*** A guide to the in vitro reconstitution of Cdc42 GTPase activity and its regulation ***
Authors: S. Tschirpke, F. van Opstal, R. van der Valk, W. K-G. Daalman, L. Laan
Delft University of Technology, Faculty of Applied Sciences, Department of Bionanoscience
Delft University of Technology, Kavli Institute of Nanoscience
Corresponding author: L. Laan
Contact Information:
l.laan@tudelft.nl
Delft University of Technology, Faculty of Applied Sciences, Department of Bionanoscience
Van der Maasweg 9, 2629 HZ Delft, The Netherlands

***General Introduction***
This dataset contains data collected for the publication 'A guide to the in vitro reconstitution of Cdc42 GTPase activity and its regulation' (doi: doi.org/10.1101/2023.04.24.538075).
The data in this data set was collected in the Laan lab of the Delft University of Technology - Faculty of Applied Sciences, Department of Bionanoscience, between 2021 and 2022.
This research project was made possible by funding from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement 758132) and funding from the Netherlands Organization for Scientific Research (Nederlandse Organisatie voor Wetenschappelijk Onderzoek) through a Vidi grant (016.Vidi.171.060). 

***Purpose***
The purpose of these experiments was to the GTPase activity of the S.cerevisae GTPase Cdc42, alone and in combination with its GEF Cdc24. We used the GTPase assay 'GTPase-Glo assay' by Promega. The data consists of assay data for variously tagged Cdc42 constructs and purification batches.

***Equipment***
All assays measured on a Synergy HTX plate reader (BioTek) in luminescence mode. 

***Description of the data in this data set***
The data is organised by per assay type (Cdc42 alone/ Cdc42 + Cdc24) in different tabs of 'Cdc42-tags.xlsx'.
Each Cdc42 construct is named with a letter code:
F: H-Cdc42-F
L: S-Cdc42-H
R: S-Cdc42-BC-H
AG: H-Cdc42-SS
AB: F-Cdc42-H
AK: H-SS-Cdc42-F
W: SS-Cdc42-H
AV: H-Cdc42:CTIS
AW: H*-Cdc42:CTIS
AWT: *-Cdc42:CTIS

The tab headers state the Cdc42 letter codes, followed by a purification batch number. They are followed by '_C' in Cdc24 was also present in the assay.  
e.g. F1 stands for data of H-Cdc42-F purification batch 1. 
e.g. F1_C indicates that this tab shows assay data of H-Cdc42-F purification batch 1 in combination with Cdc24. 
In the first tab 'Proteins' the construct abbreviations are summarised. 

Per tab the data of one Cdc42 construct is stated.
Headers:
Run: assay/experiment number
Time: incubation time
GTP_remaining: Amount of remaining GTP (normalised to the buffer, see below)
Error: error for the GTP_remaining values
Buffer_error: error of the buffer wells that were used for normalisation
Cdc42_conc: Cdc42 concentration in uM.
Cdc24_conc: Cdc24 concentration in uM. This is ONLY added IF Cdc24 was present (i.e. in all '..._C' tabs).

The data shows the amount of remaining GTP for various Cdc42 constructs. The amount of remaining GTP correlates with the measured luminescence. Wells without protein (’buffer’) were used for the normalization and represent 0% GTP hydrolysis (Eq. 3 in doi.org/10.1101/2023.04.24.538075). Reactions were carried out with at least 4 replicates (wells) per assay, and the average and standard deviation of each set was used to calculate 'GTP_remaining' and its error ('Error'), and 'Buffer_error' of each set. 

The file 'data_quantitative_cdc42_cdc24.xlsx' shows additional data that describes the quantitative behaviour of the Cdc42 and Cdc24 activity. 
The tab 'Cdc42 (F2)' shows data of Cdc42 alone. The tab 'Cdc42-Cdc24' shows data for the Cdc42-Cdc24 interaction. The header names are the same as stated above. 

