Polyubiquitin ligand-induced phase transitions are optimized by spacing between ubiquitin units

Galagedera S, Dao T, Enos S, Chaudhuri A, Schmit J, Castañeda C
Proceedings of the National Academy of Sciences 120(42) (2023 Oct 12)

doi: 10.1073/pnas.2306638120

Submitted to SASBDB: 2023 Mar 7 Published in SASBDB: 2023 Mar 17

SASDR37 – HOTag6 tetramerization domain followed by a Gly-Ser linker fused to human monoubiquitin Sample: HOTag-GS-Ubiquitin tetramer, 51 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Feb 16 RgGuinier 3.7 nm Dmax 14.0 nm VolumePorod 74 nm3

SASDR47 – HOTag6 tetramerization domain followed by a (Gly-Ser)2 linker fused to human monoubiquitin Sample: HOTag6-(GS)2-Ubiquitin tetramer, 52 kDa Homo sapiens protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2023 Feb 21 RgGuinier 3.7 nm Dmax 13.7 nm VolumePorod 69 nm3

SASDR57 – HOTag6 tetramerization domain followed by a (Gly-Ser)4 linker fused to human monoubiquitin Sample: HOTag-(GS)4-Ubiquitin tetramer, 53 kDa Homo sapiens protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Feb 16 RgGuinier 3.8 nm Dmax 14.3 nm VolumePorod 76 nm3

SASDR67 – HOTag6 tetramerization domain followed by a (Gly-Ser)10 linker fused to human monoubiquitin Sample: HOTag-(GS)10-Ubiquitin tetramer, 56 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Feb 16 RgGuinier 4.0 nm Dmax 14.8 nm VolumePorod 84 nm3

SASDR77 – HOTag6 tetramerization domain followed by a (Gly-Ser)25 linker fused to human monoubiquitin Sample: HOTag6-(GS)25-Ubiquitin tetramer, 65 kDa Homo sapiens protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2023 Feb 21 RgGuinier 4.7 nm Dmax 19.3 nm VolumePorod 117 nm3

SASDR87 – HOTag6 tetramerization domain followed by a (Gly-Ser)50 linker fused to human monoubiquitin Sample: HOTag-(GS)50-Ubiquitin tetramer, 80 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Feb 16 RgGuinier 5.6 nm Dmax 22.7 nm VolumePorod 193 nm3

SASDR97 – HOTag6 tetramerization domain followed by a Pro-Ala linker fused to human monoubiquitin Sample: HOTag-PA-Ubiquitin tetramer, 51 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Feb 16 RgGuinier 3.7 nm Dmax 12.9 nm VolumePorod 73 nm3

SASDRA7 – HOTag6 tetramerization domain followed by a (Pro-Ala)2 linker fused to human monoubiquitin Sample: HOTag6-(PA)2-Ubiquitin tetramer, 52 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Nov 28 RgGuinier 3.8 nm Dmax 15.0 nm VolumePorod 74 nm3

SASDRB7 – HOTag6 tetramerization domain followed by a (Pro-Ala)4 linker fused to human monoubiquitin Sample: HOTag-(PA)4-Ubiquitin tetramer, 53 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Jun 8 RgGuinier 4.1 nm Dmax 14.4 nm VolumePorod 78 nm3

SASDRC7 – HOTag6 tetramerization domain followed by a (Pro-Ala)10 linker fused to human monoubiquitin Sample: HOTag-(PA)25-Ubiquitin tetramer, 68 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Jun 8 RgGuinier 5.1 nm Dmax 18.4 nm VolumePorod 106 nm3

SASDRD7 – HOTag6 tetramerization domain followed by a Gly-Ser linker fused to human monoubiquitin with 200 mM NaCl Sample: HOTag6-(GS)-Ubiquitin tetramer, 51 kDa Homo sapiens protein Buffer: 20 mM sodium phosphate, 200 mM NaCl, 0.5 mM EDTA, 0.02% NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2023 Feb 19 RgGuinier 3.6 nm Dmax 12.6 nm VolumePorod 70 nm3

SASDRE7 – HOTag6 tetramerization domain followed by a (Gly-Ser)4 linker fused to human monoubiquitin with 200 mM NaCl Sample: HOTag6-(GS)4-Ubiquitin tetramer, 53 kDa Homo sapiens protein Buffer: 20 mM sodium phosphate, 200 mM NaCl, 0.5 mM EDTA, 0.02% NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2023 Feb 19 RgGuinier 3.8 nm Dmax 12.8 nm VolumePorod 114 nm3

