Reconstructing with full particles following local refinement


I am trying to generate a reconstruction of my protein using the alignments from a local refinement job and the original particles. However, when I do this I still get the masked volume as an output.

I have used the Homogeneous Reconstruction Only job type. The 3D particle alignments come from a local refinement job, and the particles are those used as the input for the particle refinement job (particle subset job). Below are the input parameters.

I have tried using the particle stack from the local refinement job and replacing the blob file with that of the original particles and got identical results.

Am I missing some other steps?

Best, Sarah

Welcome to the forum @SVB.
Please can you illustrate your question with

  • the job type

  • a screenshot of the expanded Inputs section of the job’s Inputs and Parameters tab

  • the job types that created the inputs

Here are the parameters

Did the workflow involve ant particle subtraction? If so, at what point with respect to J331 and J352?

Can you illustrate the problem by a 3-way comparison of map slices from

  1. consensus refinement
  2. local refinement
  3. reconstruction with locally refined alignments3D

(each accompanied by the corresponding expanded Inputs).

Hi Sarah - when you say you want to use the original particles, as opposed to the ones used in local refinement, what is the difference between the two sets? Were the particle images modified in some way, e.g by subtraction? Because otherwise I would expect the reconstruction to look the same as the local (as you are using the same alignment parameters).

Also, the output of local refinement should not be masked I think - a mask is used in refinement, but not applied to the output volume. It may look masked though, as the alignments will be a lot more accurate inside the mask, blurring out the density outside of it.


1 Like