This readme.txt file was generated on 20230801 by Haw Yang and Xun Sun ------------------- GENERAL INFORMATION ------------------- Title of Dataset: Raw Data for GrsA Sub-domain Dynamics Data for "Sub-Domain Dynamics Enable Chemical Chain Reactions in Nonribosomal Peptide Synthetases" Author Information: Principal Investigator Name: Haw Yang ORCID: 0000-0003-0268-6352 Institution: Princeton University Address: Department of Chemistry, Princeton University, Princeton, NJ 08544, USA Email: hawyang@princeton.edu Associate or Co-investigator: Name: Henning D. Mootz ORCID: 0000-0002-3385-0234 Institution: University of Muenster Address: Institute of Biochemistry, Department of Chemistry and Pharmacy, University of Muenster, Münster, Germany Email: Henning.Mootz@uni-muenster.de Alternate Contact(s): N.A. Date of data collection (single date, range, approximate date): 20110701-20230515 Geographic location of data collection: Princeton, NJ, USA; Cornell, NY, USA; Muenster, Germany and Hsinchu, Taiwan. Information about funding sources or sponsorship that supported the collection of the data: HFSP award (No. RGP0031/2010-C202) Princeton University -------------------------- SHARING/ACCESS INFORMATION -------------------------- Licenses/restrictions placed on the data, or limitations of reuse: CC-BY 4.0 Recommended citation for the data: DOI:10.34770/6ehx-sr32 Sun, X., Alfermann, J., Li, H., Watkins, M. B., Chen, Y.-T., Chu, J. W., & Ando, N. (2023). Raw Data for GrsA Sub-domain Dynamics [Data set]. Princeton University. https://doi.org/10.34770/6EHX-SR32 Citation for and links to publications that cite or use the data: (url will be added after publication) Links to other publicly accessible locations of the data: N.A. Links/relationships to ancillary or related data sets: N.A. -------------------- DATA & FILE OVERVIEW -------------------- File list (filenames, directory structure (for zipped files) and brief description of all data files): Below is a list of folder grouped according to the type of data as indicated by the self-explanatory folder name. 1_MD/, 2_smFRET/, 3_SEC_SAXS/, 4_multi_seq_alignment/, 5_gels/. See DATA-SPECIFIC INFORMATION below for additional details. Relationship between files, if important for context: N.A. Additional related data collected that was not included in the current data package: N.A. If data was derived from another source, list source: N.A. If there are there multiple versions of the dataset, list the file updated, when and why update was made: N.A. -------------------------- METHODOLOGICAL INFORMATION -------------------------- Description of methods used for collection/generation of data: 1. Molecular dynamics (MD) simulations data were generated per Chem. Phys. 396, 61–71 (2012). 2. Single-molecule Förster resonance energy transfer (smFRET) data were collected per J. Phys. Chem. A 110, 5191–5203 (2006). 3. Size-exclusion chromatography small-angle X-ray scattering (SEX-SAXS) data were collected per J. Am. Chem. Soc. 138, 6506–6516 (2016) and Nat. Protoc. 9, 1727–1739 (2014). 4. Multiple sequences were taken from UniProt database https://www.uniprot.org. 5. Native polyacrylamide gel electrophoresis (PAGE) data were collected per FEBS J. 277, 1159–1171 (2010). Methods for processing the data: 1. The MD data were processed per Chem. Phys. 396, 61–71 (2012). 2. The smFRET data were processed using the Maximum-information (MIM) analysis per Biophys. J. 86, 4015–4029 (2004) and using the motional narrowing analysis per Proc. Natl. Acad. Sci. USA 104, 18055–18060 (2007) and Chem. Phys. Lett. 288, 225–229 (1998). 3. The SEC-SAXS data were processed using the evolving factor analysis per J. Am. Chem. Soc. 138, 6506–6516 (2016) and Nat. Protoc. 9, 1727–1739 (2014). 