Raffaele Coray at 12:39, 2 December 2024

2024-12-02T12:39:10Z

Daniel Castaño: /* Use a surface to impart orientation */

2019-09-02T18:30:10Z

Use a surface to impart orientation

Daniel Castaño at 18:13, 2 September 2019

2019-09-02T18:13:26Z

Daniel Castaño at 18:09, 2 September 2019

2019-09-02T18:09:22Z

Daniel Castaño at 18:06, 2 September 2019

2019-09-02T18:06:03Z

Daniel Castaño: Created page with "Category:Walkthroughs This walkthrough uses a small size example based on a real tomogram to cover several tasks. == The example data set == The data is a fraction of a..."

2019-09-02T18:04:23Z

Created page with "Category:Walkthroughs This walkthrough uses a small size example based on a real tomogram to cover several tasks. == The example data set == The data is a fraction of a..."

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← Older revision		Revision as of 12:39, 2 December 2024
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−	[[Category:~~Walkthroughs~~]]	+	[[Category:WalkthroughsSpecific]]

	This walkthrough will be used during the [[EMBO workshop 2019]] uses a small size example based on a real tomogram to cover several tasks.		This walkthrough will be used during the [[EMBO workshop 2019]] uses a small size example based on a real tomogram to cover several tasks.

@@ Line 291: / Line 291: @@
   <nowiki>Volume 1. Matching models: 1 (total: 1)
-/Users/casdanie/dynamo/examples/fhvParis/fhv/tomograms/volume_1/models/mySurface.omd</nowiki>
+/i/embo2019/u/emboX/Prac-4/fhv/tomograms/volume_1/models/mySurface.omd</nowiki>
 now, we can read the model file into our memory space to operate with it:
-  <tt>m = dread(output.files{1});</tt>
+  <tt>\m = dread(output.files{1});</tt>
 Now the model <tt>m</tt> is in our space of memory and can be used to impart an orientation to the points in our table.

← Older revision		Revision as of 18:13, 2 September 2019
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	Please adapt it to the location of Chimera in your file system.		Please adapt it to the location of Chimera in your file system.

