Mfp 3d afm controllers

Measurements were performed
in Ca-TBS buffer using custom built AFM instruments (driven vertically
by PI-731 piezo actuators and laterally by a 25 × 25 mm piezomotor
(U-751) in combination with a 100 × 100 nm (P-734) stage, Physik
Instrumente, Germany) in conjunction with MFP-3D AFM controllers (Asylum
Research, Santa Barbara, CA). Upon approaching the sample surface
with the cantilever tip, the complex between CohE and either CttA-XDoc
or ScaB-XDoc was formed and the cantilever was retracted from the
surface at constant velocities of 100, 200, 400, 800, 1600, 3200,
and 6400 nm/s. After each force–extension curve was acquired,
the sample was moved laterally by 100 nm in order to probe a different
molecule. Every several hundred measurements, the glass slide was
moved laterally between protein spots, such that alternatingly CohE-ScaB-Doc
and CohE-CttA-Doc complexes were probed throughout the measurement.
In this manner, thousands of force–extension curves were automatically
acquired over a measurement time of 24–72 h. Single-molecule
interaction traces were identified by filtering the data sets using
contour length analysis, and identifying only those traces in which
two CBM unfolding events were observed.^{11 (link)} Traces exhibiting two CBM unfolding length increments were then
analyzed to create rupture event scatter plots describing the rupture
of the XDoc:CohE complexes.

All data were obtained
using Ca-TBS. Measurements were taken with custom-built instruments
(driven by PI-731 piezo actuators, Physik Instrumente, Germany) in
conjunction with MFP-3D AFM controllers (Asylum Research, Santa Barbara,
USA). Upon approaching the sample surface with the cantilever tip,
the complex between cohesin/dockerin (C/D) was formed, and the cantilever
was retracted from the surface at a constant velocity of 800 nm s^–1 while recoding the distance and cantilever deflection
at a sampling rate of 12 500 Hz. After each force–extension
curve was recorded, the sample was moved laterally by 100 nm to probe
a different molecule. For data analysis, force–distance curves
were transformed into contour length space using a freely rotating
chain model with quantum mechanical corrections for peptide backbone
stretching^{21 (link)} and then sorted by contour
length increments.^{22 (link)} Loading rates prior
to domain unfolding or complex dissociation were extracted by applying
a linear fit to the last 3 nm before the respective event and then
used in fitting the rupture-force histograms with the Bell–Evans
model.^{23 (link)}