4. The multiple sequence alignment data were aligned per manufacturer's instruction for MegAlign (DNASTAR, https://www.dnastar.com/manuals/MegAlignPro/17.3.1/en/topic/welcome-to-megalign-pro). 5. Native PAGE gels were not processed. The raw tif files were provided. Software- or Instrument-specific information needed to interpret the data, including software and hardware version numbers: 1. VMD (v1.9.3, from https://www.ks.uiuc.edu/Research/vmd/) can be used to view the MD data (psf and dcd). 2. The MIM (v1.0) and motional narrowing analysis (v5.1) codes from the Yang lab were deposited at https://github.com/PrincetonUniversity/smFRET_MaximumInformationMethod and publicly available. The processed time-distance data (.xc file) can be viewed using MATLAB (any version after 2012, https://www.mathworks.com/products/matlab.html). 3. The processed SAXS data can be viewed using MATLAB (any version after 2012, https://www.mathworks.com/products/matlab.html) and ATSAS (any version after 2.8.0, https://www.embl-hamburg.de/biosaxs/software.html). 4. WebLogo (2.8.2 from http://weblogo.berkeley.edu) can be used to view the sequence alignment. 5. Any photo viewing software can be used to view the unprocessed native PAGE gels. No instrument/hardware specific information was needed for data interpretation for any of the data. Standards and calibration information, if appropriate: The extinction coefficients of Alexa 555 and Alexa 647 were calibrated using the standard values from "The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies", 11 edn, (Invitrogen Inc., 2010). Environmental/experimental conditions: smFRET, SAXS and native PAGE sample incubation were performed in 50 mM Tris/HCl, 300 mM NaCl, and 10 mM MgCl2 at pH 8.0 at room temperature. Describe any quality-assurance procedures performed on the data: 1. The constrained MD data were constrained using smFRET results and cross compared with the SAXS data using the Ensemble Optimization Method (J. Am. Chem. Soc. 129, 5656–5664 (2007) and IUCrJ 2, 207–217 (2015)) for internal consistency. 2. The quality of smFRET data was assured per the statistical tests per J. Chem. Phys. 128, 214101 (2008) and J. Phys. Chem. B 112, 13962–13970 (2008) and per protocol in Chem. Phys. 396, 61–71 (2012). 3. The SAXS data quality was ensured per J. Am. Chem. Soc. 138, 6506–6516 (2016). 4. 151 homologous protein sequences were used in the alignment file. 5. The native PAGE gels show expected mobility due to binding of a ligand described in FEBS J. 277, 1159–1171 (2010). People involved with sample collection, processing, analysis and/or submission: Sample collection: 1_MD: Yi-Tsao Chen 2_smFRET: Xun Sun, Hao Li 3_SEC_SAXS: Maxwell B. Watkins, Hao Li, Nozomi Ando 4_multi_seq_alignment: Jonas Alfermann 5_gels: Xun Sun Processing and analysis: 1_MD: Yi-Tsao Chen, Jhih-Wei Chu 2_smFRET: Xun Sun, Hao Li, Haw Yang 3_SEC_SAXS: : Maxwell B. Watkins, Nozomi Ando 4_multi_seq_alignment: Jonas Alfermann, Henning D. Mootz 5_gels: Xun Sun, Haw Yang Data submission: Xun Sun, Haw Yang -------------------------- DATA-SPECIFIC INFORMATION -------------------------- Here are the raw smFRET trajectories, SAXS data and MD simulation trajectories for the paper titled "Sub-Domain Dynamics Enables Chemical Chain Reactions in Nonribosomal Peptide Synthetases". 1. The CHARMM formatted DCD files and the associated PSF files are organized by conformation-dye. For example, X-416-CC1 means the R416-based X-conformation with CC1 labeling sites for aCC1. Unconstrained MD simulations are noted in folder names and the rest are constrained MD simulations. For example, unconstrained-A-CC1 means unconstrained MD simulations for aCC1 in A-state. 