After cryo_fit2, secondary structures of my input molecule tend to be broken.

• Doo Nam observed that when he provided a little less ideal helix (i.g. one H-bond length within helix is rather long), then cryo_fit2 tends to "loosen/unwind" that "kink" area.
• When he provided a longer distance_cut_n_o (i.g. 4~4.5), the geometry of helix was kept well (it maintained the helical kink spot).

• For example, phenix.cryo_fit2 <user.pdb> <user.map> resolution=<reso> secondary_structure.protein.distance_cut_n_o=4.5

• Alternatively, a user can specify a lower map_weight_multiply.

• For example, phenix.cryo_fit2 <user.pdb> <user.map> resolution=<x> map_weight_multiply=0.2.

• Alternatively, a user can use phenix.model_idealization after cryo_fit2 to restore broken secondary structures (if any).

How can I provide gaussian filtered maps?

• Many people in flexible fitting field know that often providing gaussian filtered maps results in better fitting.

• For example, flexibly fit with a 4 angstrom filtered map.
• Then with the resultant atomic model and a 2 angstrom filtered map, do flexible fitting.
• Then with the resultant atomic model and a non-filtered map, do final flexible fitting.

• UCSF Chimera can generate these filtered maps.

• Tools -> Volume Data -> Volume Viewer -> Tools -> Volume Filter -> (Set Width, e.g. 4,2) -> Filter
• File -> Save map as -> (name a filtered map) -> Save

How to extract a relevant map region?

• Question: I only want to fit the monomer because only one monomer of the trimer map is the best. Do you have any idea of how to generate the monomer map that cryo_fit can use?
• Answer: Please use phenix.map_box to extract a local map near entered atomistic model.

• Alternatively, a user can try phenix.map_to_model instead.

How to improve initial cc?

• phenix.dock_in_map, UCSF Chimera's 'Fit in Map (Tools -> Volume Data -> Fit in Map)', UCSF Chimera's manual fitting (using a mouse) often improve initial cross-correlation (cc) between map and model.

• Note that these options do rigid-body fitting only. Therefore, it is useful for global fitting before cryo_fit2. When overall orientation is already well fitted, these often are not that useful for cryo_fit2.

• UCSF ChimeraX's isolde may improve initial cc as well.

How to maximize cc?

• Generally, cryo_fit2 which is followed by phenix.real_space_refine tends to maximize cc.
• However, if there is a room/chance to idealize secondary structure further even after cryo_fit2, then cryo_fit2 + phenix.model_idealization + phenix.real_space_refine combination resulted in the maximum cc.
• When the fitting needs to pass through a local minimum energetically for a perferct fit (half of the molecule is already fitted, the other half is not fitted), running phenix.dock_in_map or UCSF Chimera's 'fit in map' before cryo_fit2 is recommended.

• Note that these options do rigid-body fitting only. Therefore, it is useful for global fitting before cryo_fit2. When overall orientation is already well fitted, these often are not that useful for cryo_fit2.

• When fitting protein molecule to a map requires a significant conformational change,

  • enforcing stronger map_weight (higher map_weight_multiply) tends to fit better (obviously).

  • For example, phenix.cryo_fit2 <user.pdb> <user.map> resolution=<x> map_weight_multiply=15
  • At this high map_weight_multiply, the secondary structures may be broken (slightly or seriously) right after cryo_fit2 running.
  • However, phenix.model_idealization which is followed by phenix.real_space_refine perfectly restored ideal secondary structures and fitted very well.

  • Alternatively, HE_top_out=True sometimes (not always) boosts cc well even with moderately high map weight (confirmed with 6 benchmark cases).

  • For example, phenix.cryo_fit2 <user.pdb> <user.map> resolution=<x> map_weight_multiply=10 HE_top_out=True
  • HE_top_out=True uses top_out potential (rather than harmonic potential) to helix (H) and sheet (E) for more flexible fitting.

I have a SAXS based cryo-EM map.

• Please use map_weight < 0.2 for SAXS (small angle x-ray scattering) data based cryo-EM map
  • A user can transform saxs data into cryo-EM map (mrc/sit) by Situs SAXS
  • However, this is nothing but central points by SAXS, although UCSF Chimera will visualize this "cryo-EM" map (mrc/sit) as regular cryo-EM map

I have a very small molecule to fit.

• When Doo Nam tried to fit a very small molecule (i.e. 11 residues long), both real_space_refine and cryo_fit2 couldn't fit to cryo-EM map.
• The reason could be that effect of sidechains is too large (in this kind of small molecule), so that global fitting is hindered.
• Doo Nam confirmed that phenix.dock_in_map fits globally much better for this case.

Is there a command line option for cryo_fit2 processing on multiple cores?

• (Please be advised that using multiple cores for cryo_fit2 is only useful when explore=True)
• Like other PHENIX applications, specify nproc.

• For example, phenix.cryo_fit2 nproc=40 model.pdb model.map resolution=3

• If nproc is not specified, cryo_fit2 will use 'available number of cores'/3

My input (before cryo_fit2) and output (after cryo_fit2) pdb files have different molecule sizes

Although pdb text files show similar SCALES, input (before cryo_fit2) and output (after cryo_fit2) pdb files may show different molecule sizes in Pymol when Doo Nam used map that was made from phenix.map_box

Troubleshooting is ongoing.

In the meantime, please use UCSF Chimera to visualize bio-molecules instead.

My model is shifted after phenix.model_idealization.

• To prevent this, it is recommended to add map as well.

• For example, phenix.model_idealization <user.pdb> <user.map that was used for cryo_fit2> (resolution information is not required)

"RuntimeError: cctbx Error: Miller index not in structure factor map"

• When a user sees above message, consider to enter a correct resolution.

• For example, Doo Nam uses either EMDB reported resolution (preferred) or phenix.mtriage derived resolution to properly run cryo_fit2.

• When a user is not sure about correct resolution, often using a lower resolution works.

• For example, when Doo Nam saw an error with resolution=3, then using resolution=5 often solved the problem.

"Sorry: Crystal symmetry mismatch between different files."

• Sometimes, Doo Nam observed,

• "Sorry: Crystal symmetry mismatch between different files.

  (378, 378, 378, 90, 90, 90) P 1

  (103.95, 91.35, 89.25, 90, 90, 90) P 1"

• The first line (378, ...) shows unit cell parameters from model.

• The second line (103.95, ...) shows unit cell parameters from map.

• Solutions

1. Just erase CRYST1 header information from an input pdb file. Then, provide that input file into cryo_fit2.

• Cryo_fit2 will automatically extract CRYST1 header from map (both from original map and phenix.map_box derived map), and prepend (write at the first line) to the pdb file.

2. (Alternatively) Just leave correct CRYST1 header only in an input pdb file.

• For example, when a wrong CRYST1 header exists above a correct CRYST1 header, above error message appeared.

When cryo_fit2 is useful?

• To model dynamic conformational change, cryo_fit2 is useful.
• To simply fit to cryo-EM map, cryo_fit seems to be useful when initial cc_mask and cc_box < 0.6. When initial cc_mask and cc_box >= 0.6, phenix.real_space_refine tends to do similar fitting with faster speed.

What to do after cryo_fit?

• If there is a room/chance to idealize secondary structure further even after cryo_fit, then run phenix.model_idealization which is followed by phenix.real_space_refine.
• Otherwise, just run phenix.real_space_refine to improve overall geometry (such as rotamers).