Tutorial: Solving a structure with cryo-EM data using model-building

This example is density modification of a cryo-EM map, followed by structure determination by automatic model-building, fixing a mis-traced segment, sequence assignment and refinement

The data are half-maps for apoferritin (EMD entry 20026, 1.8 A)

The files are:

• emd_20026_half_map_1_box.ccp4 emd_20026_half_map_2_box.ccp4 -- half maps
• seq.dat -- sequence file
• run_denmod_model_building.csh -- script to run all these commands

The maps are all cut out (boxed) from the deposited maps (just to make the files smaller for this demo).

1. DENSITY MODIFICATION OF HALF-MAPS

To run one cycle of density modification from the command line you can paste this in to a terminal window. From the GUI just set all the same parameters and no need to set the output_directory (it will go in resolve_cryo_em_0 or a similar directory name):

phenix.resolve_cryo_em emd_20026_half_map_1_box.ccp4

emd_20026_half_map_2_box.ccp4 resolution=2.2 dm_resolution=1.8 seq_file=seq.dat cycles=1 nproc=4 box_before_analysis=False density_modify_unsharpened_maps=Trueoutput_directory=denmod output_files.denmod_map_file_name=denmod_map.ccp4 initial_map_file_name=initial_map.ccp4

In about 15 minutes this will produce (in a denmod/ or

resolve_cryo_em_xx directory) the file: denmod_map.ccp4

2. STRUCTURE SOLUTION BY AUTOMATED MODEL-BUILDING

We are going to build a model into our density-modified map,

refine it, fix the sequence and refine again.

1. Getting map symmetry We will want a symmetry file describing the symmetry operators that have been used in our map. We can get them automatically in about 30 seconds with:

phenix.map_symmetry denmod/denmod_map.ccp4 resolution=2

symmetry_out=denmod_map.ncs_spec

Now denmod_map.ncs_spec has the 24 symmetry operators for octahedral symmetry in the appropriate location of this map.

2. Automated model-building using a density-modified map

We can build a model into our map with map_to_model:

phenix.map_to_model denmod/denmod_map.ccp4 resolution=2 nproc=4

ncs_file=denmod_map.ncs_spec pdb_out=map_to_model.pdb seq_file=seq.dat

In a half hour or so you should get the model 1aew_A_built_in_denmod_map.pdb.

3. Cut out a piece of density-modified map and compare to build_in model You can cut out a piece of your density modified map and look at it along with your model this way:

phenix.map_box denmod/denmod_map.ccp4 map_to_model.pdb

ignore_symmetry_conflicts=true prefix=denmod_map_A

This will produce denmod_map_A.ccp4 and denmod_map_A.pdb which you can look at in Coot or Chimera. The model is mostly pretty good but may need some fixing, depending on your version of Phenix

4. Refine the model against the density-modified map

We can refine the build_in model either against the original or cut-out map.

Let's use the cut-out map. We can also refine just chain A or the whole 24-mer. The difference is if you use the whole 24-mer you are

going to include inter-chain contacts and if you use a single chain you

will ignore them. Let's use just one chain for now:

phenix.real_space_refine map_to_model.pdb denmod_map_A.ccp4 resolution=2

ignore_symmetry_conflicts=True

After a couple minutes we have the output model: map_to_model_real_space_refined.pdb. If you look at this model in Coot or Chimera you'll see it has moved some side chains into density, but that the model still doesn't match the density for residues 154-162.

5. If your model has parts that do not fit, try something like the following. First remove the bad part (here the bad part is residues 154 through 162):

phenix.pdbtools map_to_model_real_space_refined.pdb

remove="resseq 154:162" output.file_name=map_to_model_real_space_refined_del.pdb

6. Redo the sequence assignment and see if we can fill in the gap. Here the keyword run_assign_sequence=True is used to reassign the sequence and then fill in the gap:

phenix.sequence_from_map run_assign_sequence=True

model_file=map_to_model_real_space_refined_del.pdb seq_file=seq_unique.dat map_file=denmod_map_A.ccp4 pdb_out=sequence_from_map.pdbresolution=2

This should produce the file sequence_from_map.pdb in a couple minutes.

7. We can fix one other part of the model that does not fit well (residues

6-9):

phenix.pdbtools sequence_from_map.pdb

remove="resseq 6:9" output.file_name=sequence_from_map_del_6-9.pdb

8. Redo the sequence assignment and see if we can fill in the gap as in 6 above:

phenix.sequence_from_map run_assign_sequence=True

model_file=sequence_from_map_del_6-9.pdb seq_file=seq_unique.dat map_file=denmod_map_A.ccp4 pdb_out=sequence_from_map_2.pdb resolution=2

This should produce the file sequence_from_map_2.pdb in a couple minutes.

9. Let's refine one more time:

phenix.real_space_refine sequence_from_map_2.pdb denmod_map_A.ccp4

resolution=2 ignore_symmetry_conflicts=True

Now have a look at sequence_from_map_2_real_space_refined.pdb and denmod_model_A.ccp4 in Coot or Chimera. The model should fit the map quite well.