LiteFold/DisProt huggingface dataset:
Protein: DP04341 (UniProt P08551)
Region: residues 399	to 439
Annotation: identical protein binding (Molecular function)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
Using synchrotron small-angle x-ray scattering, we find that the uncharged domain of the NFLt induces attractive interactions that cause it to self-assemble into star-like polymer brushes.
We found that the sequence heterogeneity differentiates between the structures of the entire WT NFLt and a variant lacking the N-terminal domain. In particular, the WT variant self-assembles into star-like structures, while the ΔN42 one remains isolated in all measured cases.
Protein: DP04341 (UniProt P08551)
Region: residues 399	to 439
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
Fig. 1 SAXS measurements of WT and ΔN42 at different salinity (Cs). a For increasing Cs, the WT variant shows increased small-angle scattering, a signature for aggregation.
With increasing Cs, the hump at the lower q range becomes a sharper peak accompanied by a scattering rise at the higher q range. Such behavior indicates that the aggregation coexists with the WT variant’s highly dynamic and disordered regions.
Dominantly at the low wave-vector (q) region, the WT variant scattering (I) rises with added NaCl salt. Such an increase at low q implies high molecular mass particles due to aggregation of the WT variant.
Protein: DP04341 (UniProt P08551)
Region: residues 398	to 543
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
All measurements are conducted in the presence of a 20 mM Tris, pH 8.0, to fully deprotonate the histidine residue. All S segments and the full NFLt are validated to be fully disordered by circular dichroism (SI Appendix, Fig. S4).
Protein: DP04341 (UniProt P08551)
Region: residues 399	to 439
Annotation: intermediate filament polymerization (Biological process)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
Such SAXS resembles that of self-assembled decorated spherical micelles [36]. Variations of micelle models are shown to fit the data (Figs. 1, S4–S6). Sufficiently low aggregation number and core size distill the description of the spherical micelle into a ‘star-like’ brush.
a Schematic of the system’s structure variation with salinity (Cs). While ΔN42 remains disordered and segregated, the WT variant aggregates to a star-like polymer with a higher aggregation number at higher Cs.
Protein: DP04341 (UniProt P08551)
Region: residues 398	to 543
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
(A) Kratky plot of the SAXS measurements. Each line color is a different S segment (which is 20 amino acids long). The Kratky plot indicates that all segments are unfolded as Iq2/I0 intensity diverges at large q.
Overall, the result by both SAXS and trFRET shows that the NFLt consists of intramolecular structural heterogeneity—different domains within the IDP occupy space differently, although all display disorder properties.
Protein: DP04343 (UniProt P0DOW4)
Region: residues 1	to 445
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
In the Kratky plot, the scattering curves do not have the bell shape, inherent to globular proteins, but instead continuously increase with increasing of scattering vector q (Fig. 4b). Such type of scattering curves is characteristic for non-globular, flexible and disordered proteins, therefore further we described Dsup structure in terms of the Gaussian (random) coil suitable for IDPs.
The Flory`s coefficient ν for Dsup in all types of used solutions is far from the values corresponding to the globular state of proteins (νglobular = 0.33). In case of PBS, the ν is close to 0.5, which corresponds to a random coil structure.
The pair distance distribution function P(r) is Fourier transform of the small angle scattering profile and represents the distribution function of distances inside the particle, thus it describes the shape and dimensions of this particle. The P(r) functions for Dsup are asymmetric with a tailing profile for the longer distances that indicates a rather elongated shape of Dsup (Fig. 5).
Protein: DP04343 (UniProt P0DOW4)
Region: residues 1	to 445
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Circular dichroism (CD) spectroscopy was applied for the determination of secondary structure content of Dsup in PBS and H2O under native conditions and in presence of chemical denaturant (urea 4.5 M) (Fig. 8).
The results obtained for Dsup in PBS and H2O indicate the presence of disordered regions (62–66%), α-helixes (5–7%) and β-sheets (30–31%) (Table 2).
