LiteFold/DisProt huggingface dataset:
Protein: DP04273 (UniProt P33609)
Region: residues 1	to 330
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The CD spectra showed that p180 (1–330) was a random coiled protein without a typical alpha helix or beta sheet structure.
Protein: DP04273 (UniProt P33609)
Region: residues 220	to 330
Annotation: protein binding (Molecular function)
Evidence: yeast 2-hybrid evidence used in manual assertion
Supporting statements:
Mouse POT1a was associated with the N-terminal region (1–330) of p180 (Figure 1a).
The N-terminally truncated POT1a mutant (307–640; Figure 1b) and the N-terminal fragment of p180 (220–330; Figure 1c) interacted with a similar affinity to the interaction between full-length POT1a and p180 (1–330). We conclude that POT1a (307–640) and p180 (220–330) are the interaction domains required for the POT1a-p180 interaction.
Protein: DP04273 (UniProt P33609)
Region: residues 1	to 330
Annotation: protein binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
His-p180 (1–330) associated with GST-POT1a (314–640) and GST- POT1b (313–640), but not the GST tag alone, despite being present in excess (Figure 1d). Hence, each of the C-terminal fragments of POT1a (314–640) or POT1b (313–640) and the N-terminal fragment (1–330) of p180 were physically associated in vitro.
Protein: DP04274 (UniProt Q9Y2G2)
Region: residues 1	to 162
Annotation: disorder (Structural state)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
CARD8 only has a predicted N-terminal ~160-amino-acid-long unstructured region followed by a FIIND and a CARD (Figure 1A; Figure S1B).
(A) Diagram of human CARD8 (above) and its predicted disorder (below) as determined by the Sequence-Based Prediction of Disordered Residues for Proteins (SPOT-Disorder) program (Hanson et al., 2017).
Consistent with these computational assessments, we observed that limited proteolysis of full-length MTMR1, D2HGDH, and CARD8 with trypsin or proteinase K resulted in rapid protein digestion (Figure 4B). In contrast, truncated proteins lacking the predicted disordered N-terminal regions were more stable under protease treatment (Figure 4B).
When the protein is lacking the first 162 residues it becomes more stable to proteolysis. The fact that this N-terminal region is prone to proteolysis, combined with the result of disorder predictors allows us to identify this region as disordered.
Protein: DP04274 (UniProt Q9Y2G2)
Region: residues 1	to 162
Annotation: flexible N-terminal tail (Disorder function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
CARD8 only has a predicted N-terminal ~160-amino-acid-long unstructured region followed by a FIIND and a CARD (Figure 1A; Figure S1B).
(A) Diagram of human CARD8 (above) and its predicted disorder (below) as determined by the Sequence-Based Prediction of Disordered Residues for Proteins (SPOT-Disorder) program (Hanson et al., 2017).
Consistent with these computational assessments, we observed that limited proteolysis of full-length MTMR1, D2HGDH, and CARD8 with trypsin or proteinase K resulted in rapid protein digestion (Figure 4B). In contrast, truncated proteins lacking the predicted disordered N-terminal regions were more stable under protease treatment (Figure 4B).
Protein: DP04274 (UniProt Q9Y2G2)
Region: residues 1	to 162
Annotation: positive regulation of inflammatory response (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
However, VbP induced GSDMD cleavage and lactate dehydrogenase (LDH) release only in cells expressing full-length CARD8 (Figures 1B and 1C), indicating that the predicted disordered region of CARD8 plays a functional role in VbP-induced inflammasome activation.
Protein: DP04274 (UniProt Q9Y2G2)
Region: residues 1	to 162
Annotation: protein destabilization (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
We discovered that VbP only induced the degradation of the full-length proteins (Figure 4C), demonstrating that the predicted disordered regions are required for the degradation of all three proteins.
Protein: DP04275 (UniProt Q84T68)
Region: residues 177	to 191
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
No observable electron density for the region.
