScript | ||
Peptide, recombinant protein | IC216-260 | Azenta Life Sciences | ||
Chemical compound, drug | Ammonium- 15N chloride | Sigma Aldrich | Catalog number: 299251 | |
Chemical compound, drug | D-glucose 13C6 | Sigma Aldrich | Catalog number: 389374 | |
Software, algorithm | Origin 7.0 | OriginLab | ||
Software, algorithm | SEDFIT | Open-source | ||
Software, algorithm | TopSpin 3.6 | Bruker Biospin Corporation | RRID:SCR_014227 | |
Software, algorithm | CcpNmr analysis (CCPN) | Ref. 80 | RRID:SCR_016984 | |
Software, algorithm | NMRPipe | Ref. 78 |
ITC results for intermediate chain interactions with p150CC1B and NudECC at 25°C.
aFor the p150CC1B experiments, a two-site binding model was used as it provided much better fits when compared to a single site model. bFitting of this experiment was done with set N values due to the amount of extra noise in the data and thus the N values are reported without error. cThis experiment did not show evidence of binding and thus the data was not fit to a binding model.