−	~~== Project to find membrane orientations==~~
−
−	~~We first need to create files with the average and the table that we have till now in memory:~~
−
−	~~<nowiki>dwrite(t,'raw.tbl');~~
−	~~dwrite(oa.average,'rawTemplate.em');</nowiki>~~
−
−	~~We can create a project directly thorugh the command line:~~
−	~~<tt>dcp.new('first','d','particlesData','template','rawTemplate.em','masks','default','t','particlesData/crop.tbl');</tt>~~
−
−	~~...and go directly to the panel of numerical parameters to create a project adapted for catching the general orientation of the membrane:~~
−	~~[[File:FhvFirstParameters.png\|thumb\|center\|500px\| Numerical parameters for just membrane orientation]]~~
−
−	Also, remember to visit the window for execution environment, and select the appropriate settings. If you are working with Matlab, select ''Matlab''(for one single core) or "Matlab_parfor" (if you are going to several cores). If working with the standalone, use the [[standalone]] option. In both cases, select the number of cores for alignment (green box in the picture below) and averaging (blue box) .
−
−	~~[[File:FhvEnvironment.png\|thumb\|center\|500px\| Selecting the execution modus]]~~
−
−	~~You can operate the project management operations from the GUI or the command line. First checking:~~
−
−	~~<tt>dvcheck first</tt>~~
−
−	~~then unfolding~~
−
−	~~<tt>dvunfold first</tt>~~
−
−	~~and finally running it by invoking the name of the execution script. If you are running in Matlab use:~~
−
−	~~<tt>first</tt>~~
−
−	~~If you are running ''Dynamo'' in standalone modus, write into an activated Linux/MacOS shell:~~
−
−	~~<tt>./first.exe</tt>~~
−
−	~~After running the project, we check the created average:~~
−
−	~~<tt>ddb first:a -v </tt>~~
−
−	This command asks the database (hence <tt>db</tt>) of the project <tt>first</tt> to look for the item of type <tt>average</tt>, by default accessing the last computed one. The flag <tt>-v</tt> sends the output of the database query to the depiction GUI called <tt>dview</tt>.
−
−	~~[[File:FhvFirstAverage.png\|thumb\|center\|500px\|First average, no azimuthal rotation was tested. Shown in <tt> dview</tt> program]]~~
−
−	Apparently, the project finds the membranes and centers the particles correctly. If you show the volume in Chimera (by clicking in the ''Export>Send to Chimera'' option in the <tt>dview</tt> window ).
−
−	~~[[File:FhvFirstChimera.png\|thumb\|center\|500px\|First average, no azimuthal rotation was tested ]]~~
−
−	~~We can check the individual particles:~~
−
−	~~<tt>ddbrowse -d first:data -t first:rt </tt>~~
−
−	~~[[File:FhvDdbrowseFirstZ.png\|thumb\|center\|700px\| ddbrowse explores the aligned particles, each one represented as a projection. ]].~~
−
−	~~== Aligning the axis of symmetry ==~~
−
−	We would like to bring the axis of symmetry to the ''z''direction. This will create an intuitive separation between the two first euler angles that represent "change of orientation of the main axis", and the third one that represents "azimuthal rotation about the new axis". We want that azimuthal rotations occur about an axis of possible symmetry, which by convention is located along z. There are several ways to achieve this. A simple one is to create a model of the membrane/crown geometry, align our average to it (with <tt>dalign</tt>), and use the alignment parameters to create a new table. With this new table and average we will be able study possible symmetries with a new project.
−
−
−	~~=== Creating a template aligned with z ===~~
−
−	Geometrical shapes of many types can be created with <tt>dmask,dsphere,dcylinder,dshell</tt> and <tt>dtube</tt>, and combined with <tt>dynamo_mask_combine</tt>. However, there are also some available utilities to create templates representing frequently occurring membraneous geometries:
−
−	~~<nowiki>mr = dpktomo.examples.motiveTypes.MembraneWithRod();~~
−	~~mr.sidelength = 128;~~
−	~~mr.rodRadius = 20; mr.rodShift = [0,0,10]; mr.rodHeight= 20;~~
−	~~mr.getMask();~~
−	~~template = mr.mask;</nowiki>~~
−
−	~~Note that you can check the list of properties inside the object <tt>mr</tt> just by typing it without the semicolon and pressing [Return]~~
−
−	~~We can check the result with:~~
−
−	~~<tt>mr.viewMask();</tt>~~
−
−	~~[[File:FhvTemplateZ.png\|thumb\|center\|700px\| A simple template with rotational symmetry axis aligned along z]]~~
−
−	~~=== Aligning the previous average along z ===~~
−	The mask is white on black by definition. The tomogram data in this case represents bright protein on dark background, so that in this case we want to align the previously computed average to the mask of the <tt>mr</tt> object.
−
−	The alignment does not need to scan azimuthal rotations, so that our 'inplane_range' will be 0. The 'cone_range' will be the full sphere, and we take and spacing of 30 degrees. As we are looking for coarse orientations, we can bin the two volumes twice (yielding volumes with a sidelength of 32 pixels). We will also "nail" the new center to the physical center of the box (parameter <tt>limm</tt> set to 1 below).
−
−	~~Now we read the average computed by the project <tt>first</tt> into a workspace variable which we arbitrarily call <tt>previousAverage</tt>~~
