Hooke is a large, flexible beast, and the number of available commands can be intimidating. In this section, we’ll lay out the procedure for analyzing a number of common experiment types. The experiment types are arranged in the order that they were developed, citating the first known paper in each class. If we’ve got a citation wrong, please submit a bug report (Troubleshooting).
Developed by Rief et al. [1], velocity clamp spectroscopy measures domain unfolding forces as the tip is moved away from the surface at a constant velocity.
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Velocity clamp example plot.
Basic analysis is something like:
hooke> zero_surface_contact_point --block retract
hooke> flat_filter_peaks --block retract --min_points 1
hooke> zero_surface_contact_point --block retract
... --ignore_after_last_peak_info_name 'flat filter peaks'
hooke> convert_distance_to_force --block retract
... --deflection_column 'surface deflection (m)'
hooke> remove_cantilever_from_extension --block retract
hooke> flat_peaks_to_polymer_peaks --block retract
hooke> polymer_fit_peaks --block retract
hooke> block_info --block retract --output data.yaml name 'polymer peak [0-9]*'
The unfolding forces should be grouped by pull velocity, and the resulting distributions can be fit to specific unfolding models by comparison with monte-carlo simulations (see, for example, sawsim).
[1] | M. Rief, M. Gautel, F. Oesterhelt, J.M. Fernandez, H.E. Gaub. “Reversible Unfolding of Individual Titin Immunoglobulin Domains by AFM.” Science, 1997. doi: 10.1126/science.276.5315.1109 |
Developed by Fernandez and Li [2], force clamp spectroscopy measures the lifetime of a domain’s folded state while subject to a constant tension.
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Force clamp example plot.
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Force clamp example analysis.
[2] | J.M. Fernandez, H. Li. “Force-Clamp Spectroscopy Monitors the Folding Trajectory of a Single Protein.” Science, 2004. doi: 10.1126/science.1092497 |
Developed by Clarke et al. [3],...
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TCCS explanation.
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TCCS example plot.
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TCCS example analysis.
[3] | R.W. Clarke, A. Orte, D. Klenerman. “Optimized Threshold Selection for Single-Molecule Two-Color Fluorescence Coincidence Spectroscopy.” Anal. Chem., 2007. doi: 10.1021/ac062188w |