Dataset DOI: 10.5061/dryad.5mkkwh7jf

Description of the data and file structure

This dataset includes the data and quantification files for generating the figures in the related manuscript.

All the files generated from RF-diffusion peptide are also included to help reproducing.

Microscopy files that used for generate example figures in the manuscript (aec0818, in press) a also included, but for the other raw microscopy files, due to the size, are not uploaded. For requesting them, please write to longlong.wang@pharma.ethz.ch or longlongwang@outlook.com

Files and variables

File: output_of_UBiP_design.zip

Description: file for evaluating the design; this file contains the selected 11 novel ubiquitin binding protein (UBiP) candidates, designed by RF-diffusion/ProteinMPNN. The UBiPs are referred to as "UBP01" through "UBP11" in folder/file names.

For each UBiPs, the folder contains the command for design in the included .sh files, and also the generated Excel file for evaluating the design. The variables in the "results" CSV files are described below, in the section titled "Variables in tabular files".

Relevant FASTA and/or molecular model (.pdb, .pse) files are included. Where relevant, .png images of molecular models are also provided. If multiple models or designs were tested for a single UBiP, the related files are stored within subfolders. Starting models have "starting model" in the file name.

File: AF2_prediction_for_ipTM_in_table_1.zip

Description: AlphaFold2 predictions of 10 of the 11 Ub–UBiP complexes (excluding UBP05), from which the ipTM values in Table 1 were taken. Organised by UBiP; each folder contains the predicted complex structures (.pdb/.cif), the input sequences/alignments (.fasta, .sto), and a "result" subfolder with the confidence metrics (.json / summary table). Higher ipTM (>0.8) indicates a confidently predicted Ub–UBiP interface.

Additional description of predictions, analyses, and results can be found in the "results.html" files in each subdirectory. These files can be found within the "predictions" subfolder of each UBiP-named directory.

File: Fig_1.zip

Description: Affinity maturation of the HDAC6 ZnF domain for mono-Ub.

Subfolders by panel:

•    Fig_1c — isothermal titration calorimetry (ITC) of mHDAC6_(1007–1149) (and mutants) against mono-Ub: raw .itc plus exported fitting reports.

•    Fig_1f to h/PCA analysis — processed yeast deep-mutational-scanning tables, one per assay (BindingPCA, AbundancePCA), one row per variant.
The variables in these files are described below, in the section titled "Variables in tabular files". The file "Collapsed_reads_per_sample_abundance_screen_meta.csv" contains metadata about each input and output variable (the counts of sequencing reads before and after selection).

•    Fig_1i — ITC of the V1091L mutant against mono-Ub.

In the folders for Fig 1c and Fig 1i, associated Origin Project (.opj) files are provided alongside the ITC raw files. These can be opened in Origin Lab's free viewer software.

File: Fig_2.zip

Description: In silico design of the UBiPs and their affinity for mono-Ub.

Subfolders by panel:

•    Fig_2c, Fig_2e, Fig_2f — ITC raw .itc files and exported fitting reports for UBiP05, UBiP10 (folders Fig 2c and 2e), and the two UBiP10 mutants (folder Fig 2f). Associated Origin Project (.opj) files are provided alongside the ITC raw files. These can be opened in Origin Lab's free viewer software.

File: Fig_3.zip

Description: selectivity of UBiP10 for unanchored Ub

Subfolders by panel:

•    Fig_3b — raw mass-spectrometry files (in .xy format) and exported peak tables of the Lb^pro clipping experiment (PDFs).

•    Fig_3c, 3d, 3f, 3g, 3h, 3i, 3j, 3k — uncropped, unadjusted scans of every western blot (.tif/.pdf), one file per membrane/antibody, with lane and antibody key.

•    Fig_3e — ITC of UBiP10 against K48- and K63-linked di-Ub (raw .itc files + reports in OPJ format, with relevant plots included as TIFFs).

•    Fig_3l — AP-MS search result tables used for the volcano plot.

File: Fig_4.zip

Description: detection and imaging of unanchored Ub in cells.

Subfolders by panel:

•    Fig 4a and b — microscopy acquisitions and exported example images for the four cell lines and three treatments, with the ImageJ quantification tables and the Prism file used for the bar plot and statistics. Folders are named according to cell line (A549, Hela, MEF, and SW1353).

•    Fig 4c — microscopy acquisitions and example images of influenza virions (channels: viral HA, unanchored Ub, merge).

•    Fig 4d and Fig 4e — microscopy acquisitions for GST-UBiP05, GST-UBiP10 and GST staining, plus intensity tables and the Prism file.

File: Fig_5_new.zip

Description: an unanchored Ub coat around the aggresome.

Subfolders by panel:

•    Fig 5a and Fig 5b — microscopy acquisitions and example images, plus the ImageJ line-profile exports (distance vs grey value) for the two enlarged aggresomes. The distance vs grey value exports are stored in the four CSV files in this folder. For each, the two variables are Distance_(microns) and Gray_Value.

•    Fig 5c and g — free (unanchored) Ub intensity quantification at the three positions, as tables plus the Prism file with the plot and ANOVA.

•    Fig 5d — uncropped western blot scans for the myosin-10 KD and HDAC6 KO validation.

•    Fig 5e and Extendend Data Fig 6c — microscopy acquisitions and example images of scRNA, siMyo and HDAC6-KO cells, with the ImageJ line-profile and intensity tables (in CSV format; variables are Distance_(microns) and Gray_Value).

