questions about using experimental phases in phenix.refine
I'm trying to use experimental phases derived from an cryoEM density map do carry out reciprocal space refinement in phenix.refine. I used phenix.map_box to get the map coefficients, and then the CCP4 program chltofom (with the -colin-phifom flag) to create the HL coefficients from F/PHI computed from the electron density map. 1. If I ask for the mlhl target, I get quite odd target values, e.g. for a 2.9 Å data set: | normalized target function (mlhl) (work): -5862.311069 | | target function (mlhl) not normalized (work): -6438670043.811632 and attempted refinements more or less destroy my protein and increases both the mlhl target value and the R-factors. Is there an example data set I could access to help me find out what I'm doing wrong? (Using the ml target, as suggested in DeMaio et al, Nature Methods 10:1102, 2013, seems to work well, and has a postive target function, but seems sub-optimal if phase information is available.) 2. Is there a way to do a "simple" least-squares refinement, minimizing sum( w_i * |Fobs_i - Fcalc_i|**2 ) where both Fobs and Fcalc are treated as vectors? Does the ls target perhaps do this if use_experimental_phases=True? Usual apologies if there is some great tutorial on this that I've not found. ...thanks...dave case
Hi Dave, I’d suggest using the Phenix program phenix.map_to_structure_factors to convert a map to an MTZ file. This will generate HLs with reasonable FOMs. I suspect the problem with what you did before is that the FOMs are all 1, and this isn’t handled well by the MLHL target (this is a guess on my part). We don’t have a vector LS target in phenix.refine - we encourage people to use real space refinement for cryo-EM data. Cheers, Paul
On May 15, 2021, at 10:29 AM, David A Case
wrote: I'm trying to use experimental phases derived from an cryoEM density map do carry out reciprocal space refinement in phenix.refine.
I used phenix.map_box to get the map coefficients, and then the CCP4 program chltofom (with the -colin-phifom flag) to create the HL coefficients from F/PHI computed from the electron density map.
1. If I ask for the mlhl target, I get quite odd target values, e.g. for a 2.9 Å data set:
| normalized target function (mlhl) (work): -5862.311069 | | target function (mlhl) not normalized (work): -6438670043.811632
and attempted refinements more or less destroy my protein and increases both the mlhl target value and the R-factors. Is there an example data set I could access to help me find out what I'm doing wrong? (Using the ml target, as suggested in DeMaio et al, Nature Methods 10:1102, 2013, seems to work well, and has a postive target function, but seems sub-optimal if phase information is available.)
2. Is there a way to do a "simple" least-squares refinement, minimizing sum( w_i * |Fobs_i - Fcalc_i|**2 ) where both Fobs and Fcalc are treated as vectors? Does the ls target perhaps do this if use_experimental_phases=True?
Usual apologies if there is some great tutorial on this that I've not found.
...thanks...dave case
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-- Paul Adams Division Director, Molecular Biophysics & Integrated Bioimaging, LBL (http://biosciences.lbl.gov/divisions/mbib) Principal Investigator, Computational Crystallography Initiative, LBL (http://cci.lbl.gov) Vice President for Technology, the Joint BioEnergy Institute (http://www.jbei.org) Principal Investigator, ALS-ENABLE, Advanced Light Source (http://als-enable.lbl.gov) Division Deputy for Biosciences, Advanced Light Source (https://als.lbl.gov) Laboratory Research Manager, ENIGMA Science Focus Area (http://enigma.lbl.gov) Adjunct Professor, Department of Bioengineering, U.C. Berkeley (http://bioeng.berkeley.edu) Member of the Graduate Group in Comparative Biochemistry, U.C. Berkeley (http://compbiochem.berkeley.edu) Building 33, Room 250 Building 978, Room 4126 Building 977, Room 180C Tel: 1-510-486-4225 http://cci.lbl.gov/paul http://cci.lbl.gov/paul ORCID: 0000-0001-9333-8219 Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 33R0345 Berkeley, CA 94720, USA. Executive Assistant: Ashley Dawn [ [email protected] ][ 1-510-486-5455 ] --
Hi David,
I'm trying to use experimental phases derived from an cryoEM density map do carry out reciprocal space refinement in phenix.refine.
I used phenix.map_box to get the map coefficients, and then the CCP4 program chltofom (with the -colin-phifom flag) to create the HL coefficients from F/PHI computed from the electron density map.
1. If I ask for the mlhl target, I get quite odd target values, e.g. for a 2.9 Å data set:
| normalized target function (mlhl) (work): -5862.311069 | | target function (mlhl) not normalized (work): -6438670043.811632
and attempted refinements more or less destroy my protein and increases both the mlhl target value and the R-factors. Is there an example data set I could access to help me find out what I'm doing wrong? (Using the ml target, as suggested in DeMaio et al, Nature Methods 10:1102, 2013, seems to work well, and has a postive target function, but seems sub-optimal if phase information is available.)
this is odd.. Normally you should use (example) phenix.map_to_structure_factors map.mrc d_min=3.4 to convert map to structure factors. The MTZ file from the command above should be ready for phenix.refine. It contains "Fobs", free-R flags and HL coefficients that represent map phase information. MLHL target will be used in this case, it is implemented exactly as described in the original paper: Pannu, N. S., Murshudov, G. N., Dodson, E. J. & Read, R. J. (1998). Acta Cryst. D54, 1285–1294.
2. Is there a way to do a "simple" least-squares refinement, minimizing sum( w_i * |Fobs_i - Fcalc_i|**2 ) where both Fobs and Fcalc are treated as vectors? Does the ls target perhaps do this if use_experimental_phases=True?
No, this is not implemented. Pavel
participants (3)
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David A Case
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Paul Adams
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Pavel Afonine