Particle picking and Screening (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.