Welcome to what might be called the "Behind the scenes" or "Making of" page.

Here you will find detailed explanations of how we made the three sample movies featured on the eMovie title page, along with the movies themselves. Movies are made according to the eMovie recommended workflow.

The movies.

	Sample Movie 1 (small; full size) Tour of Aricept binding to acetylcholinesterase (AChE). Show me how you made this!
	Sample Movie 2 (small; full size) Active site gorge of AChE: Looking down the gorge and zooming in. Show me how you made this!
	Sample Movie 3 (small; full size) Conformational change of AChE upon binding of bis(5)-tacrine. Show me how you made this!

The detailed explanations.

Sample Movie 1: Tour of Aricept binding to aceytlcholinesterase (AChE).

	Sample Movie 1 (small; full size) Tour of Aricept binding to acetylcholinesterase (AChE).

The detailed explanation of how we planned and made this movie can be found in the eMovie Paper in TiBS.

Sample Movie 2: Active site gorge of AChE: Looking down the gorge and zooming in.

Sample Movie 2 (small; full size) Active site gorge of AChE: Looking down the gorge and zooming in.

1a) Preparations: Define what you want to show (on paper).

We want to show the overall fold of acetylcholinesterase (AChE) and the fascinating fact that the catalytic site of such a rapidly-acting enzyme is buried deep within a gorge in its structure as shown in Sussman, J.L., et al. (1991) "Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein." Science 253, 872-879.

1b) Preparations: Sketch conceptual plot or storyboard (on paper).

We plan the following conceptual storyboard: show the 3D structure of AChE as a simple surface representation, illustrating the deep gorge leading to its active site highlighted in a different color. Slowly rock the molecule left and right slightly. The view should look down the active site gorge. Now zoom into the active site gorge, and again perform the rocking motion. Finally, zoom back out again to the initial view.

1c) Preparations: Translate storyboard into eMovie actions (on paper).

The conceptual storyboard we have just created requires only two actions: rotations and zooms. The rocking action in our conceptual storyboard will be acheived by eMovie's 'Rotation' action, and the zooming in action in our conceptual storyboard will be acheived by eMovie's 'Zoom' action. Our movie will have AChE rotate a little to the right, then a little to the left, then zoom into the gorge, then rotate again a little to the right and a little to the left, and then zoom back out again. We will later decide on the exact parameters for our rotations and zooms.

1d) Preparations: Create the morphs (Using PyMOL and eMovie).

This movie requires no morphs.

1e) Preparations: Create all of the objects and selections in one PyMOL session (in PyMOL only).

First we load 1ea5.pdb (the PDB file containing native acetylcholinesterase) into PyMOL and give the molecule the name AChE. Next, we color AChE green using PyMOL's GUI. We know our active site is defined by a particular list of residues, so we select them either manually by clicking on each residue, or by typing a PyMOL command similar to 'select resi 70+72+80' where each number represents a residue to be included in the selection. We then color the active site selection white using the PyMOL GUI. Using the PyMOL GUI again, we display the molecule, AChE, in a surface representation. Now we manipulate the view using the PyMOL GUI to choose a view that looks down the active site gorge.

2) Create the scenes (in PyMOL and eMovie).

We don't change colors or representations during this movie, and the only view change that we desire we acheive using rotations and zooms, so we do not need to create scenes.

3) Insert actions into eMovie (using eMovie).

The first rotation is a 15 degree rotation about the y axis that spans 15 frames. We add this rotation to frame 1 of the movie using the 'Rotation' button of eMovie. This rotation spans 15 frames, so we start the next rotation at frame 16. The next rotation to add should also be a rotation about the y axis, but this time should rotate 30 degrees in the opposite direction, so we specify -30 degrees using the 'Rotation' button. We also choose an action length of 30 frames. For the third rotation command we specify: Axis: y; Degrees: 15; Start Frame: 46; and Action Length: 15. We add a zoom of 60 Angstroms starting at frame 66 and spanning 5 frames. Then we add 3 more rotations about the y axis in the same way that we added the first 3 rotations: A rotation of 15 degrees starting at frame 71 spanning 15 frames, a rotation of -30 degrees starting at frame 86 spanning 30 frames, and a final rotation of 15 degrees starting at frame 116 spanning 15 frames. Below is a tabular version of the actions inserted into eMovie.

Frame(s)		Action		Information
1-15		Rotation		Axis: y; Degrees: 15
16-45		Rotation		Axis: y; Degrees: -30
46-60		Rotation		Axis: y; Degrees 15
66-70		Zoom		Angstroms: 60
71-85		Rotation		Axis: y; Degrees 15
86-115		Rotation		Axis: y; Degrees -30
116-130		Rotation		Axis: y; Degrees 15

4) Review the movie (using eMovie).

