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.