De novo 3D model generation

relion-4.0 uses a gradient-driven algorithm to generate a de novo 3D initial reference from the 2D particles. As of release 4.0, this algorithm is different from the SGD algorithm in the CryoSPARC program [PRFB17]. Provided you have a reasonable distribution of viewing directions, and your data were good enough to yield detailed class averages in 2D classification, this algorithm is likely to yield a suitable, low-resolution model that can subsequently be used for 3D classification or 3D auto-refine.

Running the job

Select the Select/job014/ file on the I/O tab of the 3D initial reference jobtype. Everything is aready in order on the CTF. Fill in the Optimisation tab as follows (leave the defaults for the angular and offset sampling):

Number of VDAM mini-batches:


(The VDAM algorithm will loop over mini-batches, which contain only hundreds to thousands of particles each.)

Regularisation parameter T:


(The default is 4 for 3D runs.)

Number of classes:


(Sometimes, using more than one class may help in providing a ‘sink’ for sub-optimal particles that may still exist in the data set. In this case, which is quite homogeneous, a single class should work just fine.)

Mask diameter (A):


(The same as before).

Flatten and enforce non-negative solvent:




Run in C1 and apply symmetry later?:


(The actual refinement will be run in C1, which has been observed to converge better than running VDAM in higher symmetry groups. After the refinement, the relion_align_symmetry program is run automatically to detect the symmetry axes and the symmetry will be applied.)

On the Compute tab, set:

Use parallel disc I/O?:


Number of pooled particles:


Pre-read all particles into RAM?:


(Again, this is only possible here because the data set is small. For your own data, you would like write the particles to a scratch disk instead, see below.)

Copy particles to scratch directory:

Combine iterations through disc?:


Use GPU acceleration?:


Which GPUs to use:


On the Running tab, set:

Number of MPI procs:


(Remember that the gradient-driven algorithm cannot be run with multiple MPI processes.)

Number of threads:


Using the settings above, this job took 2 minutes on our system.

Analysing the results

You can look at the output map in 2D slices through the 3D map by selecting InitialModel/job015/initial_model.mrc from the Display: button. We like looking at 3D maps in 2D slices, as it is a good way to assess artifacts, for example streaks in the solvent region. You may also want to look at your map in 3D, with a 3D viewer like UCSF chimera.