We play the movie a few times to see how it looks. We can also view the storyboard for this movie to see the list of actions that make up our movie and selectively delete actions and reinsert them with new parameters if we desire.

5) Save the movie (using eMovie).

Of course we've been saving our movie using the 'Save eMovie' button throughout the movie-making process in order to prevent accidental loss of work, but now, having finished our movie, we save the final version using the 'Save eMovie' button.

Sample Movie 3: Conformational change of AChE upon binding of bis(5)-tacrine.

Sample Movie 3 (small; full size) Conformational change of AChE upon binding of bis(5)-tacrine.

1a) Preparations: Define what you want to show (on paper).

In this movie we want to show significant conformational changes in specific regions of AChE upon binding of an inhibitor of the enzyme. This is of particular interest as AChE had been thought to be an unusually rigid enzyme. We want to show a interpolated transition between structures of two different conformations of AChE. It includes AChE's substrate, acetylcholine (ACh), and an inhibitor, an alkylene-linked tacrine dimer called bis(5)-tacrine, whose binding results in a significant conformational change of a portion of AChE. For more details see Rydberg, E.H., et al. (2006) Complexes of alkylene-linked tacrine dimers with Torpedo californica acetylcholinesterase: Binding of bis(5)-tacrine produces a dramatic rearrangement in the active-site gorge. J. Med. Chem. 49, 5491-5500.

1b) Preparations: Sketch conceptual plot or storyboard (on paper).

To display the structure of two different conformations of AChE in this movie and visualize a transition between them, we created this conceptual storyboard: Start with AChE with a model of the substrate ACh bound to the active site, then transition to the AChE with the inhibitor bis(5)-tacrine bound to the active site. As this conformation change occurs, slowly fade ACh out and fade bis(5)-tacrine in. Then, play the conformation change in reverse, including the fading of the substrate and inhibitor. Now change to a view that looks at the active site from close-up and highlights the important active site side-chains, and repeat the conformation change forwards and backwards, with substrate and inhibitor fading.

1c) Preparations: Translate storyboard into eMovie actions (on paper).

This movie will require a morph, fading, a pause, and a couple scenes. Conformation changes will be acheived using a morph from the native AChE to the inhibitor-bound AChE. Switching between displaying ACh in the AChE active site and displaying the inhibitor bis(5)-tacrine in the active site will be acheived by fading actions. A pause will be useful at the end of the movie to provide a time gap before the movie loops to the beginning again (if we have movie-looping on in PyMOL or Quicktime). Finally, we have two different combinations of views, representations, and colors (a normal perspective and our close-up perspective) that we will store each as a distinct scene and then insert the scenes into the movie where we need them.

1d) Preparations: Create the morphs (Using PyMOL and eMovie).

We make our morph. We start by loading our two molecules to morph (in this case from PDB files), native AChE (1ea5.pdb) and AChE with bis(5)-tacrine bound (2cmf.pdb), through the PyMOL GUI, by clicking on File/Open... . Now we use eMovie's 'Make Morph' button to make the morphing between native AChE and inhibitor-bound AChE. After entering the parameters (morph from AChE, to AChE with inhibitor bound, refinement 20, place on AChE) and clicking 'OK', eMovie proceeds to make the morph, and prompts us with a window that asks us to press 'OK' again once the PyMOL Tcl/TK GUI reports that RigiMOL has finished creating the morph. This prompt window is crucial to the timing of the internal commands that 'Make Morph' executes, and serves as a user-interaction-dependent pause. After we press 'OK' at this prompt window, morph refinement begins. Soon the morph is finished, available to add into the movie using 'Add Morph', and we proceed onward.

1e) Preparations: Create all of the objects and selections in one PyMOL session (in PyMOL only).

We already have our molecules loaded into PyMOL after having created the morph. The next step is to set appearances and selections. We show the morph as the cartoon representation and color it grey using the PyMOL GUI. We again use the PyMOL GUI to set the background color to white. We create a selection of the active site gorge of the protein, and we rename the selection using the PyMOL GUI (this automatically saves the selection so that it is no longer transient). Next, we choose to show the side chains of the gorge using the stick representation. Having watched the morph previously, we know of two areas of the protein which move a lot, seemingly as wholes, so we select each one separately and color them differently. Residues 279-291 we call 'area1' and color marine, while residues 325-400 and 520-535 we call 'area2' and color cyan. The ACh we color green, the bis(5)-tacrine magenta, and we show ACh in the stick representation and bis(5)-tacrine in ball-and-stick representation. Now we have finished preparing the appearance of our molecules, and we reflect on the overall session and decide we are satisfied.