Protein: DP04343 (UniProt P0DOW4)
Region: residues 1	to 445
Annotation: DNA binding (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Differential CD spectra were obtained for the range of wavelength as the difference between spectra of Dsup-DNA complex and the sum of spectra of Dsup and DNA components (Fig. 8b). A slight signal shift (< 5%) indicates insignificant transitions in the secondary structure of the Dsup protein upon binding with dsDNA. Based on the consistent content of disordered regions in the free Dsup protein and DNA-bound Dsup protein, the Dsup/DNA complex was classified as a fuzzy complex.
Protein: DP04344 (UniProt Q7BTX0)
Region: residues 121	to 141
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The final model included residues 3–120 of SycH and 33–50 of YscM2. Despite the high temperature factors, the structure of SycH was quite well resolved. However, side chains could only be assigned to the helical portion of YscM2. The remainder of this polypeptide was poorly ordered in the crystal.
The three C-terminal residues of SycH (139–141) were also efficiently removed by thermolysin in this experiment. Therefore, all SycH expression vectors were subsequently designed to produce residues 1–138.
Cross-references: PDB:1TTW
Protein: DP04344 (UniProt Q7BTX0)
Region: residues 121	to 141
Annotation: flexible C-terminal tail (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The final model included residues 3–120 of SycH and 33–50 of YscM2. Despite the high temperature factors, the structure of SycH was quite well resolved. However, side chains could only be assigned to the helical portion of YscM2. The remainder of this polypeptide was poorly ordered in the crystal.
The three C-terminal residues of SycH (139–141) were also efficiently removed by thermolysin in this experiment. Therefore, all SycH expression vectors were subsequently designed to produce residues 1–138.
Protein: DP04347 (UniProt P28360)
Region: residues 1	to 172
Annotation: disorder (Structural state)
Evidence: author inference based on intrinsic disorder prediction used in manual assertion
Supporting statements:
VL3 in PONDR analysis revealed that the RD-containing N-terminal region in MSX1 is a largely unfolded IDR (Fig. 1A and Supplementary Fig. 1A)20.
AlphaFold prediction also agrees with this region being disordered. Authors refer to this region as IDR throughout the text.
Protein: DP04348 (UniProt Q96PD5)
Region: residues 164	to 208
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
The fluorescence of FAM-Enh II puncta recovered slowly after photobleaching
(Fig. 3F), indicating that PGLYRP2, by inducing phase separation involving HBV DNA, likely sequesters the viral DNA from cellular machinery, thereby potentially obstructing its role in viral replication (Fig. 3G).
Purified DsRed-tagged PGLYRP2IDR/209–377
triggers the formation of membraneless condensates that colocalize
with HBV DNA FAM–Enh II. Meanwhile, condensates induced by
DsRed-tagged PGLYRP2IDR do not include HBV DNA FAM–Enh
II, suggesting that phase separation induced by PGLYRP2IDR might
underpin the viral suppression function of PGLYRP2209–377 (Figure
3E).
Protein: DP04348 (UniProt Q96PD5)
Region: residues 164	to 208
Annotation: disorder (Structural state)
Evidence: author inference based on intrinsic disorder prediction used in manual assertion
Supporting statements:
The intrinsically disordered region (IDR) of PGLYRP2, predicted using PONDR, spans amino acids 164-208 and is adjacent to the PGLYRP2209-377 20 domain (Fig. 3D).
AlphaFold prediction and MobiDB predictions also agree with this region being disordered.
Protein: DP04349 (UniProt Q8N114)
Region: residues 126	to 240
Annotation: disorder (Structural state)
Evidence: author inference based on intrinsic disorder prediction used in manual assertion
Supporting statements:
 However, structure prediction by the AlphaFold program indicated that the PRD of SCOTIN does not have a strong structural signature36 (Figure 4D). Instead, it was intrinsically disordered, as illustrated by computational predictions for the unstructured low complexity of proteins (Figure 4E).
(D) AlphaFold predicted structure of SCOTIN. Orange color indicates unstructured residues.
