In this
session, we will work through one electron micrograph (cryo-EM),
selecting particles and applying alignment, from which we can build an
initial low-resolution density model. Before we start, create a working
directory and move into it. This place will be the playground of this
practice session. Download the dataset
"/gcdata1/jzchen/temp/demo/demo.tar" to your working directory. Then,
at
the command-line, type
tar
xfv demo.tar
Launch the program by the command
signature
&
Particle
Selection
- Define a new project space (Menu : Project : New)
in the working directory and name it "TEST". A directory "TEST.ems" will be created,
together with a set of sub-directories. The "Project" label at the upper right
corner of the GUI window becomes "TEST".
- Load the micrograph "6605.mrc" (in "data/") from Menu : Data :
Micrograph : Load MRC. The "Film" label at the upper right
corner of the GUI window becomes "6605".
- The intensity histogram
of the micrograph will be displayed in Image
Quality Monitor. To observe the spectrum, activate the SPEC
button and use the mouse cursor to select a region (left-click) for
display. The size of the region can be customized at Box
Size. Adjust the intensity and contrast dials to achieve the
optimal visualization.
- In Micrograph
Masking box, press Apply
for automatic masking. When that is done, use the drawing tools to refine the mask
manually.
- Take some notes at Keynote
if you like. It will be saved into the project space automatically.
- Load the template images "templates.mrc" (in "data/") from Menu : Data : Template
: Load MRC. In Circular
Masking box, set LCF
profile radius to 0.7, and SCF
profile radius to 0.6, then click Apply.
- In Rotational Search
box, set Interval
to 5 degrees.
- Save this particle screening project from Menu : Project : Save :
Screening PRJ. It will be internally named and managed by the
system.
- Click the Particle
button at the bottom of the GUI window, Particle
OpPanel appears.
- In Particle
Parameters box, set Resize
to 4. In order to save time, we will skip the full-range screening
(please see the user manual for the operational detail) and apply only
the test-run. Click the button
, the system becomes busy, as
indicated by the red light at the bottom right corner of the GUI
window.
Once the task is completed, the indicator becomes green. This should
take less than one minute.
- The number of identified particles is displayed at Particle
Count under the monitor. Change the LCF
and SCF
threshold slidebars at will and observe the effect. After each
adjustment, press
again to update.
- Manual selection: CTRL+Left-click to select,
SHIFT+Left-click to delete. More detail can be found here.
- This step is
optional: to inspect the particle selection through the
monitors,
click the Accept
(CTRL+a) and/or Reject
(CTRL+r) buttons. Undo
(CTRL+u) will take one step back. The Score
bar displays the best matching score of the current selection with the
template images. The automatically selected particles are marked by red
crosses, and the accepted selections are marked by green crosses. Set
the display scale and blurring to various levels to achieve the best
visual perception. Remember to press ENTER to trigger the update.
Toggle
the cross-hair display in the monitors by the +
button under the monitor. The distance between the two pairs of
short-bars indicates the particle's diameter as specified at Size
in Particle
Parameters box. The distance display is also affected by the
pixel size of the image, which is defined in Micrograph
OpPanel.
- Save the particle selection into an MRC stack (Menu
: Particle : Save Image Frames). The file will be named
"TEST.ems/particles/6605.mrc". Then load the particle set into Image
Stack Editor. (Alternatively, press the button
, the
particle set will be automatically saved and loaded into Image
Stack Editor.)
- Apply Normalization
function, set the proper display Scale,
and browse the particle image set in the editor. Select unwanted
particles (CTRL+left-click) and, at the end, press the Delete
button. When prompted to synchronize with the micrograph, make sure to
answer "Yes".
Particle
Alignment
Assuming that you have collected enough particles. The next step is to
alignment them. In order to achieve precise particle alignment, the
projections from a low-resolution model will be used as the template
images. With the micrograph "6605.mrc" and the particle stack
"particles/6605.mrc" are in place (continuing from the previous
section),
- Load the density "model.mrc" (in "data/") from Menu
: Data : Density Map. The system will be automatically switched
to the 3D
Model OpPanel, and the density model will be displayed in the
main display window.
- In Model
Projection box, take all default values and press Apply.
A set of 2D projections will be generated and saved to
"modeling/projections.mrc" in the project space.
- Inspect these 2D projections in Image
Stack Editor. There should be 25 frames. We notice that they
are white particles, while those in the original micrograph are black.
Apply the Intensity
Scaling function with the Multiplier
parameter set to "-1". Then save the new image stack to
"TEST.ems/template/templates25.mrc" and load it as the template from Menu
: Data : Template : LoadMRC.
- Activate Menu
: Function : Particle Alignment. Then save this alignment
project from Menu
: Project : Save : Alignment PRJ. Now, if you press the Apply
button, particle alignment will commence and might take quite a while
(please see the user manual for the operational detail). We will have
to
skip this part and use a pre-processed dataset instead.
- Copy "data/6605.aln", "data/6605_stack.ptk" and
"data/6605_particle.mrc" to "TEST.ems/particles". Rename
"6605_particle.mrc" to "6605.mrc". And, copy "data/parameter.ctf" to
"TEST.ems/micrograph".
- Open "TEST.ems/particles/6605.mrc" in Image
Stack Editor. Make sure that the Precision
value is set to "5" degrees (located in Menu
: Function : Particle Alignment). Then save this alignment
project from Menu
: Project : Save : Alignment PRJ.
- Now go to Menu
: Function : Particle Alignment and press the Apply
button to start particle alignment. The pre-processed alignment
parameters will be loaded and displayed in two charts: "Correlation
Coefficient Distribution" and "Shift Distribution". Adjust the
corresponding slidebars in the OpPanel
to set the proper thresholds. Press Apply
once again, those particles with higher C.C. and lower Shift values
will
be selected. The result of thresholding can be observed in Image
Stack Editor.
- A Frealign-style PAR file can be exported for the selected
particles: Menu
: Particle : Export Frealign PAR. This parameter file can be
read directly by the Frealign program for model reconstruction and
model
refinement.
- Clear the current particle selection from Menu
: Particle : Clear Selection. Then move back to Particle Alignment
page, press the Sync
button, the alignment shift of each particle will be compensated in its
coordinates and the updated positions will be displayed in Particle
OpPanel.
- Press the
button, a better centered particle
stack will be appear in Image
Stack Editor. This is the final particle stack from this
micrograph.
Model
Reconstruction
Now that the particle dataset has been aligned, we can directly build a
new model using the Frealign program. Go to the directory
"TEST.ems/frealign" and make copy the file "data/mparameters" and
"data/mreconstruct.com". You will need to modify the file "mparameters"
to define the working directory properly. Then type at the command-line
ln
-s ../particles/6605.mrc .
mv
6605.par toyModel_1.par
mreconstruct.com
1 500 1
When the Frealign program terminates, a new model "toyModel_1.mrc" will
be generated. Load this density map into Signature from Menu
: Data : Density Map. Because of the limited number of
particles used in this reconstruction, the model is naturally quite
noisy. We can improve the visualization by applying a low-pass
filtering:
- In Image
Processing toolbox, choose Band-pass
Filter function.
- Set Low-pass
to 20.0 Å and High-pass
to 100.0 Å, press Apply.
A filtered density model shows up.
By the time tens of thousands of particles have been collected
following the same procedure, the Frealign refinement can start
directly
from here.
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