LiteFold/DisProt huggingface dataset:
Protein: DP03003 (UniProt P0DTC3)
Region: residues 1	to 39
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N-terminus (amino acids 1–39), C-terminus (amino acids 239–275), and a short cytoplasmic loop (amino acids 175–180) are either not observed or weakly resolved in the density map, presumably due to conformational differences between particles or because they are disordered.
Cross-references: PDB:7KJR
PDB:6XDC
Protein: DP03003 (UniProt P0DTC3)
Region: residues 239	to 275
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The N-terminus (amino acids 1–39), C-terminus (amino acids 239–275), and a short cytoplasmic loop (amino acids 175–180) are either not observed or weakly resolved in the density map, presumably due to conformational differences between particles or because they are disordered.
Cross-references: PDB:7KJR
PDB:6XDC
Protein: DP03003 (UniProt P0DTC3)
Region: residues 1	to 41
Annotation: cytoplasmic side of plasma membrane (Cellular component)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
The 3a N-terminus is involved in subcellular localization
These results are consistent with the N-terminal region of 3a being a determinant of its subcellular localization without influencing channel properties.
The N-terminal ~41 residues of each chain constitute the majority of the extracellularly or lumenally exposed 3a protein and thus could be involved in retention of 3a to internal membranes in cultured cells. To test this, we generated an N-terminal deletion construct lacking the first 41 amino acids (3aΔN) and compared its localization to wild-type 3a in HEK cells. Indeed, 3aΔN-EGFP shows reduced localization to internal membranes and bright foci and increased plasma membrane expression (Fig. 4A, S12). While we were unable to model the N-terminal 39 residues of 3a, we note an unassigned density feature in the cryo-EM maps that stretches between subunits just above the extracellular entrance to the pore that could correspond to a portion of these unmodeled N-terminal residues (Fig. 4B). We speculated that if the N-terminal region was stably positioned above the mouth of the pore it could influence channel properties.
Protein: DP03003 (UniProt P0DTC3)
Region: residues 36	to 40
Annotation: molecular function activator activity (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Definition of a TRAF binding domain in ORF3a required for activation of NF-κB and IL-1β secretion.
A TRAF-binding motif, PLQAS, consistent with the consensus sequence PxQx(T/S/D) in which x could be any residue, was located at aa 36–40 of ORF3a. To define their necessity for the NF-κB- and inflammasome-activating activity of ORF3a, 3 point mutants designated M-T, M-I, and M-C were created to disrupt the TRAF binding, ion channel, and caveolin binding domains, respectively (Fig. 4A). In M-T, 3 conserved residues (P, Q, and S) in the TRAF-binding motif were changed into A. In M-I, 3 conserved C residues required for ion channel activity (14, 24) were changed into S. Finally, 3 conserved Y residues in the caveolin-binding motif were changed into A in M-C.
Interestingly, the M-C and M-I mutants were fully competent in the activation of NF-κB and the NLRP3 inflammasome (Fig. 4B, bars 5–8 vs. 3 and 4; Fig. 4C, lanes 2 and 3 vs. 1). In sharp contrast, the M-T mutant lost its ability to activate either IL-8–Luc or IL-1β secretion (Fig. 4B, bars 9 and 10 vs. 3 and 4; Fig. 4C, lane 4 vs. 1). In other words, TRAF binding was essential for NF-κB and NLRP3 inflammasome activation by ORF3a. Surprisingly, neither caveolin binding nor the ion channel was required for this activity.
Protein: DP03003 (UniProt P0DTC3)
Region: residues 36	to 40
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
When we immunoprecipitated TRAF2, 3, and 6 with an anti-FLAG antibody, ORF3a but not its M-T mutant was detected in the precipitate (Fig. 5A, lanes 1, 3, and 5 vs. 2, 4, and 6). These results indicated that ORF3a was capable of interacting with TRAF2, 3, and 6, but this ability was abrogated in the M-T mutant.