SASDRF7 – HOTag6 tetramerization domain followed by a (Gly-Ser)10 linker fused to human monoubiquitin with 200 mM NaCl Sample: HOTag6-(GS)10-Ubiquitin tetramer, 56 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 200 mM NaCl, 0.5 mM EDTA, 0.02% NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2023 Feb 19 RgGuinier 4.1 nm Dmax 14.2 nm VolumePorod 86 nm3

SASDRG7 – HOTag6 tetramerization domain followed by a Pro-Ala linker fused to human monoubiquitin with 200 mM NaCl Sample: HOTag6-(PA)-Ubiquitin tetramer, 51 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 200 mM NaCl, 0.5 mM EDTA, 0.02% NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2023 Feb 19 RgGuinier 3.7 nm Dmax 12.4 nm VolumePorod 71 nm3

SASDRH7 – HOTag6 tetramerization domain followed by a (Pro-Ala)4 linker fused to human monoubiquitin with 200 mM NaCl Sample: HOTag6-(PA)4-Ubiquitin tetramer, 53 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 200 mM NaCl, 0.5 mM EDTA, 0.02% NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2023 Feb 19 RgGuinier 4.1 nm Dmax 14.7 nm VolumePorod 80 nm3

SASDRJ7 – HOTag6 tetramerization domain followed by a (Pro-Ala)10 linker fused to human monoubiquitin with 200 mM NaCl Sample: HOTag6-(PA)10-Ubiquitin tetramer, 57 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 200 mM NaCl, 0.5 mM EDTA, 0.02% NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2023 Feb 19 RgGuinier 5.1 nm Dmax 19.0 nm VolumePorod 105 nm3

SASDRK7 – M1-linked tetraubiquitin, M1(1-72)-Ub4 Sample: Polyubiquitin-B monomer, 33 kDa Homo sapiens protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Nov 28 RgGuinier 3.2 nm Dmax 12.5 nm VolumePorod 41 nm3

SASDRL7 – M1-linked tetraubiquitin, M1(1-73)-Ub4 Sample: Polyubiquitin-B monomer, 33 kDa Homo sapiens protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Nov 28 RgGuinier 3.3 nm Dmax 12.8 nm VolumePorod 41 nm3

SASDRM7 – M1-linked tetraubiquitin, M1(1-74)-Ub4 Sample: Polyubiquitin-B monomer, 34 kDa Homo sapiens protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Nov 28 RgGuinier 3.2 nm Dmax 12.2 nm VolumePorod 43 nm3

SASDRN7 – M1-linked tetraubiquitin Pro-Ser/Gly-Ser fusion, M1(1-76)-PS(GS)4-Ub4 Sample: Tetraubiquitin, M1(1-76)-PS(GS)4-Ub4 monomer, 36 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Nov 28 RgGuinier 3.4 nm Dmax 13.9 nm VolumePorod 50 nm3

SASDRP7 – M1-linked tetraubiquitin Glu-Ala-Lys fusion, M1(1-74)-A(EA3K)3A-Ub4 Sample: Tetraubiquitin, M1(1-74)-A(EA3K)3A-Ub monomer, 39 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Nov 28 RgGuinier 4.2 nm Dmax 18.2 nm VolumePorod 62 nm3

SASDRQ7 – M1-linked tetraubiquitin Glu-Ala-Lys fusion, M1(1-74)-A(EA3K)6A-Ub4 Sample: Tetraubiquitin, M1(1-74)-A(EA3K)6A-Ub monomer, 43 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2022 Nov 28 RgGuinier 5.2 nm Dmax 21.2 nm VolumePorod 108 nm3

SASDRK8 – HOTag6 tetramerization domain followed by a (Pro-Ala)25 linker fused to human monoubiquitin Sample: HOTag-(PA)25-Ubiquitin tetramer, 68 kDa synthetic construct protein Buffer: 20 mM sodium phosphate, 0.5 mM EDTA, 0.02 % NaN3, pH: 6.8 Experiment: SAXS data collected at BioCAT 18ID, Advanced Photon Source (APS), Argonne National Laboratory on 2023 Mar 22 RgGuinier 6.3 nm Dmax 24.9 nm VolumePorod 180 nm3