Cross-references: PDB:3HKM
Protein: DP04276 (UniProt Q13613)
Region: residues 1	to 94
Annotation: disorder (Structural state)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
MTMR1, D2HGDH, and other significantly depleted proteins have predicted disordered regions (Figure 4A; Figure S4A). Consistent with these computational assessments, we observed that limited proteolysis of full-length MTMR1, D2HGDH, and CARD8 with trypsin or proteinase K resulted in rapid protein digestion (Figure 4B). In contrast, truncated proteins lacking the predicted disordered N-terminal regions were more stable under protease treatment (Figure 4B).
When the protein is lacking the first 94 residues it becomes more stable to proteolysis. The fact that this N-terminal region is prone to proteolysis, combined with the result of disorder predictors allows us to identify this region as disordered.
Protein: DP04276 (UniProt Q13613)
Region: residues 1	to 94
Annotation: flexible N-terminal tail (Disorder function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
Consistent with these computational assessments, we observed that limited proteolysis of full-length MTMR1, D2HGDH, and CARD8 with trypsin or proteinase K resulted in rapid protein digestion (Figure 4B). In contrast, truncated proteins lacking the predicted disordered N-terminal regions were more stable under protease treatment (Figure 4B).
When the protein is lacking the first 94 residues it becomes more stable to proteolysis. The fact that this N-terminal region is prone to proteolysis, combined with the result of disorder predictors allows us to identify this region as disordered.
Protein: DP04276 (UniProt Q13613)
Region: residues 1	to 94
Annotation: protein destabilization (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
We discovered that VbP only induced the degradation of the full-length proteins (Figure 4C), demonstrating that the predicted disordered regions are required for the degradation of all three proteins.
Protein: DP04277 (UniProt Q8N465)
Region: residues 1	to 51
Annotation: disorder (Structural state)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
MTMR1, D2HGDH, and other significantly depleted proteins have predicted disordered regions (Figure 4A; Figure S4A). Consistent with these computational assessments, we observed that limited proteolysis of full-length MTMR1, D2HGDH, and CARD8 with trypsin or proteinase K resulted in rapid protein digestion (Figure 4B).
When the protein is lacking the first 51 residues it becomes more stable to proteolysis. The fact that this N-terminal region is prone to proteolysis, combined with the result of disorder predictors allows us to identify this region as disordered.
Protein: DP04277 (UniProt Q8N465)
Region: residues 1	to 51
Annotation: flexible N-terminal tail (Disorder function)
Evidence: combinatorial experimental and author inference evidence contained in single publication used in manual assertion
Supporting statements:
MTMR1, D2HGDH, and other significantly depleted proteins have predicted disordered regions (Figure 4A; Figure S4A). Consistent with these computational assessments, we observed that limited proteolysis of full-length MTMR1, D2HGDH, and CARD8 with trypsin or proteinase K resulted in rapid protein digestion (Figure 4B).
When the protein is lacking the first 51 residues it becomes more stable to proteolysis. The fact that this N-terminal region is prone to proteolysis, combined with the result of disorder predictors allows us to identify this region as disordered.
Protein: DP04277 (UniProt Q8N465)
Region: residues 1	to 51
Annotation: protein destabilization (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
We discovered that VbP only induced the degradation of the full-length proteins (Figure 4C), demonstrating that the predicted disordered regions are required for the degradation of all three proteins.
Protein: DP04278 (UniProt Q9UMY1)
Region: residues 1	to 94
Annotation: disorder (Structural state)
Evidence: combinatorial experimental and author inference evidence used in manual assertion
Supporting statements:
Both of a per residue model confidence score (pLDDT) and predicted aligned error (PAE) supported by AlphaFold expected that N-terminal region of human PQBP3/NOL7 was unstructured (Fig. 2b), similarly to the prediction by the IUPred2A.
Consistent with the predictions that PQBP3/NOL7 is an IDP, HS-AFM revealed that a large portion of the purified PQBP3/NOL7 protein outside of globular region (Fig. 3a), which moved dynamically in live imaging (Fig. 3b, Supplementary Movie 1).