The TRAF binding domain is required for the association of ORF3a with TRAF2, 3, and 6 and ASC.
Protein: DP03003 (UniProt P0DTC3)
Region: residues 36	to 40
Annotation: protein binding (Molecular function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
A TRAF-binding motif, PLQAS, consistent with the consensus sequence PxQx(T/S/D) in which x could be any residue, was located at aa 36–40 of ORF3a. To define their necessity for the NF-κB- and inflammasome-activating activity of ORF3a, 3 point mutants designated M-T, M-I, and M-C were created to disrupt the TRAF binding, ion channel, and caveolin binding domains, respectively (Fig. 4A). In M-T, 3 conserved residues (P, Q, and S) in the TRAF-binding motif were changed into A. In M-I, 3 conserved C residues required for ion channel activity (14, 24) were changed into S. Finally, 3 conserved Y residues in the caveolin-binding motif were changed into A in M-C.
Interestingly, the M-C and M-I mutants were fully competent in the activation of NF-κB and the NLRP3 inflammasome (Fig. 4B, bars 5–8 vs. 3 and 4; Fig. 4C, lanes 2 and 3 vs. 1). In sharp contrast, the M-T mutant lost its ability to activate either IL-8–Luc or IL-1β secretion (Fig. 4B, bars 9 and 10 vs. 3 and 4; Fig. 4C, lane 4 vs. 1). In other words, TRAF binding was essential for NF-κB and NLRP3 inflammasome activation by ORF3a. Surprisingly, neither caveolin binding nor the ion channel was required for this activity.
Protein: DP03004 (UniProt P58004)
Region: residues 296	to 309
Annotation: flexible linker (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Sestrin2 is a 55 kDa, monomeric, all α-helical, globular protein that contains distinct N-terminal [NTD, residues 66–220] and C-terminal [CTD, residues 339–480] domains connected by a partially disordered, partially helical linker region [Linker, residues 221–338] 
Disordered residues not present in the crystal structure (1 to 65, 242 to 255, 272 to 280, 296 to 309) are shown as dashed lines
Cross-references: PDB:5DJ4
Protein: DP03004 (UniProt P58004)
Region: residues 240	to 255
Annotation: flexible linker (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Region 240-255 is intrinsically disordered and is a flexible linker connecting Sesn-B and Sesn-C domains
The overall structure is well-defined except for residues 1–65, 221–224, 240–255, 272–279, 295–307, 329–332 and 479–480
"The structure of full-length hSesn2 indicates that the protein contains three domains separated by two unstructured flexible linker regions (Fig. 1b)"
Cross-references: PDB:5CUF
Protein: DP03004 (UniProt P58004)
Region: residues 295	to 307
Annotation: flexible linker (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Region 295-307 is intrinsically disordered and is a flexible linker connecting Sesn-A and Sesn-B domains
The overall structure is well-defined except for residues 1–65, 221–224, 240–255, 272–279, 295–307, 329–332 and 479–480.
