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Demonstrations

REGALS can be applied to SAXS datasets from a variety of experimental setups. Demos 1-4 included here reproduce the analysis in the REGALS paper ([Meisburger, Xu & Ando, 2021]). Each demo is contained in a live notebook and requires an input data file provided in the data folder. Both MATLAB and python versions are included. See below for a list of files and reference information for each dataset.

The easiest way to run the demos is to clone (or download) the REGALS repository and run the live notebooks in place. The jupyter notebooks can also be viewed directly within GitHub.

Meisburger, S.P., Xu, D. & Ando, N. (2021). REGALS: a general method to deconvolve X-ray scattering data from evolving mixtures. IUCrJ 8(2). https://doi.org/10.1107/S2052252521000555

1. AEX-SAXS

Experiment: SAXS with in-line anion exchange chromatography (AEX) collected on as-isolated B. subtilis NrdE, the large subunit of ribonucleotide reductase (from Parker et al. 2018).

Files:

  • data/NrdE_mix_AEX.mat - input data (MATLAB-formatted hdf5)
  • NrdE_mix_AEX.ipynb - jupyter notebook
  • NrdE_mix_AEX.mlx - MATLAB live notebook

Data is included here with permission from the authors. If you use it please cite the original publication:

Parker, M. J. et al. (2018). An endogenous dAMP ligand in Bacillus subtilis class Ib RNR promotes assembly of a noncanonical dimer for regulation by dATP. Proc Natl Acad Sci USA 115, E4594–E4603. https://doi.org/10.1073/pnas.1800356115

2. Ligand titration

Experiment: Phenylalanine hydroxylase (PheH) with varying amounts of allosteric ligand, phenylalanine (from Meisburger et al. 2016).

Files:

  • data/PheH_titration.mat - input data (MATLAB-formatted hdf5)
  • PheH_titration.ipynb - jupyter notebook
  • PheH_titration.mlx - MATLAB live notebook

Data is included here with permission from the authors. If you use it please cite the original publication:

Meisburger, S. P. et al. (2016). Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering. J Am Chem Soc 138, 6506–6516. https://doi.org/10.1021/jacs.6b01563

3. Time-resolved mixing

Experiment: Stopped-flow mixing of ATP with nucleotide binding domain (NBD) from membrane transporter MsbA (from Josts et al. 2020).

Preprocessing: Downloaded scattering curves from the SASBDB (ID: SASDGV5) and reformatted.

Files:

  • data/MsbA_time_resolved.mat - preprocessed input data (MATLAB-formatted hdf5)
  • MsbA_time_resolved.ipynb - jupyter notebook
  • MsbA_time_resolved.mlx - MATLAB live notebook

If you use this data please include the SASBDB ID and cite the original publication:

Josts, I. et al. (2020). Structural Kinetics of MsbA Investigated by Stopped-Flow Time-Resolved Small-Angle X-Ray Scattering. Structure 28, 348-354.e3. https://doi.org/10.1016/j.str.2019.12.001

4. Temperature-jump

Experiment: SAXS/WAXS measurement following rapid temperature jump (IR laser pulse) to 29.9 degC (from Thompson et al. 2019).

Preprocessing: downloaded raw data from the NIH Figshare Archive (Fraser, Anfinrud & Thompson, 2019), calculated difference profiles (laser on - laser off), scaled in WAXS regime, subtracted buffer blanks, and truncated to q < 1 A-1 (see Thompson et al. 2019 and [Meisburger, Xu & Ando, 2021] for details).

Files:

  • data/CypA_Tjump.mat - preprocessed input data (MATLAB-formatted hdf5)
  • CypA_Tjump.ipynb - jupyter notebook
  • CypA_Tjump.mlx - MATLAB live notebook

If you use this data please cite the Figshare entry and the original publication:

Thompson, M. C. et al. (2019). Temperature-Jump Solution X-Ray Scattering Reveals Distinct Motions in a Dynamic Enzyme. Nature Chemistry 11, 1058–1066. https://doi.org/10.1038/s41557-019-0329-3

Fraser, J. Anfinrud, P. & Thompson, M. (2019). X-ray scattering curves (SAXS/WAXS) used for the analysis described in Temperature-Jump Solution X-ray Scattering Reveals Distinct Motions in a Dynamic Enzyme. The NIH Figshare Archive. Dataset. https://doi.org/10.35092/yhjc.9177143.v1

EFA demo

Experiment: SAXS with in-line size-exclusion chromatography (SEC) collected on Phenylalanine hydroxylase (PheH) in 0 mM phenylalanine (from Meisburger et al. 2016).

Files:

  • data/PheH_0phe_SEC.mat - input data (MATLAB-formatted hdf5)
  • PheH_0phe_SEC.ipynb - jupyter notebook
  • PheH_0phe_SEC.mlx - MATLAB live notebook

Data is included here with permission from the authors. If you use it please cite the original publication:

Meisburger, S. P. et al. (2016). Domain Movements upon Activation of Phenylalanine Hydroxylase Characterized by Crystallography and Chromatography-Coupled Small-Angle X-ray Scattering. J Am Chem Soc 138, 6506–6516. https://doi.org/10.1021/jacs.6b01563