Multiple bioinformatics analyses predict that N-terminal region of PQBP3 is unstructured. High-speed atomic force microscopy (HS-AFM) reveals that N-terminal region of PQBP3 is dynamically changed in the structure consistently with the predictions of the bioinformatics analyses.
Figure 4 shows the region the authors describe as an IDR is the 1-94 N-terminal region of the protein.
Protein: DP04278 (UniProt Q9UMY1)
Region: residues 1	to 94
Annotation: flexible N-terminal tail (Disorder function)
Evidence: combinatorial experimental and author inference evidence used in manual assertion
Supporting statements:
Both of a per residue model confidence score (pLDDT) and predicted aligned error (PAE) supported by AlphaFold expected that N-terminal region of human PQBP3/NOL7 was unstructured (Fig. 2b), similarly to the prediction by the IUPred2A.
Consistent with the predictions that PQBP3/NOL7 is an IDP, HS-AFM revealed that a large portion of the purified PQBP3/NOL7 protein outside of globular region (Fig. 3a), which moved dynamically in live imaging (Fig. 3b, Supplementary Movie 1).
Multiple bioinformatics analyses predict that N-terminal region of PQBP3 is unstructured. High-speed atomic force microscopy (HS-AFM) reveals that N-terminal region of PQBP3 is dynamically changed in the structure consistently with the predictions of the bioinformatics analyses.
Figure 4 shows the region the authors describe as an IDR is the 1-94 N-terminal region of the protein.
Protein: DP04279 (UniProt P49815-5)
Region: residues 50	to 126
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues of TSC1 (876–971), TSC2 (127–936, 1015–1082, 1182–1245, 1494–1732), and TBC1D7 (21–287) were traced and modeled with a TSC2a region (residues 127–315) and TSC1 (residues 746–875) being replaced by poly alanine due to the relatively weak cryo-EM density (Fig. 1a and Supplementary Fig. 4).
Other TSC1 regions were invisible in our cryo-EM map due to flexibility.
Cross-references: PDB:7DL2
EMDB:30708
Protein: DP04279 (UniProt P49815-5)
Region: residues 644	to 682
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues of TSC1 (876–971), TSC2 (127–936, 1015–1082, 1182–1245, 1494–1732), and TBC1D7 (21–287) were traced and modeled with a TSC2a region (residues 127–315) and TSC1 (residues 746–875) being replaced by poly alanine due to the relatively weak cryo-EM density (Fig. 1a and Supplementary Fig. 4).
Other TSC1 regions were invisible in our cryo-EM map due to flexibility.
Cross-references: PDB:7DL2
EMDB:30708
Protein: DP04279 (UniProt P49815-5)
Region: residues 937	to 970
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues of TSC1 (876–971), TSC2 (127–936, 1015–1082, 1182–1245, 1494–1732), and TBC1D7 (21–287) were traced and modeled with a TSC2a region (residues 127–315) and TSC1 (residues 746–875) being replaced by poly alanine due to the relatively weak cryo-EM density (Fig. 1a and Supplementary Fig. 4).
Other TSC1 regions were invisible in our cryo-EM map due to flexibility.
Cross-references: PDB:7DL2
EMDB:30708
Protein: DP04279 (UniProt P49815-5)
Region: residues 1039	to 1137
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues of TSC1 (876–971), TSC2 (127–936, 1015–1082, 1182–1245, 1494–1732), and TBC1D7 (21–287) were traced and modeled with a TSC2a region (residues 127–315) and TSC1 (residues 746–875) being replaced by poly alanine due to the relatively weak cryo-EM density (Fig. 1a and Supplementary Fig. 4).
Other TSC1 regions were invisible in our cryo-EM map due to flexibility.
Cross-references: PDB:7DL2
EMDB:30708
Protein: DP04279 (UniProt P49815-5)
Region: residues 1202	to 1426
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues of TSC1 (876–971), TSC2 (127–936, 1015–1082, 1182–1245, 1494–1732), and TBC1D7 (21–287) were traced and modeled with a TSC2a region (residues 127–315) and TSC1 (residues 746–875) being replaced by poly alanine due to the relatively weak cryo-EM density (Fig. 1a and Supplementary Fig. 4).