File: supplementary_figures_new.zip

Description: One folder per supplementary figure (Fig_S1 … Fig_S7), with panel subfolders inside.

•    Fig_S1 — affinities of the various HDAC6 constructs for mono-Ub. Structural detail of ZnF selectivity (A) and the scheme of the yeast mutagenesis assay (D); ITC of hHDAC6_(1073–1215) (B), full-length hHDAC6 (C) and the mouse mutants Y1039R, A1040K, S1064H and S1064H/V1091L (E–H).

Files: raw .itc and exported reports (OPJ format) per construct, plus the structure files for A and I.

•    Fig S2 and Table S1 — summary of the design campaign: six starting models were used and UBiP01–UBiP11 selected by RMSD to the RF-diffusion design.

Files: one folder per UBiP with the .sh command, the RF-diffusion/ProteinMPNN result .csv and the models. The variables in the "results" .csv files are described below, in the section titled "Variables in tabular files".

•    Fig S3 — purification of UBiP01–UBiP10 and their affinities for mono-Ub (with tUI for comparison), the modelled UBiP05–Ub interface, and purification of the UBiP10 mutants.

Files: one folder per UBiP with raw .itc and reports (OPJ format), plus gel scans and structure files.

•    Fig_S4 — interaction of UBiP10 with unanchored Ub chains: AlphaFold3 models of UBiP05/UBiP10 with Ub lacking its C-terminal tail, with linear di-Ub, and of tUI–Ub (A, B, F, G); purification of K48/K63 di-Ub (C); superposition with di-Ub structures (D, E); immunoblots of HA-tagged construct pulldowns and of GST pulldowns from di-Ub-supplemented and Poly (I:C)-treated lysates (H–J); IAV uncoating assay measured as the M1-spreading cell ratio (K).

Files: structures, uncropped blot scans, and the uncoating quantification table with its Prism file.

•    Fig_S5 — validation of the UBiPs for immunofluorescence: staining workflow (A), background of the GST control (B), absence of aggregate staining by UBiP10 (C), ELISA on immobilised 3xUb-RARA/mono-Ub mixtures probed with GST-UBiP05 or GST-UBiP10 (D), linear fluorescence response up to 10 µg/mL Ub and its calibration (E, F), quantification of the Ub-antibody signal from Fig. 4D (G), and the estimated unanchored fraction of total Ub (H).

Files: microscopy acquisitions and example images, the ELISA raw plate readings, and the quantification tables with Prism files.

•    Fig_S6 — unanchored Ub during aggresome formation: a bortezomib time course imaged with anti-Ub and GST-UBiP10 (A); the Fiji mask workflow used for the three-position quantifications of Fig. 5C and 5G (B: aggresome = Mask 1, peri-aggresomal ring = dilated minus original, remote area = cell outline minus eroded outline); and the control scRNA condition (C).

Files: microscopy acquisitions, example images, and quantification tables reporting distance (microns) and gray value.

•    Fig_S7 — the aggresome experiment repeated in differentiated SH-SY5Y cells (bortezomib 2 µM, 24 h): imaging (A), line profiles (B), three-position quantification of ca. 40 aggresomes (C), and a comparison of UBiP10 with a TUBE reagent (D).

Files: microscopy acquisitions, example images, quantification tables and the Prism file.

Variables in tabular files:

Design / prediction score tables (design output, fig. S2, Table 1):

• design/model = design identifier;
• seq = designed sequence;
• mpnn = ProteinMPNN sequence score (dimensionless, lower = better);
• plddt = AlphaFold per-residue confidence (0–100, some files 0–1);
• ptm = fold confidence (0–1);
• i_ptm/iptm = interface confidence (0–1, the value in Table 1);
• pae = predicted aligned error (Å, lower = better);
• rmsd = deviation between the AlphaFold2 prediction and the RF-diffusion design (Å, the ranking criterion).
• Remaining columns (seed, contig, sample number, recycles) are pipeline bookkeeping.

BindingPCA / AbundancePCA tables (Fig. 1F–H): one row per single amino-acid variant —

• Input1–6 / Input1–4 — read counts before selection (the library as built)
• Output1–6 / Output1–4 — counts after selection; the ratio to input is the signal
• Zinc finger variant — the 216 nt (72 codon) mutagenised region carried by that variant
• Barcode — 20 nt tag; 41,722 unique barcodes, median ~10 per variant
• Variant Name — WT amino acid + position + mutant, * = stop codon
• logFC — log2(output/input), the raw PCA fitness score. Negative = depleted = destabilised (abundance) or reduced binding (interaction).
• Relative logFC (dLFC) —It's logFC minus the wild-type's logFC, so WT sits at 0.
• P-Value / Adjusted P-Value — test of logFC ≠ 0, and its FDR-corrected version
• Standard Error logFC — uncertainty on the estimate; use it to filter or weight low-confidence variants
• Wildtype Amino Acid / Mutant Amino Acid / Protein Position — just parsed out of the mutation name for convenience

Code/software

No code was generated, and for graph plotting, we used graphpad prism 10, and for microscopy picture processing, we used ImageJ. ver 1.54p.

.sto files: Stockholm multiple-sequence alignment format; can be read using HMMER, Jalview, UGENE, or a text editor. UGENE explicitly supports Stockholm .sto files.

.itc files: are raw isothermal titration calorimetry (ITC) data files and can be opened and analyzed using the MicroCal PEAQ-ITC Analysis Software or compatible MicroCal ITC analysis software.

All the code used in this manuscript have been cited.