(E) Prediction of the IDR from five different predictors in the D2P2 resource.37 The threshold for all predictors except protein disorder prediction server (PrDOS) (threshold for PrDOS is shown as dotted line) is 0.5 (bold line). Gray box indicates the region of 150–177.
Protein: DP04349 (UniProt Q8N114)
Region: residues 126	to 240
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
The puncta-like structure of SCOTIN disappeared when SCOTIN(ΔPRD)-expressing cells were tested (Figure 4B). In contrast, overexpression of the SCOTIN(PRD) or SCOTIN(TMPRD) was sufficient to induce the formation of the puncta structure, indicating that the PRD of SCOTIN was needed to assemble this puncta structure (Figures 4B, 4C, S4A, and S4B).
These results together indicate that the intrinsically disordered region (IDR) of SCOTIN possesses the ability to form punctate structures in cells.
Protein: DP04349 (UniProt Q8N114)
Region: residues 150	to 177
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: chimeric protein phenotypic evidence used in manual assertion
Supporting statements:
Overexpressed Sec61β-mEmerald was detected in the tubular ER, without any evident puncta. Upon addition of SCOTIN(PRD), but not the PRD(Δ150–177), distinct punctate structures of sphere shape clearly appeared (Figures 4H and 4I).
Protein: DP04349 (UniProt Q8N114)
Region: residues 150	to 177
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: genetic transformation evidence used in manual assertion
Supporting statements:
To further assess whether condensation occurs under endogenous protein levels as well as ectopic overexpression, a tet-ON SCOTIN-MYC cell line was generated in the SCOTIN KO and stimulated with doxycycline to reach a SCOTIN-MYC protein level similar to that of endogenous SCOTIN (Figure 4J). SCOTIN-MYC formed puncta under this condition (Figures 4K and 4L). In contrast, SCOTIN(Δ150–177)-MYC was diffusely localized when it was expressed at endogenous levels (Figures 4K and 4L).
Protein: DP04349 (UniProt Q8N114)
Region: residues 126	to 240
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: differential interference contrast microscopy evidence used in manual assertion
Supporting statements:
Under a physiological salt concentration and neutral pH, purified SCOTIN(PRD) assembled into droplets with the aid of the molecular crowding agent polyethylene glycol (PEG)-8000 (Figure 6D). The formation of SCOTIN(PRD) droplets depended on the protein concentration and pH (Figure 6E). SCOTIN(PRD) droplets coalesced into larger droplets without any energy source (Figure S5B).
SCOTIN condensates exhibit liquid- to gel-like properties
Protein: DP04349 (UniProt Q8N114)
Region: residues 150	to 177
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Moreover, the SCOTIN(PRDΔ150–177) protein did not form droplets when tested under the same conditions (Figure 6D), confirming that residues 150–177 within the PRD of SCOTIN govern its ability to form condensates.
SCOTIN condensates exhibit liquid- to gel-like properties
Protein: DP04349 (UniProt Q8N114)
Region: residues 150	to 177
Annotation: regulation of ER to Golgi vesicle-mediated transport (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
As expected, ER-to-Golgi transport was significantly delayed by overexpression of SCOTIN but not by SCOTIN(Δ150–177) or Sec61β (Figures 7B and 7C), suggesting that the ability of SCOTIN to form condensates is required to exert an inhibitory effect.
The inhibitory effect of SCOTIN on VSV-G trafficking was separately confirmed using the EndoH assay. It distinguishes the fractional abundance of EndoH-sensitive (on ER) or Endo H-resistant (within Golgi or beyond) forms (Figure 7D). In empty vector- or Sec61β-overexpressing control cells, the EndoH-resistant VSV-G protein appeared with biotin treatment, suggesting its proper transport to the Golgi. In SCOTIN-expressing but not SCOTIN(Δ150–177)-expressing cells, the EndoH-resistant VSV-G protein was not increased, again confirming that the inhibition of vesicle transport by SCOTIN requires the ability of SCOTIN to form condensates.