Other TSC1 regions were invisible in our cryo-EM map due to flexibility.
Cross-references: PDB:7DL2
EMDB:30708
Protein: DP04279 (UniProt P49815-5)
Region: residues 1689	to 1740
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues of TSC1 (876–971), TSC2 (127–936, 1015–1082, 1182–1245, 1494–1732), and TBC1D7 (21–287) were traced and modeled with a TSC2a region (residues 127–315) and TSC1 (residues 746–875) being replaced by poly alanine due to the relatively weak cryo-EM density (Fig. 1a and Supplementary Fig. 4).
Other TSC1 regions were invisible in our cryo-EM map due to flexibility.
Cross-references: PDB:7DL2
EMDB:30708
Protein: DP04279 (UniProt P49815-5)
Region: residues 1689	to 1740
Annotation: flexible C-terminal tail (Disorder function)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues of TSC1 (876–971), TSC2 (127–936, 1015–1082, 1182–1245, 1494–1732), and TBC1D7 (21–287) were traced and modeled with a TSC2a region (residues 127–315) and TSC1 (residues 746–875) being replaced by poly alanine due to the relatively weak cryo-EM density (Fig. 1a and Supplementary Fig. 4).
Other TSC1 regions were invisible in our cryo-EM map due to flexibility.
Cross-references: PDB:7DL2
EMDB:30708
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
SEC-coupled small-angle X-ray scattering (SAXS) analyses revealed that both p-FRQ and np-FRQ shared characteristics of highly flexible proteins, but that p-FRQ was more extended than np-FRQ (Table 1).
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: disorder (Structural state)
Evidence: author inference based on intrinsic disorder prediction
Supporting statements:
Analysis of an AlphaFold 2.0 (Jumper et al., 2021) model of full-length FRQ indicates that FRQ lacks a defined arrangement of large folded domains; instead, most of the molecule is unstructured (Figure 1B), in agreement with previous studies predicting that FRQ mostly comprises intrinsically disordered regions (Hurley et al., 2013).
Four regions distributed throughout the length of FRQ (residues 194–212, 312–329, 709–719, and 801–817) have at least 10% helical propensity, and these helical regions mostly agree with those of the AlphaFold model (Figure 1B).
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: protein homodimerization activity (Molecular function)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
Four regions distributed throughout the length of FRQ (residues 194–212, 312–329, 709–719, and 801–817) have at least 10% helical propensity, and these helical regions mostly agree with those of the AlphaFold model (Figure 1B).
Authors refer as p-FRQ to phosphorylated FRQ (p-FRQ), whereas non-phosphorylated FRQ is mention as np-FRQ.
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: phosphorylation display site (Disorder function)
Evidence: mass spectrometry evidence used in manual assertion
Supporting statements:
To generate phosphorylated FRQ, the protein was co-expressed with (untagged) CK1, which forms a stable complex with FRQ and acts as its primary kinase (Querfurth et al., 2011).
The latter revealed a network of >80 phosphorylation sites (Figure 1—figure supplement 2), most of which agree with those found on natively expressed FRQ (Baker et al., 2009; Tang et al., 2009).
The phosphorylation sites are well distributed across the entire protein.
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: disorder (Structural state)
Evidence: electron paramagnetic resonance evidence used in manual assertion
Supporting statements:
cw-ESR reveals that FRQ has regions of varying order that agree with computational predictions and that some of these sites undergo phosphorylation-dependent conformational changes (Figure 3C).
Such traces are indicative of long inter-spin distances and conformational flexibility, which would be consistent with the large mobility and intrinsic disorder of these peripheral regions (Figure 3—figure supplement 2, Figure 4—figure supplement 1).
