Collaborators frequently ask us how to write a X-ray methods section. For your convenience, we have provided a template below for muscle diffraction experiments. Please update the parameters based on your specific experiment. Please rewrite these sessions to avoid plagiarism.

Beamline hardware section:

X-ray diffraction experiments were done using the small angle X-ray diffraction instrument on the BioCAT beamline 18ID at the Advanced Photon Source, Argonne National Laboratory (Fischetti et al., 2004). The X-ray beam energy was set to 12KeV (0.1033 nm wavelength) at an incident flux of ~10¹³ photons per second in the full beam. The X-ray beam was focused to ~250 μm x 250 μm at the sample position and to ~150 x 30 µm at the detector. The specimen to detector distance was about ### m (usually between 2-4 m).

Example of using permeabilized porcine myocardium:

Left ventricular myocardium samples were prepared as described previously (Ma et al., 2022; Ma，W et al., 2023). Briefly, samples of frozen left ventricular myocardium were permeabilized in relaxing solution (2.25 mM Na₂ATP, 3.56 mM MgCl₂, 7 mM EGTA, 15 mM sodium phosphocreatine, 91.2 mM potassium propionate, 20 mM Imidazole, 0.165 mM CaCl₂, creatine phosphate kinase 15 U/ml) containing 15 mM 2,3-Butanedione 2-monoxime (BDM) and 1% Triton-X100 and 3% dextran at pH 7) at room temperature for 2-3h). Muscles were then washed with fresh relaxing solution for ~ 10 minutes, repeated 3 times. The tissue was dissected into ~300 μm diameter fiber bundles and clipped with aluminum T-clips. The preparations were then stored in cold (4°C) relaxing solution with 3% dextran for the day’s experiments. The muscles were incubated in a customized chamber, and experiments were performed at 28-30 °C.

To minimize radiation-induced muscle damage, the X-ray intensity was attenuated to ### of the full beam using 20 µm thick Al-films and the muscle samples were oscillated along their horizontal axes at a velocity of 1 – 2 mm/s. The irradiated areas were moved vertically after each exposure to avoid overlapping X-ray exposures. The force data were also collected during the X-ray experiment.

Using the Eiger 2 9M Detector:  A high sensitivity, high readout speed (up to 500 Hz), Eiger 2 9M detector (Dectris Inc.) with an active area of 233 x 245 mm2 with 75 ´ 75 μm pixels was used to collect the X-ray patterns with ### s exposure time and ### s exposure periods.

Using the MarCCD Detector: A Mar 165 CCD detector (Rayonix Inc., Evanston Il) was used to collect the X-ray patterns with a ###s exposure time.

Data analysis if using MuscleX:

The data were analyzed using the MuscleX software package developed at BioCAT (Jiratrakanvong et al., 2024). The equatorial reflections were measured by the “Equator” routine in MuscleX to calculate the equatorial intensity ratio, I_1,1/I_1,0, and inter-filament lattice spacing, d_1,0, as described previously (Ma et al., 2018). The intensities and spacings of meridional and layer line reflections were measured using the “Projection Traces” routine in MuscleX after the patterns were quadrant folded and the diffuse scattering subtracted to improve the signal-to-noise ratio as described previously (Ma et al., 2018; Ma et al., 2020). Three to four patterns were collected under each condition, and spacings and intensities of X-ray reflections extracted from these patterns were averaged

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References

Fischetti, R., S. Stepanov, G. Rosenbaum, R. Barrea, E. Black, D. Gore, R. Heurich, E. Kondrashkina, A.J. Kropf, S. Wang, K. Zhang, T.C. Irving, and G.B. Bunker. 2004. The BioCAT undulator beamline 18ID: a facility for biological non-crystalline diffraction and X-ray absorption spectroscopy at the Advanced Photon Source. J. Synchrotron Radiat. 11,:399-405.

Jiratrakanvong, J., J. Shao, J. Li, M. Menendez Alvarez, X. Li, P. Das, G. Nikseresht, N. Miskin, R. Huo, J. Nabon, T. Leduc, E. Zhang, W. Ma, G. Agam, and T.C. Irving. 2024. MuscleX: data analysis software for fiber diffraction patterns from muscle. Journal of synchrotron radiation.

Ma, W., M. Childers, J. Murray, F. Moussavi-Harami, H. Gong, R. Weiss, V. Daggett, T. Irving, and M. Regnier. 2020. Myosin dynamics during relaxation in mouse soleus muscle and modulation by 2′-deoxy-ATP. The Journal of physiology. 598:5165-5182.

Ma, W., H. Gong, and T. Irving. 2018. Myosin Head Configurations in Resting and Contracting Murine Skeletal Muscle. Int J Mol Sci. 19.

Ma, W., H. Gong, V. Jani, K.H. Lee, M. Landim-Vieira, M. Papadaki, J.R. Pinto, M.I. Aslam, A. Cammarato, and T. Irving. 2022. Myofibril orientation as a metric for characterizing heart disease. Biophysical journal. 121:565-574.

Ma，W, T. McMillen, M. Childers, H. Gong, M. Regnier, and T. Irving. 2023. Structural OFF/ON transitions of myosin in relaxed porcine myocardium predict calcium activated force. Proceedings of the National Academy of Sciences of the United States of America. 120:e2207615120.