Intrinsically disordered region-mediated condensation of IFN-inducible SCOTIN/SHISA-5 inhibits ER-to-Golgi vesicle transport
Protein: DP04349 (UniProt Q8N114)
Region: residues 150	to 177
Annotation: regulation of COPII vesicle uncoating (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Condensation is required for the ER-to-Golgi transport delay and the disassembly of COPII by SCOTIN
Finally, we verified whether SCOTIN condensates inhibit COPII-mediated vesicle formation from the ER membrane using in vitro budding assays (Figure 7H). The inhibition of COPII budding by SCOTIN was not observed after SCOTIN(Δ150–177) overexpression. These results together suggest that SCOTIN condensates preferentially recruit Sec31 and interfere with COPII vesicle formation on the ER membrane, thereby delaying transport from the ER to the Golgi.
Protein: DP04349 (UniProt Q8N114)
Region: residues 150	to 177
Annotation: positive regulation of endoplasmic reticulum unfolded protein response (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Based on these results, we concluded that the ability of SCOTIN to condense on the ER is essential for activation of the UPR.
In contrast to SCOTIN-MYC overexpression, SCOTIN(Δ150–177)-MYC overexpression barely induced the UPR (Figure 2A).
Significantly fewer IRE1 foci were detected in SCOTIN(Δ150–177)-MYC-overexpressing cells than in SCOTIN-MYC-overexpressing cells, confirming that SCOTIN(Δ150–177)-MYC overexpression barely induces IRE1 oligomerization or the UPR (Figures 2B and 2C).
SCOTIN condensation is critical for inducing ER stress
Protein: DP04349 (UniProt Q8N114)
Region: residues 126	to 240
Annotation: positive regulation of response to endoplasmic reticulum stress (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
The most significant differences in ER stress activation were observed with the Δ150–177 mutant (Figure 6H). In all cases (C1, C2, and C3), the addition of the Δ150–177 mutation led to a marked reduction in both XBP1 and CHOP production, confirming that SCOTIN condensation via the cytosolic PRD is essential for BiP sequestration and induction of ER stress responses.
Our data thus far demonstrate that cytosolic condensation of the SCOTIN PRD results in the transduction of signals to the luminal side of the ER, resulting in the sequestration of BiP within SCOTIN condensates, ultimately triggering the ER stress response.
We thus sought to determine whether the mere presence of the 150–177 aa is sufficient to induce ER stress. To investigate this possibility, we generated mutant constructs by adding the 150–177 aa to the SCOTIN(ΔPRD) or SCOTIN(1–149) mutant, neither of which could induce ER stress (Figures 7A–7C).
Although adding 150–177 aa to the SCOTIN(ΔPRD) mutant resulted in a slight increase in the proportion of cells expressing F-XBP1ΔDBD-Venus, it was insufficient for full restoration to a proportion comparable to that observed among cells expressing full-length SCOTIN (Figures 7B and S4A).
These data suggest that the intrinsic activity of the entire PRD of SCOTIN is necessary for activating the ER stress response.
Protein: DP04352 (UniProt A0A564ZT73)
Region: residues 29	to 56
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Again, no electron density was observed for the N- and C-terminal extensions (residues 30–60 and 150–230, respectively).
Modified boundaries as missing residues in the deposited structure correspond to 29-56.
Cross-references: PDB:8RWU
Protein: DP04352 (UniProt A0A564ZT73)
Region: residues 148	to 229
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Again, no electron density was observed for the N- and C-terminal extensions (residues 30–60 and 150–230, respectively).
Modified boundaries as missing residues in the deposited structure correspond to 148-229.
Cross-references: PDB:8RWU
Protein: DP04368 (UniProt Q99PZ6)
Region: residues 1	to 25
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The structure of OspG in the complex is well defined by the electron density with the exception of a 10x‐His tag and 25 native residues at the N‐terminus that are disordered and may encode a sequence necessary for recognition by the Type III secretion apparatus.
