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Supplementary data to RSOB-22-0369

Code and data associated with our study "Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH" published in Open Biology with the ID code RSOB-22-0369. An earlier version of our study has been posted on BioRxiv on February 05, 2021.

The associated structure of NTF2 in complex with the Caprin-1 derived SLiM has been deposited on 2019-10-29 and released on 2021-05-12 with PDB code 6TA7

Here, we also thank the critical reviewers during the inital submission at Molecular Cell for suggesting

  • to re-analyze the imaging data to take into account the variable GFP-G3BP1 (and GFP-Caprin-1) expression
  • to establish in vitro reconstituted condensate assays

Data used to generate the figures have also been depostited at DRYAD.

Analysis and visualization scripts of the data are separated by method, and listed here.
Note: the scripts are not annotated thoroughly. So you need to be familiar with R and the tidyverse.
Plots were saved as postscript files, and edited into publication-quality figures using Adobe Illustrator. The legend to the pre-figures in the markdown document contain information for which figure they were used in the manuscript.
The bookdown document was created based on bookdown, and rtemps.

Please reach out to me if you spot errors, have probles with the scripts/data, or want to develop the scripts into something more useful for the community etc.

ITC

related to Figures 2 and 4.

Data were obtained using an ITC200 calorimeter (GE Healthcare). Data and binding parameters were pre- analyzed using the MicroCal PeakITC software (Malvern). Final analysis and global fits were performed using NITPIC and Sedphat (Brautigam et al., 2016; Scheuermann and Brautigam, 2015).

Link to analysis/visualisation script: https://github.com/derpaule/RSOB-22-0369_ITC

Github page: https://derpaule.github.io/RSOB-22-0369_ITC/

nanoDSF

related to Figure 4.

Data were obtained using Prometheus (Nanotemper) and pre-analyzed using supplier's software. Data were exported for further analysis and visualization.

Link to analysis/visualisation script: https://github.com/derpaule/RSOB-22-0369_nanoDSF_IP

Github webpage: https://derpaule.github.io/RSOB-22-0369_nanoDSF_IP/

BLI

related to Figure 2.

Measurements were performed on an Octet RED instrument (ForteBio). Raw data were pre-processed by subtracting the reference from the sample surfaces and applying a Savitzky-Golay filter as implemented in the Octet RED analysis software.

Link to analysis/visualisation script: https://github.com/derpaule/RSOB-22-0369_BLI

Github webpage: https://derpaule.github.io/RSOB-22-0369_BLI/

Cellular and reconstituted condensate assays

Images were captured using a four-megapixel sCMOS digital camera with the manufacturer’s software MetaXpress, and raw TIF files were analyzed using CellProfiler (CP), ImageJ and Rstudio (McQuin et al., 2018; Schindelin et al., 2012; Wickham, 2017, 2016; Wickham and Grolemund, 2017). Note: original images _w3 were offset from w2 by 4 pixels which was corrected using Fiji using batch processing - the corresponding macro file was deposited on DRYAD.
Output tables were analysed in R.

High-throughput cellular imaging datasets

related to Figures 5 and 7.

DRYAD data related to Figure 5 (G3BP1 mutants)

DRYAD data related to Figure 7 (Caprin-1 chimera)

Raw images/western blots

related to Figures 2, 4, 5, 6, 7.

The images/blots shown as Figures in the main manuscript and the supplement have been deposited as ZIP file at DRYAD. The image files are organized in folders/subfolders according to Figure number.