Supplementary data for dissertation: The Evolution and Regulation of Morphological Complexity in the Vibrios Nicholas Martin (nrm2@princeton.edu) and Zemer Gitai (zgitai@princeton.edu), Princeton University Department of Molecular Biology Recommended citation for this dataset: Martin, N., & Gitai, Z. (2021). Supplementary data for thesis: The Evolution and Regulation of Morphological Complexity in the Vibrios [Data set]. Princeton University. https://doi.org/10.34770/GK6N-GJ34 Data collected 2020-2021 "Supplementary Video 5.avi" Corresponds to referenced video in chapter III of dissertation Representative time lapse videos of CrvB-GFP structures in a genetic background containing a crvA CT deletion. Images were taken every 10 seconds. Top to bottom rows of images are phase contrast, GFP fluorescence, and merge, respectively. Columns represent different fields of view. "Supplementary Video 6.avi" Corresponds to referenced video in chapter III of dissertation Representative time lapse videos of CrvA-GFP structures in the indicated genetic backgrounds. Images were taken every 20 seconds. Top to bottom rows of images are phase contrast, GFP fluorescence, and merge, respectively. Columns within an image group represent different fields of view of the same strain. "Supplementary Video 7.avi" Corresponds to referenced video in Appendix B of dissertation Representative time lapse videos of CrvA*-mCherry class B structures. Images were taken every 5 seconds. "Table C.2.xlsx" Full list of hits from mass spectrometry of α-FLAG co-immunoprecipitation. Sheets included in workbook: "Full Samples Report" Data report exported from Scaffold4 software including DECOY proteins. Rows 1-40 contain metadata and algorithm parameters for the experiment. Row 44 contains headers for columns of tabular data which compose the remaining rows of the sheet. "20200911_CE_947_WT_SDB", "20200911_CE_947_CrvAFLAG_SDB", and "20200911_CE_947_CrvBFLAG_SDB" in columns J-L label the normalized total spectral counts of each protein in the three different experimental samples. "Simplified Data" A subset of columns from the tabular data in "Full Samples Report" Row 3 contains headers for columns of tabular data. "WT", "CrvA", and "CrvB" in columns F-H replace "20200911_CE_947_WT_SDB", "20200911_CE_947_CrvAFLAG_SDB", andm"20200911_CE_947_CrvBFLAG_SDB", respectively, as simplified titles for each of the three samples. Columns J and K are the ratio of the values of each cell from "CrvA" and "CrvB" divided by the corresponding value from "WT" Note that due to the lack of detected peptides from some proteins in WT, the formula returns an error due attempted division by zero. "Proteins enriched twofold" A subset of rows from the tabular data in "Simplified Data" containing those proteins with fold changes greater than 2 in either CrvA or CrvB (Rows 4-41) or both CrvA and CrvB (Rows 45-53). All relevant methodological information can be found in the Princeton University PhD dissertation: The Evolution and Regulation of Morphological Complexity in the Vibrios and is repeated here: Overnight cultures of strains with 3xFLAG-tagged proteins were grown in 50ml LB shaking in 250mL flasks and cells were harvested at 10000 x g for 10 min. Cell pellets were rinsed twice with 2 ml Buffer 1 (50 mM NaPO4 pH7.4, 150 mM NaCl, 1 mM EDTA) without resuspending the pellet. Pellets were then resuspended in 1mL Buffer 2(50 mM Na3PO4 pH7.4, 150 mM NaCl, 1 mM EDTA, 10 mM MgCl2, 0.5% Dodecyl maltoside). Cells in this suspension were lysed by adding 0.12μL Ready-Lyse lysozyme (4200 units), 15μL of DNAse I (30 units), and 10μL of 100x protease inhibitor cocktail (1x final concentration) and incubated at room temp for 30 min. Cellular debris was removed by centrifugation at 10000xg for 5 min at 4°C. 50μL of α-FLAG magnetic beads (Sigma-Aldrich Cat #M8823) was washed three times with 1 ml of Buffer 2 using magnets to produce the pellet. 1ml of lysate was added to beads and gently rock at 4°C for 2 hours. Beads were pelleted and washed three times by resuspending in 500μL Buffer 2 and rotating at 4 C for 10 min. After washes, an additional 500μL Buffer 2 was added briefly and removed with special care to remove all liquid. Proteins were eluted from the beads by incubating in 100μL of 3X FLAG elution buffer (3μL of 5 mg/ml 3X FLAG peptide (Sigma-Aldrich Cat #F4799) solution in 100μL of Buffer 2) and rocking at 4 C for 30 min.Spreadsheet containing the normalized total spectra from co-immunoprecipitation experiment. To submit samples for mass spectrometry, 30μL eluate was run briefly by SDS-PAGE so that the dye front migrated about 1cm into the resolving gel. The entire lane above the dye front was then cut out and submitted for liquid chromatography/mass spectrometry. In-gel digestion of protein bands using trypsin was performed as in Shevchenko et al. Trypsin digested samples were dried completely in a SpeedVac and resuspended with 20ul of 0.1% formic acid pH 3 in water. 2ul (~ 360ng) was injected per run using an Easy-nLC 1200 UPLC system. Samples were loaded directly onto a 45cm long 75um inner diameter nano capillary column packed with 1.9um C18-AQ resin (Dr. Maisch, Germany) mated to metal emitter in-line with an Orbitrap Fusion Lumos (Thermo Scientific, USA). Column temperature was set at 45’C and two-hour gradient method with 300nl per minute flow was used. The mass spectrometer was operated in data dependent mode with the 120,000 resolution MS1 scan (positive mode, profile data type, AGC gain of 4e5, maximum injection time of 54 sec and mass range of 375-1500 m/z) in the Orbitrap followed by HCD fragmentation in ion trap with 35% collision energy. Dynamic exclusion list was invoked to exclude previously sequenced peptides for 60s and maximum cycle time of 3s was used. Peptides were isolated for fragmentation using quadrupole (1.2 m/z isolation window). Ion-trap was operated in Rapid mode. Raw files were searched using Byonic and Sequest HT algorithms within the Proteome Discoverer 2.2 suite (Thermo Scientific, USA). 10 ppm MS1 and 0.4 Da MS2 mass tolerances were specified. Carbamidomethylation of cysteine was used as fixed modification, oxidation of methionine, deamidation of asparagine and glutamine were specified as dynamic modifications. Pyro glutamate conversion from glutamic acid and glutamine are set as dynamic modifications at peptide N-terminus. Acetylation was specified as dynamic modification at protein N-terminus. Trypsin digestion with maximum of 2 missed cleavages were allowed. Files were searched against UP000000584 Vibrio cholerae database downloaded from Uniprot.org. Scaffold (version Scaffold_4.11.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.9% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.