Cross-references: PDB:4BVU
Protein: DP04368 (UniProt Q99PZ6)
Region: residues 1	to 25
Annotation: flexible N-terminal tail (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The structure of OspG in the complex is well defined by the electron density with the exception of a 10x‐His tag and 25 native residues at the N‐terminus that are disordered and may encode a sequence necessary for recognition by the Type III secretion apparatus.
Cross-references: PDB:4BVU
Protein: DP04369 (UniProt A0A0H3JPC6)
Region: residues 206	to 216
Annotation: disorder to order (Structural transition)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The second loop (205–215), designated as the substrate access (SA) loop, is ordered only in the presence of UDP-GlcNAc and sterically occludes an otherwise open channel leading into the active site in the absence of the intact donor (Fig 2C). Based on these observations it may be inferred that in the native structure, the SA loop is disordered allowing binding of UDP-GlcNAc, upon which the CS loop moves closer to the active site center and the SA loop becomes ordered, occluding the active site channel.
Cross-references: PDB:5TZI
PDB:5TZJ
Protein: DP04369 (UniProt A0A0H3JPC6)
Region: residues 208	to 213
Annotation: hydrolase activity, acting on glycosyl bonds (Molecular function)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
Several constructed active-site mutants were also tested for activity, along with a designed control (D198A) chosen distal from the catalytic center (Fig 5B). Based on these results, mutations R75A, D91A, D93A, D94A, E177A, and H210A abolished activity as defined by UDP-GlcNAc hydrolysis, whereas mutations D178N, R206A, and S212A led to severe decreases in activity, validating the importance of these residues for catalysis, as discussed above.
Protein: DP04369 (UniProt A0A0H3JPC6)
Region: residues 206	to 216
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The second loop (205–215), designated as the substrate access (SA) loop, is ordered only in the presence of UDP-GlcNAc and sterically occludes an otherwise open channel leading into the active site in the absence of the intact donor (Fig 2C).
Cross-references: PDB:5TZI
Protein: DP04370 (UniProt L7N667)
Region: residues 85	to 99
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The electron density map was clearly defined throughout the structure, except for residues 85–99 in PE8 and residues 174–194 in PPE15, in line with a secondary structure prediction by Phyre2 (31), suggesting that these regions are highly disordered. This disordered region of PE8 includes the YXXXD/E secretion motif.
Cross-references: PDB:5XFS
Protein: DP04371 (UniProt P40296)
Region: residues 220	to 229
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
YscQ-C was expressed in E. coli, purified, and crystallized. The 2.25 Å resolution limit structure of YscQ-C was determined by single-wavelength anomalous dispersion (Table 1). Except for the first 11 residues, which were presumably flexible, the entirety of YscQ-C was visible and unambiguously traced.
However, the N-terminus of YscQ-C is disordered, and thus, the exact location of the start site is unlikely to be consequential.
The IDR characterized in the publication corresponds to region 220-228 of the amino acid sequence, since YscQ-C contains the residues 220–307 of YscQ.
Cross-references: PDB:3UEP
Protein: DP04372 (UniProt P0A367)
Region: residues 780	to 818
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Disordered regions indicating the location of cysteine residues are shown in gray font (Met1-Gly32, Gly780-Glu818, Glu1064-Pro1140).
The residues of Met1-Gly32, Ile369-Asn377, Gly442-Val444, Ser556-Asn559, Gly593, Thr609-Ala612, Gly780-Glu818, and Glu1064-Pro1140 could not be modelled because of missing of the corresponding electron density [25].
Cross-references: PDB:8W7N
Protein: DP04372 (UniProt P0A367)
Region: residues 1	to 32
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Disordered regions indicating the location of cysteine residues are shown in gray font (Met1-Gly32, Gly780-Glu818, Glu1064-Pro1140).
The residues of Met1-Gly32, Ile369-Asn377, Gly442-Val444, Ser556-Asn559, Gly593, Thr609-Ala612, Gly780-Glu818, and Glu1064-Pro1140 could not be modelled because of missing of the corresponding electron density [25].
Cross-references: PDB:8W7N