Hi, I'm attempting to run ResolveCryoEM, and I have a few questions about setting the job up correctly. My half-maps were reconstructed with C4 symmetry to ~2 Å (CryoSPARC), and my model was refined into the density with the "Ncs constraints" box checked (Phenix RSR). My sample is a protein-DNA complex (4 copies of a protein protomer, 4 copies of a ssDNA protomer). I'm running Phenix 1.21.1-5286-000 (the latest installation provided by SBGrid). In the first version of the run, I only supplied the two half-map files and a seq file. Question 1: My current seq.dat file looks like this (as mentioned previously in the forum, I was getting errors when I tried the version where my seq.dat file has only 1 protomer and I set the number of copies to 4 in the GUI field "Copies of sequence file in map"):
protein_protomer1 MGSRSG... protein_protomer2 MGSRSG... protein_protomer3 MGSRSG... protein_protomer4 MGSRSG... DNA_protomer1 AAAAAA DNA_protomer2 AAAAAA DNA_protomer3 AAAAAA DNA_protomer4 AAAAAA
Currently, Phenix is reading in the "AAAAAA" as a protein chain ("Chain types considered: PROTEIN"). How do I tell Phenix that those are deoxyadenosines, not alanines? Question 2: Is Phenix automatically detecting the C4 symmetry and density-modifying with that constraint? The resulting map looks symmetric in the noise, but I don't see anything in the log suggesting that it knows the symmetry. Here are some settings from the log that seem relevant (all left to default values): refine_symmetry_in_denmod = True use_symmetry_in_denmod = False symmetry_in_denmod_last_cycle_only = True fraction_ncs = None I attempted to supply a symmetry file (the output of a MapSymmetry job on the full map = "symmetry_from_map.ncs_spec"), but the file fails to load into the input ("Phenix error: Phenix did not recognize the file type for ****/symmetry_from_map.ncs_spec"). Question 3: A part of my protein is disordered and does not appear in the cryo-EM density (and was thus left unmodeled). Is it correct to leave that piece out of the seq.dat file? Question 4: If I include a model in the input, Phenix demands a symmetry file, but I couldn't get my symmetry file to load as explained above. What is the symmetry file supposed to look like? Thank you! -Josh
Hi Josh,
All good questions about resolve_cryo_em
1. Yes. supply a sequence file with 4 copies of each chain if there are 4
copies in the structure.
With your structure having protein and DNA and the sequence of the DNA is
AAAAAA, no there is no way to tell resolve_cryo_em that those A's are DNA.
Best to just say TTTTTT as it will guess that is DNA, and it is only the
number of residues that count.
2. As you supplied a map with C4 symmetry, that symmetry will remain in the
density-modified map
3. You can leave the disordered part out of your sequence file
4. Your symmetry file is probably fine...the GUI version of
resolve_cryo_em does not recognize symmetry files right now. You can run
from the command line and that should work.
Let me know if that does not do it! You can email me directly if you like.
All the best,
Tom T
On Tue, Sep 17, 2024 at 1:24 PM Josh Cofsky
Hi,
I'm attempting to run ResolveCryoEM, and I have a few questions about setting the job up correctly. My half-maps were reconstructed with C4 symmetry to ~2 Å (CryoSPARC), and my model was refined into the density with the "Ncs constraints" box checked (Phenix RSR). My sample is a protein-DNA complex (4 copies of a protein protomer, 4 copies of a ssDNA protomer). I'm running Phenix 1.21.1-5286-000 (the latest installation provided by SBGrid).
In the first version of the run, I only supplied the two half-map files and a seq file.
Question 1: My current seq.dat file looks like this (as mentioned previously in the forum, I was getting errors when I tried the version where my seq.dat file has only 1 protomer and I set the number of copies to 4 in the GUI field "Copies of sequence file in map"):
protein_protomer1 MGSRSG... protein_protomer2 MGSRSG... protein_protomer3 MGSRSG... protein_protomer4 MGSRSG... DNA_protomer1 AAAAAA DNA_protomer2 AAAAAA DNA_protomer3 AAAAAA DNA_protomer4 AAAAAA
Currently, Phenix is reading in the "AAAAAA" as a protein chain ("Chain types considered: PROTEIN"). How do I tell Phenix that those are deoxyadenosines, not alanines?
Question 2: Is Phenix automatically detecting the C4 symmetry and density-modifying with that constraint? The resulting map looks symmetric in the noise, but I don't see anything in the log suggesting that it knows the symmetry. Here are some settings from the log that seem relevant (all left to default values): refine_symmetry_in_denmod = True use_symmetry_in_denmod = False symmetry_in_denmod_last_cycle_only = True fraction_ncs = None I attempted to supply a symmetry file (the output of a MapSymmetry job on the full map = "symmetry_from_map.ncs_spec"), but the file fails to load into the input ("Phenix error: Phenix did not recognize the file type for ****/symmetry_from_map.ncs_spec").
Question 3: A part of my protein is disordered and does not appear in the cryo-EM density (and was thus left unmodeled). Is it correct to leave that piece out of the seq.dat file?
Question 4: If I include a model in the input, Phenix demands a symmetry file, but I couldn't get my symmetry file to load as explained above. What is the symmetry file supposed to look like?
Thank you! -Josh _______________________________________________ phenixbb mailing list -- [email protected] To unsubscribe send an email to [email protected] Unsubscribe: phenixbb-leave@%(host_name)s
-- Thomas C Terwilliger Laboratory Fellow, Los Alamos National Laboratory Senior Scientist, New Mexico Consortium 100 Entrada Dr, Los Alamos, NM 87544 Email: [email protected] Tel: 505-431-0010
participants (2)
-
Josh Cofsky -
Tom Terwilliger