TITLE: Processing of LRRK2-RCKW:GZD-824:E11 
AUTHORS: Amalia Villagran Suarez

[DESCRIPTION]
Protocol for processing of LRRK2RCKW bound to GZD-824 and DARPin-E11. This protocol covers everything from preprocessing to refinement.

[STEPS]
SECTION: Preprocessing data
1. Use your preferred software. The original publication used MotionCor 2 and CTFFIND4.
As a note, all of the data processed was collected on a UltrAuFoil Holey Gold 2/2 200 mesh grids and we used CryoSparc v.4.3.1 to process the data.
SECTION: Particle picking
2. Blob picker
We used cryoSparc-live blob picker option with a minimum particle diameter of 90Å to 250Å, lowpass filter to 20Å. 

 



SECTION: Particle picking
3. 2D Classification and Topaz picking
Using the extracted particles, we run 2D classification jobs in cryoSparc, using 40 iterations to clean our particle dataset. Selected particles then were use to train a Topaz model, and model was used to pick particles, followed by rounds of 2D classification. Selected particles can then be re-extracted to original pixel size (in this case 0.935Å, box size of 320pixel for monomers and 400pixel for dimers)
 

SECTION: Refinement for monomer
4. Ab-initio reconstruction and Heterogeneous Refinement
Select monomer particles then are subject to an Ab-initio reconstruction in cryoSparc asking 3 classes. Followed by a Heteregeneous Refinement. 
 

SECTION: Refinement for monomer
5. Nu-Refinement and Local refinement
Selected refinement from step 4, is subject to a Nu-refinement at C1 symmetry. Followed by a local refinement with a continuous mask surrounding the Kinase, WD40 and DARPin-E11. 


SECTION: Refinement for monomer
6. 3D Variability
To account for the hetereogeneity of the ROC-COR domains, create a mask using the Nu-refinement of step 5, and using the same amount of particles of the same job, run a 3DVA. 
 

SECTION: Refinement for monomer
6.1. 3D Variability Display
Use this jobs with the output from Step 6, to visualize the movement of the ROC-COR domain. Input the following: for output-mode "intermidiates", "11: frames, and filter resolution of "7Å".
 To visualize the movement of each component download file to chimera and use volume morph. Select component with the most movement in the ROC-COR domain.
Clone 3D Variability Display job and now select: skip reconstruction, only used this component (write which component you picked), and select Intermediates: output particle subsets. The output of this job should be the 11 particles stacks. 



SECTION: Refinement for monomer
7. Local Refinement of the ROC-COR domain
Particles of the component obtained from step 6.1, are split in half, selecting the particles that account for the movement of the ROC-COR closest to the Kinase domain.
 This are subject to a local refinement. The Nu-refinement volume from step 5 is used as the input for the local refinement, a continuous mask surrounding the ROC-COR domains is created and the selected particles of the component. 


SECTION: Refinement for trimer
8. Ab-initio and Nu-Refinement
Select trimer particles were use to run an Ab-initio reconstruction in cryoSparc. Followed by a Nu-Refinement C1 and at C3 symmetry. 
We compare both the C1 and C3 maps to check for density for the inhibitors in the protomers and overall resolution.
 

SECTION: Refinement for trimer
9. Particle expansion 
Particles from Nu-Refinement in step 8 were used for a Symmetry Expansion job in cryoSparc, based on the volume symmetry. 

SECTION: Refinement for trimer
10. Local Refinement
A local refinement was carried using the following inputs:  mask of one protomer of the trimer, expanded particles from step 9, Nu-refinement from step 8 and C1 symmetry.