1. Segmentation outward inward Slide 1
The flower motif structure is shown along with the two possible interpretations of the density corresponding to a myosin V molecule as segmented from this structure. The cargo-binding domain has been removed as its structure is not yet available for fitting. Segmentation of the densities is necessary for docking by SITUS and fitting using Normal Mode Flexible Fitting (NMFF).
inward

2. Three Conformations pre-power stroke state
in smooth muscle (PDB - 1BR1)
pre-power stroke state
in scallop muscle (PDB - 1QVI)
Slide 2
This slide illustrates the lever arm orientations of the three myoV atomic models used for the initial docking into the 3-D reconstruction of the inactive conformation. They are shown docked on F-actin to illustrate the extent of the differences which are mainly in the lever arm orientation. All have the myoV sequence and the same motor domain structure based on PDB 1W7I and the same lever arm structure, but have had their lever arms oriented according to the PDB data set indicated on the slide. This produces two pre-powerstroke models, one that is scallop myosin-like and one that is smooth muscle myosin-like. The color scheme has the motor domain in red, the calmodulins in green and the heavy chain part of the lever arm in dark blue.
Myosin-V with ADP (PDB - 1W7I)
F-actin

3. Scallop-like pre-power stroke smooth muscle-like pre-power stroke
Docking by SITUS (colores)
outward
CC= 0.64
CC= 0.53
CC= 0.42
CC= 0.58
CC= 0.41
Slide 3
This slide illustrates the initial docking achieved using the COLORES utility in SITUS. The three initial models have been docked into the two possible interpretations of the structure. The color scheme has the motor domains in red and magenta, the calmodulins in green and the heavy chain part of the lever arm in dark blue. None of the initial fits are good in the “inward” configuration. In the “outward” configuration, the quality of the initial fit is ordered by lever arm angle, rigor like being the best initial fit.
inward
ADP
myosin-V
Scallop-like pre-power stroke
smooth muscle-like pre-power stroke

4. Scallop-like pre-power stroke smooth muscle-like pre-power stroke
Docking by SITUS (colores)
outward
CC= 0.64
CC= 0.53
CC= 0.42
CC= 0.58
CC= 0.41
Slide 4
This slide illustrates the initial docking achieved using the COLORES utility in SITUS but rotated 45° about the vertical axis to show a different view of the initial docking. None of the initial fits are good in the “inward” configuration. In the “outward” configuration, the quality of the initial fit is ordered by lever arm angle, rigor like being the best initial fit.
inward
ADP
myosin-V
Scallop-like pre-power stroke
smooth muscle-like pre-power stroke

5. Docking by SITUS (myoV-ADP)
Slide 5
This slide illustrates at higher magnification the initial SITUS docking of the myoV-ADP model to the “outward” interpretation. The left panel is the same view as shown in Slide 3, the right panel is the same view shown in Slide 4.
outward
CC= 0.64
ADP
myosin-V

6. Normal Mode Flexible Fitting (myoV-ADP)
Slide 6
This slide shows at higher magnification the NMFF fitting result of the myoV-ADP model to the “outward” interpretation. By toggling back and forth between Slides 5 and 6 in “view slide show” mode, one can see the effect of the flexible fitting into the density.
outward
CC= 0.82
ADP
myosin-V

7. Scallop-like pre-power stroke
Docking by SITUS (scallop-like)
Slide 7
This slide shows at higher magnification the initial SITUS docking of the scallop-like model to the “outward” interpretation. The left panel is the same view as shown in Slide 3, the right panel is the same view shown in Slide 4.
outward
CC= 0.58
Scallop-like pre-power stroke

8. Scallop-like pre-power stroke
Normal Mode Flexible Fitting (scallop-like)
Slide 8
This slide shows at higher magnification the NMFF fitting result of the scallop-like model to the “outward” interpretation. By toggling back and forth between Slides 7 and 8 in “view slide show” mode, one can see the effect of the flexible fitting to the density.
outward
CC= 0.77
Scallop-like pre-power stroke

9. Fitted “scallop-like” on F-actin
pre-power stroke state in smooth muscle myosin (PDB - 1BR1)
pre-power stroke state in scallop myosin (PDB - 1QVI)
Slide 9
This slide illustrates the docking of the NMFF fitted “scallop-like” model onto F-actin with the cargo-binding domain added. The left panel is the docked model and the right panel repeats the view shown in Slide 2 of the three initial myoV head structures used for the fitting. After fitting, the near 90° orientation of the “scallop-like” lever arm has tilted toward the (+) end of the actin filament by ~25°. The placement of the cargo-binding domain on the motor domain after NMFF fitting is different from the cargo-binding domain location after NMFF fitting of the myoV-ADP model. A consequence of the different placement of the cargo-binding domain on the fitted “scallop-like” motor domain is a steric clash between it and the actin filament when the structure is docked onto actin.
Myosin-V with ADP (PDB - 1W7I)
F-actin
F-actin