"The structure of full-length hSesn2 indicates that the protein contains three domains separated by two unstructured flexible linker regions (Fig. 1b)"
Cross-references: PDB:5CUF
Protein: DP03004 (UniProt P58004)
Region: residues 1	to 65
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Disordered residues not present in the crystal structure (1 to 65, 242 to 255, 272 to 280, 296 to 309) are shown as dashed line
Cross-references: PDB:5DJ4
Protein: DP03004 (UniProt P58004)
Region: residues 241	to 255
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Disordered residues not present in the crystal structure (1 to 65, 242 to 255, 272 to 280, 296 to 309) are shown as dashed line
Cross-references: PDB:5DJ4
Protein: DP03004 (UniProt P58004)
Region: residues 296	to 309
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Disordered residues not present in the crystal structure (1 to 65, 242 to 255, 272 to 280, 296 to 309) are shown as dashed line
Cross-references: PDB:5DJ4
Protein: DP03004 (UniProt P58004)
Region: residues 222	to 232
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Disordered residues not present in the crystal structure (1 to 65, 242 to 255, 272 to 280, 296 to 309) are shown as dashed line
Cross-references: PDB:5DJ4
Protein: DP03004 (UniProt P58004)
Region: residues 222	to 232
Annotation: flexible linker (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
"Sestrin2 is a 55 kDa, monomeric, all α-helical, globular protein that contains distinct N-terminal [NTD, residues 66–220] and C-terminal [CTD, residues 339–480] domains connected by a partially disordered, partially helical linker region [Linker, residues 221–338] "
"Disordered residues not present in the crystal structure (1 to 65, 242 to 255, 272 to 280, 296 to 309) are shown as dashed lines"
Cross-references: PDB:5DJ4
Protein: DP03004 (UniProt P58004)
Region: residues 241	to 255
Annotation: flexible linker (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
"Sestrin2 is a 55 kDa, monomeric, all α-helical, globular protein that contains distinct N-terminal [NTD, residues 66–220] and C-terminal [CTD, residues 339–480] domains connected by a partially disordered, partially helical linker region [Linker, residues 221–338] "
"Disordered residues not present in the crystal structure (1 to 65, 242 to 255, 272 to 280, 296 to 309) are shown as dashed lines"
Cross-references: PDB:5DJ4
Protein: DP03004 (UniProt P58004)
Region: residues 295	to 307
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
"The overall structure is well-defined except for residues 1–65, 221–224, 240–255, 272–279, 295–307, 329–332 and 479–480"
Cross-references: PDB:5CUF
Protein: DP03004 (UniProt P58004)
Region: residues 240	to 255
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
"The overall structure is well-defined except for residues 1–65, 221–224, 240–255, 272–279, 295–307, 329–332 and 479–480"
Cross-references: PDB:5CUF
Protein: DP03004 (UniProt P58004)
Region: residues 1	to 65
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
"The overall structure is well-defined except for residues 1–65, 221–224, 240–255, 272–279, 295–307, 329–332 and 479–480"
Cross-references: PDB:5CUF
Protein: DP03005 (UniProt Q53TN4)
Region: residues 231	to 286
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Although full-length Dcytb (residues 1–286) was used for crystallization, the atomic model includes only residues 6–230 because the electron density map for the five N-terminal residues and the 56 C-terminal residues was disordered.
Cross-references: PDB:5ZLG
PDB:5ZLE
Protein: DP03006 (UniProt O95843)
Region: residues 186	to 209
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In both molecules, and in spite of different packing contacts, 19 amino acids from the N–terminus and 24 from the C– terminus could not be traced, presumably due to conformational flexibility.
Cross-references: PDB:2GGZ
Protein: DP03006 (UniProt O95843)
Region: residues 1	to 22
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In both molecules, and in spite of different packing contacts, 19 amino acids from the N–terminus and 24 from the C– terminus could not be traced, presumably due to conformational flexibility.

The region with missing residues in the PDB structure has a different numbering in UniProt and the publication, the region defined here follows the UniProt numbering.
Cross-references: PDB:2GGZ
Protein: DP03007 (UniProt P97784)
Region: residues 167	to 177
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The extended loop between helices α6 and α8 (Met160mCRY1 to Gly213mCRY1) is partly disordered in mCRY1 (missing residues 167 to 177 and 203/204), and the region between Cys178mCRY1 and α8 significantly differs from dCRY (Figure 3A). 
Cross-references: PDB:4K0R
Protein: DP03007 (UniProt P97784)
Region: residues 498	to 606
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
The full-length mCRY1 protein was spontaneously proteolysed to a mCRY1[1-497] fragment corresponding to the photolyase homology region (PHR).
Cross-references: PDB:4K0R
Protein: DP03007 (UniProt P97784)
Region: residues 471	to 606
Annotation: disorder (Structural state)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
The hydrodynamic (Stokes) radii determined by AUC analyses (Table 1) suggest that the mCRY and mBMAL1 proteins have somewhat elongated shapes and might be at least partially unstructured.
