Protocols for Subtomogram Averaging of Membrane Proteins in the Dynamo Software Package - PubMed (original) (raw)

Imparting orientation with a membrane surface Dynamo model. The OMM is modeled by a triangulated mesh based on a set of manually picked points. In

dynamo tomogram slice (dtmslice)

membrane points are picked using a montage (_Montage_>Create Montage of full depicted scene) to manually click on a set of membrane points. By default, this montage shows slices orthogonal to Z, extracted every 20 pixels of the non-binned tomogram (A). Montage settings can be modified by the user (_Montage_>Change montage settings). Create a new membrane model in the montage GUI following: _Model Pool_>_Create new model in pool (choose type)_>Surface. Activate the

[c]

and

[i]

montage GUI button to insert points on this interface by activating the point switcher in the montage toolbar (

c-i

button). The points are also shown in the 3D model displayed in the depiction box of

dtmslice

(B). Explore the tomogram along the Z-view in the montage GUI by clicking on the arrow buttons at the bottom. The membrane is modeled as a surface where points are manually picked by pressing

[i]

or left-clicking with the mouse. Importantly, points should be clicked in the same order from slice to slice in order to get a smother triangulation in next steps of the protocol. Points should be picked in the slice where the OMM is clearly seen, and can be deleted by pressing

[d]

or right-clicking on top of the point that will display an option menu. For semiautomatic detection of points in Z-slices above and below a reference slice, activate the Z-slice where the semiautomatic detection is desired, leave the mouse on top of the activated Z-slice and press the key

[o]

. The surface that should be modeled is the membrane where the neck of the viral spherules is inserted (Supplementary Figure S1). Importantly, defining the interior side of the membrane model is essential to impart the right orientation to the particles. Leave the mouse on the point where the center is defined and press the keys

[shift]

and

[c]

. A green and yellow point is created on the inside of the membrane (A, green and yellow dot). The model is saved into the catalogue [_Active model_>save active model into catalogue (disk) or save all current models in pool by: _Model pool_> _Save all models into catalogue_]. (A) Montage window representing the XY plane of selected tomogram slices. Blue dots represent membrane surface points linked by a red line that simulates the continuity of the modeled membrane. The green, black and yellow bull's eye point represents the inside side of a mitochondrion. (B) Tomogram slice displayed in

dtmslice

. The OMM is represented as blue dots, this is what the user visualizes in

dtmslice

when picking the membrane model points in the montage GUI. The FHV docking site particles are represented as green dots. (C) Tomogram slice displayed in

dtmslice

. The OMM is represented as a gray triangulated surface containing the FHV docking site particles modeled as green points along the membrane surface. Importantly, the goal of this figure is to visualize the two final models unequivocally [particles (green dots) and membrane]. At this point, no surface triangulation is required, albeit later in this protocol triangulation is performed (see Figure 7). (D) Oriented particles obtained by imparting orientation with the membrane using

dpktbl.triangulation.fillTable

to obtain a table containing the geometrical information of the membrane surface used to impart the correct orientation to the particles. (E) Graphical representation of the mitochondrial membrane as a red triangulated surface with the normal vector (black line) of the FHV particles (black dots) pointing in the right direction, toward the outside of the membrane. The Z orientation computed for each particle is represented by the longest semiaxis.