2) Create the scenes (in PyMOL and eMovie).

We create two scenes using eMovie's 'Scenes (views, appearances)' function: The first shows a zoomed out AChE with one set of representations and colors. The second displays a close-up of the active site with a separate set of representations and colors. Having two scenes will allow us an easy way to dynamically change representations, colors, and views during the movie. Here is a screenshot showing the creation of the second scene.

3) Insert actions into eMovie (using eMovie).

We insert the individual actions into the movie, starting with the morphs.

We click on 'Add Morph' in eMovie and then on 'Click to add a morph to movie'. Then we enter in the parameters for our morph (start at frame 1, loop with 15 frame pause). We use 'Add Morph' again to insert the same morph again but this time starting at frame 91 (loop with 15 frame pause). We insert our first scene at frame 1 and our second scene at frame 91 using 'Scenes (views, appearances)' and then 'Insert a scene into movie'. To add fading, we click on eMovie's 'Fading' button and specify that we want ACh, shown as sticks, to fade from 100% visible to 0% visible over the course of 13 frames, starting at frame 1. We use the 'Fading' button in the same way again to add the fading in of bis(5)-tacrine, but since bis(5)-tacrine is shown in ball-and-stick, we must use the fading button twice for each fading of bis(5)-tacrine, once to address fading of the sticks, and once to address fading of the balls (sphere). We use the 'Fading' button multiple times again to insert actions to fade out bis(5)-tacrine as the morph plays backwards (starting at frame 46) and to fade ACh back in. Since our morph plays again in a loop after we change the scene to a close-up view at frame 91, we add the same fading actions during the second instance of the morph in our movie. Finally, we add a pause at the end of our movie (start frame is 166, pause length is 15 frames). Below is a tabular version of the actions inserted using eMovie.

Frame(s)		Action		Information
1-75		Morph		Name: morph1; Direction: forward (30 frames), pause for 15 frames, then backward (30 frames)
91-165		Morph		Name: morph1; Direction: forward (30 frames), pause for 15 frames, then backward (30 frames)
1		Scene Set		Scene name: Overall_View
91		Scene Set		Scene name: Active_site_closeup
1-12		Fading		Molecule: ACh_model; Shown as: stick; Fade from: 100% visible; Fade to: 0% visible
19-30		Fading		Molecule: bis-5-tacrine; Shown as: stick; Fade from: 0% visible; Fade to: 100% visible
19-30		Fading		Molecule: bis-5-tacrine; Shown as: sphere; Fade from: 0% visible; Fade to: 100% visible
46-57		Fading		Molecule: bis-5-tacrine; Shown as: stick; Fade from: 100% visible; Fade to: 0% visible
46-57		Fading		Molecule: bis-5-tacrine; Shown as: stick; Fade from: 100% visible; Fade to: 0% visible
64-75		Fading		Molecule: ACh_model; Shown as: stick; Fade from: 0% visible; Fade to: 100% visible
91-102		Fading		Molecule: ACh_model; Shown as: stick; Fade from: 100% visible; Fade to: 0% visible
109-120		Fading		Molecule: bis-5-tacrine; Shown as: stick; Fade from: 0% visible; Fade to: 100% visible
109-120		Fading		Molecule: bis-5-tacrine; Shown as: sphere; Fade from: 0% visible; Fade to: 100% visible
136-147		Fading		Molecule: bis-5-tacrine; Shown as: stick; Fade from: 100% visible; Fade to: 0% visible
136-147		Fading		Molecule: bis-5-tacrine; Shown as: sphere; Fade from: 100% visible; Fade to: 0% visible
154-165		Fading		Molecule: ACh_model; Shown as: stick; Fade from: 0% visible; Fade to: 100% visible

4) Review the movie (using eMovie).

We review the movie by playing it several times and reviewing the storyboard of actions. If we need to, we delete actions from the storyboard and reinsert them with different parameters.

In this screenshot, we see the eMovie storyboard displaying the first 10 actions that make up our movie, and in the PyMOL Viewer window we see our first scene displayed. In this screenshot, the storyboard shows actions 11 through 20 with the second scene displayed in the PyMOL viewer as we are about to insert a pause to the end of the movie.

5) Save the movie (using eMovie).

Of course we've been saving our movie using the 'Save eMovie' button throughout the movie-making process in order to prevent accidental loss of work, but now, having finished our movie, we save the final version using the 'Save eMovie' button.

Please send us movies you've made using eMovie! We would love to display them on this website.

And don't forget to cite eMovie!

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