Cryo_fit1 FAQ

Contents

How can I provide gaussian filtered maps?

How long does it take to run cryo_fit?

../images/cryo_fit_FAQ_how_long1.png ../images/cryo_fit_FAQ_how_long2.png

How to enlarge map box size?

  1. cryo_fit which reports map size after step 6 before step 7 with codes of
  • from iotbx import ccp4_map
  • ccp4_map = ccp4_map.map_reader(user_input_map)
  • target_map_data = ccp4_map.map_data()
  1. EMAN2's e2iminfo
  • For example, e2iminfo.py <user>.mrc
  • (note) As of EMAN2.2, the file extension should be mrc, not ccp4
  1. VMD which can visualize current map size as in this FAQ.
  • For example, relion_image_handler --i <user>.mrc --new_box 370 --o <user_box_size_370>.mrc

How to generate and record movie?

How to improve initial cc?

I can't run phenix.superpose_pdbs with cryo_fitted pdb file

I observe cc values keep decreasing (or do not increase that much) during step 8

  1. If a user input pdb file has unreasonable structural geometry. Although the fit between atomic model and map looks good, it is a fictitious fitting without considering ideal geometry. Run cryo_fit to find decent fit to the cryo-EM map along maintaining reasonable structural geometry.
  1. Providing higher (better) resolution map tends to help this problem.
  2. Enforcing stronger map weight tends to help this problem.
  3. If cryo_fit is provided a giant cryoem map with a tiny atomic model.
    Then, the cryo_fit calculates the gradient of CC because of the large empty space not filled. The constraint forces for the model are not helping as they are very small.
3-1. Re-run cryo_fit with an atomic model that fits the majority of the map.
Fit multiple atomic models into a symmetric map or sequential fitting into a non-symmetric map. Watch Tom Goddard's lecture (2013)

3-2. Re-run cryo_fit with only relevant map region. A user can extract relevant map region by phenix.map_box (preferred) or phenix.map_to_model

  1. If the initial model is not properly aligned to a map, fit using UCSF Chimera -> Tools -> Volume Data -> Fit in Map

I see "Fatal error: A charge group moved too far between two domain decomposition steps. This usually means that your system is not well equilibrated" at my 8_cryo_fit step.

I see "Fatal error: Incomplete ring in HIS50" at my 8_cryo_fit step

  1. Open pdb file
  2. [menu]
  3. Select -> Residue -> HIS
  4. Tools -> Structure Editing -> Rotamers -> OK
  5. (select the most probable rotamer each)
  6. File -> Save PDB

I see "Fatal error: Number of grid cells is zero. Probably the system and box collapsed." at my 8_cryo_fit step.

  • Most likely, this means that initial cc is too low for MD simulation.
  • When Doo Nam ran real_space_refine first, then run real_space_refined atomic model in cryo_fit, it was solved.
  • Alternatively, improve initial cc by fitting initial atomic model into a map (see "How to improve initial cc?" in this FAQ)
  • Less likely, but still a possible case is when the map weight is too high, lowering emweight_multiply_by may help.

I see "pdb file cleaning is not done" at my step 1 (Make gro and topology file by regular gromacs)

"I edited out lipids, HEM and other hetero atoms and I verified that they are all gone. However, still my pdb file is not clean enough for gromacs_cryo_fit".

I see "state.cpt not found, step_8 may be full of stepxb_nx.pdb" at my 8_cryo_fit step

I see "step 0 correlation coefficient: nan" at my 8_cryo_fit step.

  • Therefore, please improve initial cc by fitting initial atomic model into a map (see "How to improve initial cc?" in this FAQ)

I see "step 0 correlation coefficient: nan" and "Range checking error: Explanation: During neighbor searching, we assign each particle to a grid based on its coordinates. If your system contains collisions or parameter errors that give particles very high velocities you might end up with some coordinates being +-Infinity or NaN (not-a-number). Obviously, we cannot put these on a grid, so this is usually where we detect those errors. Make sure your system is properly energy-minimized and that the potential energy seems reasonable before trying again." at my 8_cryo_fit step.

  1. Doo Nam observed this error when starting molecule is not stabilized structurally. There are two possible solutions.
  • Run phenix.real_space_refine before cryo_fit. Then, provide real_space_refined molecule into cryo_fit. Then, most problems were solved.
  • When Doo Nam skipped minimization, this error occured. Since most users minimize the starting structure by default (cryo_fit default), most users do not need care about this. Besides, as of 9/18/2019, the default value of number_of_steps_for_minimization=20000 (which is a lot).
  1. It means that the map dimensions need to be larger. Therefore, check map size and solve a problem with VMD/EMAN2/relion like followings.
  • Like other MD simulations, gromacs need enough map box size to cover the atomic model to run (ziggle and wiggle). Refer Waters seems to be out of the box

  • For example, stuck-out red oxygen atoms outside the right edge of the box are the problem.

  • ../images/cryo_fit_FAQ_sticking_out.png
  • In order to run any MD simulation (including cryo_fit), a box should be large enough like

  • ../images/cryo_fit_FAQ_not_stick_out.png
  • Make map box size larger (see "How to enlarge map box size?" in this FAQ), and run cryo_fit again. You can check map box size by VMD. Alternatively, remove sticking out atoms if these are unnecessary, then run cryo_fit again.

  • For protein modeling, I would use cryo_fit2 which is not limited by box size requirement. Most of the time, it better fits than cryo_fit1 in terms of fitting and geometry preservation anyway.

I see "The initial cell size (xxx) is smaller than the cell size limit (xxx), change options -dd, -rdd or -rcon, see the log file for details" at my 8_cryo_fit step

  1. When he provided a pdb file that has unspecified/deleted region of a molecule. Even when vmd assisted map box/cell dimension is larger than initial molecule space, this error appeared.
  2. When he provided a pdb file that has small number of residues (e.g. 5~16 amino acids). Even when vmd assisted map box/cell dimension is larger than initial molecule space, this error appeared. This error appeared regardless of the existence of CRYST1 and SCALE header.

I see "Too many LINCS warnings" at my 8_cryo_fit step

  1. Run phenix.real_space_refine before cryo_fit. Then, provide real_space_refined molecule into cryo_fit. Then, most problems were solved.
  2. When Doo Nam skipped minimization, this error occured. Since most users minimize the starting structure by default (cryo_fit default), most users do not need care about this. Besides, as of 9/18/2019, the default value of number_of_steps_for_minimization=20000 (which is a lot).
  3. If starting molecule has non-standard chemical geometry that is too extreme that are not stabilized neither by real_space_refine nor by cryo_fit minimization, then a user needs to fix those non-standard geometry manually.

I see "User's provided atomic model had 0.0 cc" in my cryo_fit.overall_log.