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: protein binding (Molecular function)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
The FFC was generated by mixing p-FRQ or np-FRQ with excess FRH and CK1, followed by SEC purification to give p-FFC or np-FFC, respectively. FRHΔN (residues 100–1106), which binds FRQ, was used to improve expression and enhance stability (Conrad et al., 2016; Hurley et al., 2013). The resulting symmetric SEC traces and SDS-PAGE gels revealed pure and homogeneous ternary complexes in each case (Figure 4A).
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: protein binding (Molecular function)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
MW estimates of the p-FFC and np-FFC from SAXS indicate a 1:1 stoichiometry of FRQ:FRH, with a likely single component of CK1 also contained within the complex (Table 1).
SEC-SAXS analysis of the FFC showed that both FRQ phospho-forms produced complexes that were more globular than FRQ alone, but still somewhat flexible, with the p-FFC being more flexible than the np-FFC (Figure 4B, Figure 2—figure supplement 1).
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: differential interference contrast microscopy evidence used in manual assertion
Supporting statements:
FRQ exhibits behavior consistent with LLPS in vitro and in vivo
We tested whether both np-FRQ and p-FRQ can undergo LLPS in vitro. In each case, upon exchanging the purified protein into their respective phase separation buffers (which lack any crowding agents), we observed the appearance of a turbid solution that contained microscopic droplets (Figure 6B). These droplets scaled in size and number with increasing protein concentration, dissolved upon the addition of 1,6-hexanediol, and were shown to dock and fuse to one another, thereby confirming their liquid-like properties (Figure 6B, Figure 7—figure supplement 1)
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: turbidity measurement evidence used in manual assertion
Supporting statements:
FRQ exhibits behavior consistent with LLPS in vitro and in vivo
UV-Vis turbidity assays as a function of temperature revealed that FRQ undergoes a low critical solution temperature (LCST) phase transition that is concentration and phosphorylation status-dependent (Figure 6C, Figure 7—figure supplement 1, Tables 6 and 7).
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: nucleus (Cellular component)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Live-cell imaging of N. crassa expressing FRQ tagged with the fluorescent protein mNeonGreen at its C-terminus (FRQmNeonGreen) revealed heterogeneous patterning of FRQ in nuclei (Figure 6D and E).
Protein: DP04280 (UniProt P19970)
Region: residues 1	to 989
Annotation: non-membrane-bounded organelle assembly (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
FRQ exhibits behavior consistent with LLPS in vitro and in vivo
Nonetheless, bright and dynamic foci-like spots were observed well inside the nucleus and near the nuclear periphery, which is delineated by the cytoplasm-facing nucleoporin Son-1 tagged with mApple at its C-terminus (Figure 6D and E, Video 1). Such foci are characteristic of phase-separated IDPs (Bartholomai et al., 2022b; Caragliano et al., 2022; Gonzalez et al., 2021; Tatavosian et al., 2019) and share similar patterning to that seen for clock proteins in Drosophila (Meyer et al., 2006; Xiao et al., 2021), although the foci we observed are substantially more dynamic than those reported in Drosophila.
Protein: DP04281 (UniProt P68431)
Region: residues 1	to 38
Annotation: disorder (Structural state)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Elevated {1H}15N heteronuclear NOE values measured for free H3(1–59) indicated that residues 39–55 are partially structured in the free peptide (Fig. 4C).
Protein: DP04281 (UniProt P68431)
Region: residues 1	to 38
Annotation: flexible N-terminal tail (Disorder function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Elevated {1H}15N heteronuclear NOE values measured for free H3(1–59) indicated that residues 39–55 are partially structured in the free peptide (Fig. 4C).
Protein: DP04283 (UniProt P69229)
Region: residues 127	to 141
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The two domains are connected together by a linker of 15 residues in length; in our electron density maps we can observe some density for the linker but the resolution is not sufficient to be built (Supporting Information Fig. S2).
Cross-references: PDB:4B09
Protein: DP04284 (UniProt Q8TDX7)
Region: residues 182	to 195
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues 20–300 of Nek7Y97F were modelled with the exception of the activation loop residues 182–195 (chain A) and 181–199 (chain B), which were disordered.
Cross-references: PDB:5DE2