Protein: DP03007 (UniProt P97784)
Region: residues 564	to 591
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
To quantify the contributions of the two mBMAL1 binding epitopes of mCRY1 and to find out which epitope is responsible for the different binding affinities of mCRY1 to mBMAL1-(577–625), mBMAL1-(490–625), and mBMAL1-(490–625)K537Q, we have synthesized peptides comprising mCRY1 residues Asn-472 to Ser-505 corresponding to the predicted coiled coil region (peptide P1) as well as residues Ser-564 to Glu-591 within the mCRY1 tail region (peptide P2). CD spectroscopy showed that peptide P1 has a high α-helical content (as predicted), whereas peptide P2 is mostly disordered (Table 2 and supplemental Fig. S3B).
Protein: DP03007 (UniProt P97784)
Region: residues 565	to 595
Annotation: molecular function regulator (Molecular function)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
To determine which mCRY residues are most critical for the mBMAL1 interaction, we have SPOT-synthesized peptides including the conserved mCRY coiled coil epitope (mCRY1 sequence 473HAEASRLNIERMKQIYQQLSRYRGLGLLASVP504) and the major part of the mCRY1 tail epitope (565QQTHSLKQGRSSAGTGLSSGKRPSQEEDAQS595). We have exchanged each amino acid in the mCRY peptides to alanine and measured the binding of the modified peptides to Cy3.5 fluorescently labeled mBMAL1-(577–625) (Fig. 4D). his experiment showed that single alanine mutations of the negatively charged residues Glu-590, Glu-591, or Asp-592 in the mCRY1 tail peptide raise its affinity to mBMAL1 drastically, whereas the exchange of any single lysine or arginine to alanine lowers it.
Protein: DP03007 (UniProt P97784)
Region: residues 565	to 595
Annotation: protein binding (Molecular function)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
To determine which mCRY residues are most critical for the mBMAL1 interaction, we have SPOT-synthesized peptides including the conserved mCRY coiled coil epitope (mCRY1 sequence 473HAEASRLNIERMKQIYQQLSRYRGLGLLASVP504) and the major part of the mCRY1 tail epitope (565QQTHSLKQGRSSAGTGLSSGKRPSQEEDAQS595). We have exchanged each amino acid in the mCRY peptides to alanine and measured the binding of the modified peptides to Cy3.5 fluorescently labeled mBMAL1-(577–625) (Fig. 4D). his experiment showed that single alanine mutations of the negatively charged residues Glu-590, Glu-591, or Asp-592 in the mCRY1 tail peptide raise its affinity to mBMAL1 drastically, whereas the exchange of any single lysine or arginine to alanine lowers it.
Protein: DP03007 (UniProt P97784)
Region: residues 564	to 591
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
Peptide P2, however, binds to mBMAL1-(577–625) and to the mBMAL1-(490–625)K537Q mutant fragment with an affinity of ∼3 μm but to the wild-type mBMAL1-(490–625) fragment with a lower affinity of ∼8 μm (Table 3 and Fig. 5, B–D). In contrast to all other mCRY-mBMAL1 interactions that we have analyzed by ITC, the interaction of peptide P2 with mBMAL1-(490–625)K537Q is exothermic and enthalpically as well as entropically favored (Fig. 5D, Table 3). This indicates that the P2-mBMAL1-(490–625)K537Q complex involves a larger number of polar contacts (e.g. hydrogen bonds) than the other mCRY-BMAL1 interactions, which are entropically but not enthalpically favored (30).
Protein: DP03007 (UniProt P97784)
Region: residues 564	to 591
Annotation: molecular function regulator (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
Peptide P2, however, binds to mBMAL1-(577–625) and to the mBMAL1-(490–625)K537Q mutant fragment with an affinity of ∼3 μm but to the wild-type mBMAL1-(490–625) fragment with a lower affinity of ∼8 μm (Table 3 and Fig. 5, B–D). In contrast to all other mCRY-mBMAL1 interactions that we have analyzed by ITC, the interaction of peptide P2 with mBMAL1-(490–625)K537Q is exothermic and enthalpically as well as entropically favored (Fig. 5D, Table 3). This indicates that the P2-mBMAL1-(490–625)K537Q complex involves a larger number of polar contacts (e.g. hydrogen bonds) than the other mCRY-BMAL1 interactions, which are entropically but not enthalpically favored (30).
