DOI: 10.5061/dryad.pk0p2ngwh
title: Data from: Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock

description: Circadian clocks are composed of molecular oscillators that pace rhythms
 of gene expression to the diurnal cycle. Therein,
 transcriptional-translational negative feedback loops (TTFLs) generate
 oscillating levels of transcriptional repressor proteins that regulate
 their own gene expression. In the filamentous fungus Neurospora crassa,
 the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH) and
 Casein-Kinase I (CK1) form the FFC complex that represses expression of
 genes activated by the White-Collar complex (WCC). A key question concerns
 how FRQ orchestrates molecular interactions at the core of the clock
 despite containing little predicted tertiary structure. We present the
 reconstitution and biophysical characterization of FRQ and the FFC in
 unphosphorylated and highly phosphorylated states. Site-specific spin
 labeling and pulse-dipolar ESR spectroscopy provides domain-specific
 structural details on the full-length, 989-residue intrinsically
 disordered FRQ and the FFC. FRQ contains a compact core that associates
 and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ
 phosphorylation increases conformational flexibility and alters oligomeric
 state but the changes in structure and dynamics are non-uniform.
 Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to
 sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ
 phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation
 by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals
 FRQ foci characteristic of condensates near the nuclear periphery.
 Analogous clock repressor proteins in higher organisms share little
 position-specific sequence identity with FRQ; yet, they contain amino-acid
 compositions that promote LLPS. Hence, condensate formation may be a
 conserved feature of eukaryotic circadian clocks. 
description: # Phosphorylation, disorder, and phase separation govern the behavior of
 Frequency in the fungal circadian clock
 Description of the data
 and file structure These files represent the raw data from the ESR, SAXS,
 UV-Vis, MALS, and microscopy experiments. The main folder titled "Raw
 Data" contains various sub-folders that are labeled by experiment
 type. 1. The "Microscopy Images" sub-folder contains all the
 microscopy images showing FRQ droplets +/- binding partners under
 different conditions. Within this folder, there are 3 sub-folders. 1.
 "Co-LLPS" contains co-phase-separation microscopy images with
 FRQ and either FRH or CK1. This folder also has the images from the CheY
 control. 2. "np-FRQ and p-FRQ Concentration Titration +_-
 hexanediol" contains microscopy images of FRQ at different
 concentrations +/- hexanediol, which is used to dissolve droplets and show
 that they are indeed liquid-like. 3. "np-FRQ Cysless
 Temperature" contains microscopy images of FRQ undergoing phase
 separation at different temperatures and correlates to the radio ATP based
 assay shown in the paper Figure 7. 2. The "Raw ESR Data" folder
 contains the raw data from all the CW-ESR and DEER experiments shown in
 the paper. 1. The "CW ESR" sub-folder contains the raw files for
 the Cw ESR experiments done with the various single cysteine mutants shown
 in Figure 3 of the paper as well as the N and C terminus of FRQ. Each
 filename contains the site of the spin-label attachment. 1. Each file is a
 “.DTA” format that is the raw output from the spectrometer, containing the
 field strength and intensity (at those field strengths) values. These
 files are processed by the easyspin software package in MATLAB. 2. The
 "DEER" sub-folder contains the raw data files from all of the
 DEER experiments shown in the paper. Each filename contains the site of
 the spin-label attachment. 1. Each file is a “.DTA” format that is the raw
 output from the spectrometer. These files contain the time points and echo
 intensity values.These files are processed by the easyspin software
 package in MATLAB. 3. The "Raw SAXS Data" folder contains the
 files used to generate the dimensionless kratky plots and p(r)
 distributions shown in the paper. The filenames reflect which type of
 plots they were used to generate. 1.
 “PhosFRQ_NonPhosFRQ_Dimensionless_Kratky.csv” contains the data used to
 generate the dimensionless Kratky plot showin in Figure 2. 1. Column 1 has
 the qRG values for np-FRQ 2. Column 2 has the qRGI^2(Q)/I0 values for
 np-FRQ 3. Column 3 has the qRG values for p-FRQ 4. Column 4 has the
 qRGI^2(Q)/I0 values for p-FRQ 2.
 “PhosFFC_NonPhosFFC_Dimensionless_Kratky.csv” 1. Column 1 has the qRG
 values for np-FFC 2. Column 2 has the qRGI^2(Q)/I0 values for np-FFC 3.
 Column 3 has the qRG values for p-FFC 4. Column 4 has the qRGI^2(Q)/I0
 values for p-FFC 4. The "Raw_SEC_MALS_Data" folder contains the
 csv files used to plot the analytical SEC trace for the FFC complex in
 Figure 4 and the MALS trace for FRQ shown in Figure 2. 1.
