============================================================ __ ________ _____ \ \ / / ____| __ \ /\ \ \ / /| |__ | | | | / \ \ \/ / | __| | | | |/ /\ \ \ / | |____| |__| / ____ \ \/ |______|_____/_/ \_\ USER'S GUIDE (Beta) for VEDA 25.10.10 ============================================================ Content : ------------------------------------------------------------ [0] Introduction [1] Target Map and Symmetry (1.1) Map (1.2) Symmetry (1.3) Crop [2] Models (2.1) Browse and Add (2.2) Models in detail [3] Molecules (3.1) Select and Add (3.2) Molecules in detail [4] Molecule placement (4.1) Main interactive keys (4.2) Build Constellation [5] Fitting Setup (5.1) Configure (5.2) Setup in detail [6] Fitting (6.1) URO Refinement (6.2) Interactive Fitting [7] Save molecules/constellations ============================================================ [0] Introduction : We suppose that VEDA has been installed (read the file VEDA_INSTALL in /VEDA/doc/). We also suppose that you have read the $VEDA/login file and set the environment variables required to execute VEDA. This Beta version of the user's guide give a simple way to perform a fit in 7 steps : 1 : Load a target map and define its intrinsic symmetry. 2 : Load models (pdb and|or map). 3 : Add molecules to the workspace. 4 : Place molecules within the target map and build their constellation (symmetry mates). 5 : Setup for fitting. 6 : Launch interactive fitting and/or refinement. 7 : Save molecules and constellations. Each of these steps refer to a section of this guide. Each section deals with : - How to pass the step rapidly. - The step in detail. - A short description about methods used in the step. ============================================================ [1] Target Map and Symmetry : We start by opening the "Target Map" wizard (whole-menu:|File > Target Map|). This wizard contains 3 tabs (Map, Symmetry, Crop) ------------------------------------------------------------ (1.1) Map Whole-menu:|File > Target Map > Map| To load a map click on button "Browse Map-File" VEDA reads the density map in EZD format used in O. MAPMAN can be used for conversion to other formats. Then click on button "Load" The Density map is depicted as contour surfaces, i.e., surfaces corresponding to a constant user-specified density value. The default "Contour Level" is 1 sigma. You may change this value within this wizard or afterwards in entry whole-menu:|Display > Map > Properties > Contour Level| The loaded map can be displayed as a standard surface, as points or as a wireframe, options available in option menu whole-menu:|File > Target Map > Map > Rendering Type| For all these representations, an opacity parameter controls the transparency of the surface depiction (entry whole-menu:|File > Target Map > Map > Opacity|) >>> WARNING : "Opacity" may not work with some graphic cards <<< Another feature is the modification of the EM magnification. Indeed, the reconstructions are not always perfectly scaled. The magnification may be changed in entry whole-menu:|File > Target Map > Map > EM Magnification| ------------------------------------------------------------ (1.2) Symmetry Several symmetries are available, among them icosahedral, octahedral, tetrahedral, dihedral, cyclic, helical and P1. In tab whole-menu:|File > Target Map > Symmetry|, chose a symmetry in the "Select Symmetry" option menu. Then click on button "Load" For the icosahedral symmetry we implemented all settings having two orthogonal axes parallel to symmetry axes. The helical symmetry may be specified by giving the CTU parameters or the elementary helix parameters plus the number of starts. >>> WARNING : "EM magnification" does not change the helical rise (see "Scale in Fitting Setup) <<< ------------------------------------------------------------ (1.3) Crop Whole menu:|File > Target Map > Crop| If the map is too big for manipulation, it can be cropped manually. Cropping takes into account the FFT restrictions. It generates and loads the new cropped map. ============================================================ [2] Models : Whole menu:|File > Models| After loading a map and setting up the symmetry, one usually loads the models (PDB coordinates or EZD maps). ------------------------------------------------------------ (2.1) Browse and Add In whole-menu:|File > Models|, click on button "Browse File". By default the browser displays files with the filter *.pdb. To load a model map, change the filter to *.ezd. Then give a name to your model or keep the automatically generated name. Then click on button "Add" The loaded models do not appear in the main render window. Indeed, the units that compose the macromolecular assembly are distinct objects in VEDA, called molecules. The conceptual difference between models and molecules is important, which will become clear rapidly. ------------------------------------------------------------ (2.2) Models in detail The models play two main roles in VEDA. First, they are used to create the molecules (i.e. their graphical representations). The second role is to allow the calculation of the Fourier transform of the density corresponding to the constellation. Indeed, the latter is calculated in terms of the molecular scattering factors, which in turn are calculated from the scattering factors corresponding to the individual models. ============================================================ [3] Molecules : Let us start by opening the "Molecules Wizard" in whole-menu |Assemblage > Molecules|. This wizard contains 2 tabs (Molecules , Constellation) ------------------------------------------------------------ (3.1) Select and Add In the wole-menu |Assemblage > Molecules > Molecules| Select fist a model on the left list of the wizard. Then give a name to your molecule or keep the automatically generated name. Then click on the button "Add". You should now see the molecule in the workspace. If not, explore the environment (the molecule may be out of the camera field of view, this is dependant of the pdb coordinates). (you can also press the key 'r'(see section 4)) ------------------------------------------------------------ (3.2) Molecules in detail There are two types of molecules : the independent ones that are necessary and sufficient to build the whole assembly by applying the symmetry operations and the second type is the symmetric copies (called sym-mates) of