Protein: DP03007 (UniProt P97784)
Region: residues 494	to 497
Annotation: selective autophagy (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
We then determined the role of each LIR motif in degradation of CRY1 by expressing FLAG-tagged WT-CRY1 plasmid and each CRY1 mutant LIR plasmid (mLIR1, mLIR2, mLIR3, and mLIR4 as in Figure 5B) in liver via tail vein injections using the jet-PEI in vivo transfection system (Chang et al., 2014) (Figure S5A).
Strikingly, mLIR1-CRY1 and mLIR4-CRY1 accumulated significantly more than mLIR2-CRY1 and mLIR3-CRY1 or WT-CRY1. Levels of mLIR2 and mLIR3 CRY1 were moderately higher than WT-CRY1, but differences between these groups remained statistically insignificant (Figure 5C). These results indicate that LIR1 (271DLYKKV276) and LIR4 (492SRYRGL497) are required for degradation of CRY1
by autophagy. Accumulation of mLIR1-CRY1 and mLIR4-CRY1 did not occur from reduced autophagy per se, indicated by equivalent LC3-II flux in livers injected with WT or mLIR-CRY1 plasmids (Figure 5D), or from changes in transfection
determined by qPCR showing equivalent Cry1 expression in
livers injected with WT and mLIR-CRY1 plasmids (Figure S5E).
CoIP in 293T cells confirmed that interactions of mLIR1-CRY1
and mLIR4-CRY1 with GFP-tagged LC3 were remarkably
reduced compared with WT-CRY1, mLIR2-CRY1, or mLIR3-
CRY1 (Figure S5F). These results are consistent with significant
accumulation of mLIR1-CRY1 and mLIR4-CRY1 and moderate
accumulation of mLIR2 and mLIR3 CRY1 in liver compared
with WT-CRY1 (Figure 5C). Consistent with our observation that accumulation of CRY1 in Atg7KO mice associates with
decreased BMAL1 levels (Figure 2J), accumulation of mLIR1-
CRY1 or mLIR4-CRY1 each associated with reduced BMAL1
levels (Figure 5E), indicating that expressing autophagy-resistant
forms of CRY1 is sufficient to disrupt the liver clock.
In the article, the functional LIR motif YRGL in positions 494-497 corresponds to LIR4.
The non-functional LIR motif YQQL in positions 488-491, and the functional LIR motif YRGL in positions 494-497, are both incorporated inside the disordered region fragment in positions 471-606.
mLIR4-CRY1 (492SRARGA497)
Figure 5. LIR Motifs Determine CRY1 Degradation and Regulation of Gluconeogenesis
(A) Murine CRY1 has 13 LIR motifs (green).
(B) Inactivation of selected LIR motifs (mLIR1-4) on CRY1 via mutagenesis of tyrosine (Y), valine (V), or leucine (L) residues (green) to alanine (A) (red).
(C) IB for FLAG in livers from male mice at 7 p.m. expressing FLAG-tagged WT-CRY1 plasmid or each FLAG-tagged CRY1 LIR mutant plasmid (mLIR1–4); n = 12.
(D) IB for LC3 in livers from male mice at 7 p.m. expressing FLAG-tagged WT-CRY1 or each CRY1 LIR mutant (mLIR1–4) and cultured in presence or absence of
Lys Inh for 2 hr; n = 7–10.
(E and F) IB for BMAL1 and G6P in livers from male mice at 7 p.m. expressing FLAG-tagged WT-CRY1 plasmid or each CRY1 LIR mutant plasmid (mLIR1–4);
n = 8–12.