 “FFC_AnalyticalSEC.csv” contains the data used to plot the FFC analytical
 SEC trace. 1. Column 1 contains the elution volume(in mLs) 2.  Column 2
 contains the corresponding UV absorbance at 280 nm for the volume in
 Column 1. 2. “PhosFRQ_NonPhosFRQ_SECMALS.csv” contains the data used to
 plot the SEC-MALS trace shown in Figure 2. 1. Column 1 has the elution
 volumes for the concentrated np-FRQ sample 2. Column 2 has the UV
 absorption values for the concentrated np-FRQ sample 3. Column 3 has the
 elution volumes for the concentrated np-FRQ sample that were chosen for
 molecular weight determination 4. Column 4 has the molecular weight
 estimates across the chosen peak region for the np-FRQ sample 5. Column 5
 has the elution volumes for the np-FRQ 4X diluted sample 6. Column 6 has
 the UV absorption values for the concentrated np-FRQ 4X diluted sample 7.
 Column 7 has the elution volumes for the concentrated np-FRQ 4X sample
 that were chosen for molecular weight determination 8. Column 8 has the
 molecular weight estimates across the chosen peak region for the np-FRQ 4X
 diluted sample 9. Column 9 has the elution volumes for the p-FRQ sample
 10. Column 10 has the UV absorption values for the concentrated p-FRQ
 sample 11. Column 11 has the elution volumes for the concentrated p-FRQ
 sample that were chosen for molecular weight determination 12. Column 12
 has the molecular weight estimates across the chosen peak region for the
 p-FRQ sample The "Temperature Concentration Phase Diagram UV-Vis
 Data" folder contains the csv files with the fit used to generate the
 temperature and concentration dependent phase diagram in Figure 6 as well
 as the results of the concentration titration turbidity assays done for
 both phosphorylated and non-phosphorylated FRQ. 1. The file “np-FRQ
 Concentration Titration (5-15 uM)” contains the following data: 1. Column
 1: The temperature at which absorbance, or scattering, at 300 nm was
 recorded. 2. Columns 2 and 3: Two replicates of np-FRQ at 5 uM and their
 absorbance values 3. Columns 4 and 5: Two replicates of np-FRQ at 10 uM
 and their absorbance values 4. Columns 6 and 7: Two replicates of np-FRQ
 at 15 uM and their absorbance values 5. Columns 8 and 9: Two replicates of
 Buffer without protein and their absorbance values used as a control and
 baseline 2.     The file “p-FRQ Concentration Titration (5-15 uM)”
 contains the following data: 1. Column 1: The temperature at which
 absorbance, or scattering, at 300 nm was recorded. 2. Columns 2 and 3: Two
 replicates of p-FRQ at 5 uM and their absorbance values 3. Columns 4: No
 data as there is no third replicate for p-FRQ at 5 uM 4. Columns 5 and 6:
 Two replicates of p-FRQ at 10 uM and their absorbance values 5.    
 Columns 7: No data as there is no third replicate for p-FRQ at 10 uM 1.
 Columns 8, 9, and 10: Three replicates of p-FRQ at 15 uM and their
 absorbance values 2. Columns 11, 12, and 13: Three replicates for Buffer
 without protein and their absorbance values used as a control and baseline
 3. The file “Nonlin fit of Temperature-Concentration Phase Diagram”
 contains information calculated by GraphPad using the data from the files
 “np-FRQ Concentration Titration (5-15 uM)” and “p-FRQ Concentration
 Titration (5-15 uM)”. 4. The "Proteome LLPS Propensity and Disorder
 Scores" folder contains two files: 1. The first file contains the
 IUPRED disorder scores for FRQ, hPER, dPER as well the proteomes for each
 of these organisms (Neurospora crassa, Drosophila melanogaster and humans)
 1. Column 1: The disorder scores for FRQ and its homologs 2. Column 2: The
 disorder scores for the *Neurospora crassa* proteome 3. Column 3: The
 disorder scores for dPER and its homologs 4. Column 4: The disorder scores
 for the *Drosophila melanogaster* proteome 5. Column 5: The disorder
 scores for hPER and its homologs 6. Column 6: The disorder scores for the
 human proteome 2. The second file contains the LLPS propensity scores for
 FRQ, hPER, dPER as well the proteomes for each of these organisms
 (*Neurospora crassa*, *Drosophila melanogaster *and humans) based on the
 method described in the following paper:
 1. Column 1: The LLPS
 propensity scores for FRQ and its homologs 2. Column 2: The LLPS
 propensity for the *Neurospora crassa* proteome 3. Column 3: The LLPS
 propensity for dPER and its homologs 4. Column 4: The LLPS propensity for
 the *Drosophila melanogaster* proteome 5. Column 5: The LLPS propensity
 for hPER and its homologs 6. Column 6: The LLPS propensity for the human
 proteome