the previous ones which compose a set called constellation. The independent molecules are the polygonal representations of the models in the VEDA's workspace. It is these entities, considered as rigid bodies, which are assembled to build macromolecular complexes. le-menu:|File > Target Map > Map > Opacity| Their positions within the EM Volume are specified internally by a rotation matrix R parametrized by the Euler angles (α,β,γ) and a translation vector T with coordinates (x,y,z). ============================================================ [4] Interactive keys and molecule placement : (4.1) Main Keys ------------------------------------------------------------ [tab] toggle between camera and molecule modes. In camera mode, mouse events affect the camera position and focal point. In molecule mode, mouse events affect the molecule that have been picked by the mouse pointer. #Mouse [Left Button] : rotate the camera around its focal point (in camera mode) rotate the molecule picked around its origin (in molecule mode). The rotation is in the direction defined from the center of the render window towards the mouse position. [Middle Button] : translate the camera (in camera mode) translate the molecule (in molecule mode). The direction of motion is the direction the mouse moves. [Right Button] : zoom the camera (in camera mode) rotate molecules in plane (in molecule mode) [Left Button] + [Ctrl] : rotate the camera in view plane (in camera mode) [Middle Button] + [Ctrl] : zup/zdown the molecules (in molecule mode). #Miscelaneous Keys [3] : toggle the render window into and out of stereo mode. By default, red-blue stereo rendering mode. [r] : reset the camera view along the current view direction. center all objects present in the workspace and move the camera so that all objects are visible. ------------------------------------------------------------ (4.2) Building Constellation Once you have placed the molecules, you can build their constellation. In whole-menu |Assemblage > Molecules > Constellation| To generate the constellations, select all the molecules (button "Select All" or by clicking in the list) and click on button "Build Constellation" You can notice that, when you move an independent molecule, the set of symmetric molecules move as well in real time according to the symmetric operation defined before. The generation of the constellation in VEDA concerns, as a first step, a symmetry operator selection, which determines which sym-mates associated to each operator will be generated, and, as a second step, the building of a list of sym-mates (called symlist) for : each independent molecule ("Individual Constellation" checked) or for the complete set of independent molecules ("Individual Constellation" unchecked). The selection of the symmetry operators consists in applying the symmetry operators to the positional variable of the center of mass of the independent molecule. If the combination sends the center of mass inside the EM volume, the operators are retained and a sym-mate can be generated at these new positional variables. You can modulate the EM volume criteria by changing the "Minimal Distance to Box Edges" (in angstrom). A positive value for this variable decreases the EM volume. A second test computes the distance between all the sym-mate pairs belonging to the same constellation and checks if any collides. The "Collision Filter" check box allows you to remove from the selection the operators that generate collision between sym-mates if checked. ============================================================ [5] Setup : The fitting setup consists of preparing all the data for the interactive/automatic fitting. ------------------------------------------------------------ (5.1) Configure In whole-menu |Fitting > Setup|. Define the resolution range. During the following fitting, you will be able to work within the range that you just defined. Then click on the button "Configure" and wait until the red color in the status bar of the main window disappears. You can, if you need, change the "EM Scale", which has nearly the same effect as the magnification but the "EM Scale" affects also the parameters of helicoidal symmetry if used. ------------------------------------------------------------ (5.2) Setup in detail The fitting setup computes the Fourier transform of the electron density corresponding to the EM reconstruction and also computes the Fourier transform of the density corresponding to the scattering factors of the models. ============================================================ [6] Fitting : ------------------------------------------------------------ (6.1) URO Refinement In the whole-menu |Fitting > URO Refinement| click on the button "Refine" the "Working Resolution Range"(low and high) is set by default to the max and min values defined in the setup. In URO, in Each cycle all the molecules are refine sequentialy i.e. one molecule is refined while the others are fixed. The "Number of Cycles", set by default to the number of molecules, indicates the number of minimisation cycles. Each molecular position is refined iteratively. the "Number of iterations" representing the number of steps in this refinement. >>> HINT(a) : if you work with more than one molecule, do not enter a big number for iterations at the beginning of the optimisation. <<< >>> HINT(b) : if your molecules move a lot after an optimisation, press "Update Sym-Mates", because the symmetry operation list involved in the fitting may have changed and must be updated <<< ------------------------------------------------------------ (6.2) Interactive Fitting In the whole-menu |Fitting > Interactive Fitting| Press the button "Start/Stop" to launch the interactive fitting, then move the molecule (cf section 4). The correlation is calculated and displayed in real time. the "Working Resolution Range"(low and high) is set by default to the max and min value defined in the setup. >>> HINT : if your molecules move a lot, the symmetry operation list involved in the fitting may have changed and must be updated <<< ============================================================ [7] Save molecules/constellations : Select in the Mol List (at the right on the main window) one Molecule. In the whole-menu |File > Save| choose molecule or constellation. ============================================================ The End