Python-based
Hierarchical
ENvironment
for
Integrated
Xtallography
This example is density modification of a cryo-EM map, followed by optional structure determination by docking a homologous structure
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
- 20026_auto_sharpen_A.ccp4 -- deposited map, half-map sharpened
- apoferritin_chainA_rsr.pdb -- model refined against sharpened map
- 1aew_A.pdb -- chain A from X-ray structure of horse apoferritin (53% identical to human apoferritin)
- run_denmod_dock.csh -- command file that will run all these things for you if you want
The maps are all cut out (boxed) from the deposited maps (just to make the files smaller for this demo).
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):
In about 15 minutes this will produce (in a resolve_cryo_em_xx directory) the file:
denmod/denmod_map.ccp4 -- density-modified map
You may want to cut out the parts of your map that match apoferritin_chainA_rsr.pdb:
We are saying "ignore_symmetry_conflicts=True" because the map in denmod/denmod_map.ccp4 may have a different "unit cell" than apoferritin_chainA_rsr.pdb. Here the "unit cell" is just a description of the box of points that are in the map. It can sometimes refer to the size of the original map and sometimes to the size of the working part of the map and this command says to ignore these differences.
In Coot or Chimera you can load in the model (apoferritin_chainA_rsr.pdb) and compare the sharpened deposited map (20026_auto_sharpen_A.ccp4) with the density modified map (denmod_map_A.ccp4). Look at residues 13 and 81 and the main chain near residue 161 for examples of substantial improvement in the map.
The file 1aew_A.pdb contains the X-ray structure of 1aew, a homologous protein from horse (53% identical, rmsd 0.5 A). We are going to dock this structure into our density-modified map, refine it, fix the sequence, refine again, and density modify using the model to get a final map.
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:
Now denmod_map.ncs_spec has the 24 symmetry operators for octahedral symmetry in the appropriate location of this map.
We can find where 1aew_A.pdb fits in our map with dock_in_map:
In a minute or two you should get the model 1aew_A_docked_in_denmod_map.pdb.
You can cut out a piece of your density modified map and look at it along with this model this way:
phenix.map_box denmod/denmod_map.ccp4 1aew_A_docked_in_denmod_map.pdb ignore_symmetry_conflicts=true
This will produce 1aew_A_docked_in_denmod_map_box.ccp4 and 1aew_A_docked_in_denmod_map_box.pdb which you can look at in Coot or Chimera. Notice that a lot of the side chains do not match the density because the model is from a homologous structure.
We can refine the docked 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 1aew_A_docked_in_denmod_map.pdb 1aew_A_docked_in_denmod_map_box.ccp4 resolution=2 ignore_symmetry_conflicts=True
After a couple minutes we have the output model: 1aew_A_docked_in_denmod_map_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 they still don't match the density because the sequence of this docked model is not the sequence of human apoferritin (look at R29 for example which should be a tyrosine).
We can redo the sequence of our docked model with sequence_from_map:
In a couple minutes you should get the file: 1aew_A_docked_rsr_seq.pdb with the sequence of human apoferritin mapped onto the real-space-refined model. Have a look at this with the map now in Coot or Chimera. You can see that some of the side chains need refinement and that a loop by P157 needs to be moved. Let's first just refine our new model.
We can refine just the same as before but with our new sequence:
phenix.real_space_refine 1aew_A_docked_rsr_seq.pdb 1aew_A_docked_in_denmod_map_box.ccp4 resolution=2 ignore_symmetry_conflicts=True
In a couple minutes we have the refined model: 1aew_A_docked_rsr_seq_real_space_refined.pdb. This model fits the map pretty well, but the loop at P157 is still way off. We need to re-fit this loop.
We can fix this loop using the fit_loops tool. We can look at the model and map and notice that the part of the model that doesn't fit is residues 155-159. Let's fix these with:
In a minute this should fit this part of the chain into the density.
We can refine one more time after fitting our loop at P157:
phenix.real_space_refine 1aew_A_docked_rsr_seq_rsr_loop157.pdb 1aew_A_docked_in_denmod_map_box.ccp4 resolution=2 ignore_symmetry_conflicts=True
Now we have 1aew_A_docked_rsr_seq_rsr_loop157_real_space_refined.pdb. Have a look at it in Coot or Chimera.