Algorithm 4: Image Cleaning

1. Input: $I_c$/* cluster cryo-EM image */

2. Return: $I_{cc}$/* cleaned cluster image */

3. ${\mathrm{I}}_{\mathrm{c}1}\leftarrow \mathrm{imopen}\left({\mathrm{I}}_{\mathrm{c}}\right)$/* apply image opening on the cluster image to enlarge small blobs */

4. $\mathrm{L}\leftarrow \mathrm{bwlabel}\left({\mathrm{I}}_{\mathrm{c}1}\right)$/* label each object in the cluster image which returns a label matrix $\mathrm{L}$ that contains 8-connected object in the cluster */

5. for $i$=1 to $L$ do/* for each object in the clustered image */

6.  ${\mathrm{I}}_{\mathrm{object}}\leftarrow \mathrm{state}\left(\mathrm{L}\left(\mathrm{k}\right)\right)$/* get each particle where $\mathrm{k}$ is the total number of objects in the cluster cryo-EM */

7.  ${\mathrm{I}}_{\mathrm{object}}\leftarrow \mathrm{remove}\left(\mathrm{state}\left(\mathrm{L}\left(\mathrm{k}\right)\right)\right)$/* remove all the connected components that are smaller than the p pixels */

8.  $\mathrm{centroid}=\mathrm{stats}\left(\mathrm{k}\right).\mathrm{Centroid}$/* mark and get the actual index numbers and the centroid of each object */

9. end for

10. ${\mathrm{obj}}_{\mathrm{number}}\leftarrow \mathrm{is}\mathrm{member}\left({\mathrm{I}}_{\mathrm{object}}\right)$/* extract the number of object (particles) */

11. $\mathrm{L}\leftarrow \mathrm{bwlabel}$/* label each object (particle) */

12. for $i$= 1 to $L$ do/* for each object (particles) */

13.  Do size filtering and roundness filtering

14.  $\mathrm{p}\leftarrow \left[\mathrm{props}.\mathrm{label}\right]$/* extract the 8-connectedlabeld object */

15.         $\mathrm{keeperObjects}\leftarrow \mathrm{props}.\mathrm{label}>\mathrm{p}/$* remove each object that less than 8-connected component in the binary image */

16.  ${\mathrm{I}}_{\mathrm{c}2}\leftarrow \mathrm{keeperObjects}$/* get actual index numbers instead of a logical vector */

17.  ${\mathrm{I}}_{\mathrm{cc}}\leftarrow \mathrm{bwareaopen}\left({\mathrm{I}}_{\mathrm{c}2}\right)$/* produce new binary image with only the small, or non-connected object */

18. end for

19. Generate and getting a new binary image