−
−	~~<tt>ddb first:a -r previousAverage;</tt>~~
−
−	~~Here, the volume <tt>previousAverage</tt> will be treated as a "particle" to be aligned onto a ''template'', which in this case is the volume contained in <tt>mr.mask</tt>.~~
−
−	~~<tt>sal = dalign(previousAverage,mr.mask,'cr',360,'cs',30,'ir',0,'dim',32,'limm',1,'lim',[10,10,10]); </tt>~~
−
−	~~The flags are the same parameters that would be used in an alignment project. You can check the shorthands with <tt>dvhelp</tt>.~~
−
−	The output result <tt>sal</tt> contains many fields. We can, for instance directly visualize how the previous average looks like when transformed through the results of this <tt>dalign</tt> operation. For this, use the field <tt>aligned_particle</tt> in the output <tt>sal</tt>:
−
−	~~<tt>dview(sal.aligned_particle);</tt>~~
−
−
−
−	~~[[File:ZAlignedAverage.png\|thumb\|center\|700px\| A simple template with rotational symmetry axis aligned along z]]~~
−
−	~~=== Applying the z-alignment to the table ===~~
−
−	We don't want just to get a z-oriented average: we also want to convert the table yield by the <tt>first</tt> project into a table that, when applied onto the original data, produces a correct, z-aligned average. For this, we use the alignment parameters coded inside the output <tt>sal</tt> (as field <tt>.Tp</tt>) and apply them onto the table.
−
−	~~<tt>ddb first:rt -r tf</tt>~~
−
−	~~The variable <tt>tf </tt> contains now the original table, which is now rotated and shifted according with the rigid body transform <tt>Tp</tt> inside <tt>sal</tt>~~
−	~~<tt>tr = dynamo_table_rigid(tf,sal.Tp); </tt>~~
−
−	now the ''rectified ''table <tt>tr</tt> should contain the metadata that makes the particles "point upward". We use now the typical sanity check to assess that a table behave as expected: we apply it to the particles in its paired [[data folder]] and check the average:
−
−	~~<tt>oz = daverage('first:data','t',tr,'fc',1); </tt>~~
−
−	~~whose results can be checked by:~~
−
−	~~<tt>dview(oz); </tt>~~
−
−	~~[[File:FhvSanityCheckZ.png\|thumb\|center\|700px\| Particles aligned with rectified table <tt>tr</tt>]]~~
−
−
−	~~We can visualize this operation by plotting a sketch with the orientation of the particles. We first show the results for the table computed in the project <tt>first</tt>~~
−
−	~~<nowiki>figure;~~
−	~~dtplot('first:rt','m','sketch','sketch_length',100,'sm',30);~~
−	~~view(-230,30);axis equal;</nowiki>~~
−
−	~~[[File:FhvSketchFirst.png\|thumb\|center\|400px\| sketch of orientations after computation of the the project <tt>first</tt>]]~~
−
−	Here, the 'z' orientation computed for each particle is represented by the longest semiaxis. Note that with the original definition of the template, the 'z' points -approximately- along the direction of the 'z' in the tomogram. If we depict the same plot with the table we just computed:
−
−	~~<nowiki>figure;~~
−	~~dtplot(tr,'m','sketch','sketch_length',100,'sm',30);~~
−	~~view(-230,30);axis equal;</nowiki>~~
−
−	~~[[File:FhvSketchZ.png\|thumb\|center\|400px\| sketch of orientations in the rectified table]]~~
−
−	we see that now the 'z' axis in each particle is pointing orthogonally(-ish) to the membrane. A different quick check would be to show the projections of the particles along ''y''. Instead of using the GUI <tt>ddbrowse</tt>, we can create this depiction with a single command:
−
−	~~<tt>figure;dslices('particlesData','t',tr,'projy','*','align',1,'labels','on'); </tt>~~
−
−	~~[[File:fhvOrientedSlicesX.png\|thumb\|center\|400px\| full ''y''projection of the particles according to the orientations in the rectified table ]]~~
−
−	Lets look at the ''z''-projection. In this case we project only the central slices around the crown and the aperture in the viral vesicle, chosing thus slices 60 to 70. if project the full particle, we won't distinguish any features.
−
−	~~<tt>figure;dslices('particlesData','t',tr,'projz',60:80,'align',1,'labels','on');</tt>~~
−
−	~~[[File:fhvOrientedSlicesZ.png\|thumb\|center\|400px\| ''z''projection of the central slices in the rectified table ]]~~
−
−	The depiction clearly shows the missing wedge of the particles. Incidentally, it also shows that averaging all the particles should produce an average that covers all the orientaions in the Fourier space.

	== Using the membrane to impart an orientation ==		== Using the membrane to impart an orientation ==

@@ Line 9: / Line 9: @@
   [[File:FhvDefinitionCrown.png|thumb|center|500px| Average for several hundreds of particles. The "crown" area is shown under a red shadow.]]
-== Downloading ==
+== Accessing the data==
+In the linux terminal, type
-During the Dynamo workshop you will find the needed tilt series in your data folder. Outside the workshop, you can download all the files related to this example with the command:
+  <tt>>cd Prac-4 </tt >
+and check the contents with
-  <tt>dpkhelp.wiki.downloadExample('fhv');</tt>
+  <tt> >ls  </tt>
+Now, open Dynamo by typing:
-If it fails under Matlab or the ''Dynamo'' command line, you can try to directly use the linux order
+  <tt> dynamo </tt>
+The Dynamo command line will open.
 ===Size check of a file===