2. The raw smFRET fr files (photon arrival times for both donor and acceptor channels, see J. Phys. Chem. A, 2006, 110, 5191-5203) and xc files (maximum-information binning using Fisher information analysis of the photon arrival times, see Biophys. J., 2004, 86, 4015-4029). The format of the fr files related to smFRET data is: Byte 4-5, unsigned short, Version number 2 Byte 8-11, unsigned long, length of channel 1 Byte 12-15, unsigned long, length of channel 2 Byte 24-31, double, clock speed Byte 56-63, double, x position of the spot Byte 64-71, double, y position of the spot Byte 72-79, double, donor bleach time Byte 80-88, double, acceptor bleach time Byte 1024, unsigned long ,channel 1 photon arrival times Byte 1024+(length of channel 1), unsigned long, channel 2 photon arrival times The format of the xc files is: column 1: bin start time (s) column 2: bin end time (s) column 3: R/R0 column 4: error in distance The raw photon arrival time can be extracted using rawload.m inside the smFRET folder. The two-state motional narrowing model is done per Proc. Natl. Acad. Sci. USA, 2007, 104,18055-18060. See https://github.com/PrincetonUniversity/smFRET_MaximumInformationMethod for a full set of analysis codes for the maximum-information method and the motional narrowing fits. The smFRET data are organized by mutant-condition. For example, CC1.apo.sf means apo CC1 substrate-free in smFRET buffer and CC2.holo.Phe.ATP means holo CC2 with 1 mM ATP and 1 mM L-Phe. The concentration of Phe-AMS was 100 µM. 3. SAXS raw intensity and P(r) data for aCC2 with and without 1 mM ATP/L-Phe. 4. Multiple sequence alignment file for GrsA with other sequences from 151 homologous proteins that were aligned by MegAlign. The enzymes included in this sequence alignment were: actinomycin synthetase I A, actinomycin_synthetase II A1-A2, actinomycin synthetase III A1-A3, ACV synthetase A1-A3, bacillibactin synthetase II A1-A2, bacitracin synthetase 1 A1-A5, bacitracin synthetase 2 A1-A2, bacitracin synthetase 3 A1-A5, bleomycin synthetase I A1-A3, bleomycin synthetase III A1-A2, bleomycin synthetase IV A1, bleomycin synthetase VI A1, bleomycin synthetase VII A1-A2, bleomycin synthetase VIII A1, CDA peptide synthetase I A1-A6, CDA peptide synthetase II A1-A3, CDA peptide synthetase III A1-A2, complestatin synthetase I A1-A2, complestatin synthetase II A1, complestatin synthetase III A1-A3, complestatin synthetase IV, A1-A2, cyclosporine synthetase A1-A11, enterobactin synthetase component E A1-A2, fengycin synthetase I A1-A3, fengycin synthetase II A1, fengycin synthetase III A1-A2, fengycin synthetase IV A1-A2, fengycin synthetase V A1-A2, gramicidin S synthetase A A, gramicidin S synthetase B A1-A4, HC-toxin synthetase A1-A4, iturin A synthetase A A1-A2, iturin A synthetase B A1-A4, iturin A synthetase C A1-A2, lichenysin synthetase A A1-A2, lichenysin synthetase B A1-A3, lichenysin synthetase C A1, microcystin synthetase I A1-A2, microcystin synthetase II A1-A2, microcystin synthetase III A1, microcystin synthetase V A1, microcystin synthetase VII A1, mycosubtilin synthetase I A1-A2, mycosubtilin synthetase II A1-A4, mycosubtilin synthetase III A1-A2, pristinamycin I synthase I, A1, pristinamycin I synthase II A1-A2, pristinamycin I synthase III A1-A4, pyochelin synthetase PchD A1, pyochelin synthetase PchE A1, pyochelin synthetase PchF A1, surfactin synthetase I A1-A3, surfactin synthetase II A1-A3, surfactin synthetase III A1, syringomycin synthetase I A1, syringomycin synthetase II A1-A8, tyrocidine synthetase I A1, tyrocidine synthetase II A1-A3, tyrocidine synthetase III A1-A6, and yersiniabactin peptide synthetase HMWP2. 5. Original native PAGE gels for Extended Data Figure 2b-c (20120108_masterGrsA.tif, from left to right lane 5-8 for CC1 and lane 9-12 for CC2) and Extended Data Figure 5h (20120327_pJA10,14,17_2.tif. from left to right lane 5-8 for CC2-K517A).