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Particle picking andScreening
(Practical work)
Vahid Abrishami
Introduction
• Particle picking is an important step to select your “particles” from a set of micrographs.
• Manual picking can be very tedious and each tool is more or less convenient depending on the sample and the personal preferences.
• In Scipion, we have integrated different picking tools, so that the user can select which better fits to its needs, or combine some of them.
EMAN Particle Picking
• Create a subset of micrographs by sorting micrographs according to their defocus values.
• Select the first, middle, and the last micrographs.
• Select a proper name for this sub-set.
EMAN Particle Picking (Cont.)
• Double click on eman2-boxer and set the input micrographs.
• Set the particle size and particle diameter to 64.
• Select the Swarm tool.
EMAN Particle Picking (Cont.)
• Select some particles and the algorithm suggests you new particles.
• You can remove particles using shift + left button mouse.
• Go to the next micrograph to pick automatically.
EMAN Particle Picking (Cont.)
• You can select the “gauss” tool.
• Set the Gaussian kernel Width.
• Play with “Threshold Low” and “Threshold High” for CCF (0.1 and 5.0, respectively).
• Click “Run”.
EMAN Particle Picking (Cont.)
• If you are not happy with the parameters, press “Clear Boxes” and set the parameters again.
• When you are happy with the results press “Done”.
XMIPP Particle Picking
• Xmipp particle picking is divided into two steps:
(1) manual/supervised picking and (2) completed automatic picking
XMIPP Particle Picking (Cont.)
• Double click on manual picking and execute the protocol.
• Set the particle size to 64.• Set the Explore (%) to 30.• You can use
Brightness/Contrast option from Filters to improve the visualization of particles.
XMIPP Particle Picking (Cont.)
• Pick at least 15 particles from the first micrograph.
• Click Autopick and make sure that you have picked all the particles inside the yellow box (if not, click No) .
• The algorithm will train a classifier and will propose some coordinates automatically.
XMIPP Particle Picking (Cont.)
• Use shift + mouse wheel to zoom in and out.
• Use mouse left button to add new particles.
• Use mouse left button + shift to remove a particle.
• To deal with larger areas of contamination use the eraser.
XMIPP Particle Picking (Cont.)
• Continue with more micrographs till you are satisfied with the performance of the classifier.
• we can register the output coordinates by clicking on the “Coordinates” red button.
• Then we can close the GUI and open the “xmipp3 – automatic” box and select the previous execution of manual/supervised as input.
RELION Particle Picking
• The protocols for the Relion auto-picking in the Scipion have been divided in two steps: (1) computing the figure-of-merit (FOM) maps.(2) Adjusting the parameters and picking the rest of the micrographs.
• You will need to go a bit ahead to the 2D section and produce some classes and go back to the Relion and use them as references.
RELION Particle Picking (Cont.)
• Open the “relion- auto-picking (1)”.
• Select the 3mics as the input for this protocol.
• Select the CTF from the previous step (XMIPP or CTFFIND).
RELION Particle Picking (Cont.)
• In references, select the averages that you imported to the project.
• “Angular sampling” is used for performing cross-correlation.
• Set inverted contrast and CTF corrected to Yes.
• Click “Execute”.
RELION Particle Picking (Cont.)
• Open “relion- auto-picking (2)”.
• Set the “relion- auto-picking (1)” as the FOM.
• Select the CTF from the previous step (XMIPP or CTFFIND).
RELION Particle Picking (Cont.)
• Play with “Picking threshold” and particle distance and use the wizard to check the results.
• Click “Execute” to automatically pick the particles from 15 micrographs.
XMIPP Consensus Particle Picking
• Select the picking results of two picking algorithms.
• Select the radius in which All coordinates presumed to correspond to the same particle.
• Click “Execute”.
Extracting Particles
• Once we have a set of coordinates, we can proceed to extract these particles with Xmipp.
• The extract protocol will allow us to extract, normalize and correct the CTF phase of your picked particles.
Extracting Particles (Cont.)
• Set the “Particle box size” to 64.
• Check the “Sort by statistics” option, to tell the protocol to sort the particles based on general statistics assigning to each particle a z-score value.
Extracting Particles (Cont.)
• The invert contrast flag: If activated, bright regions become dark regions and the other way around.
• The phase flipping flag: If activated, the protocol corrects the CTF phase of your particles.
Extracting Particles (Cont.)
• Click “Analyse Results”.• You can see the particles
with their related z-scores.
• Disable the particles with high z-scores (noises) and click “Particles”.
• You have a new set of particles.
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