Hi Vennila, I think Nat answered your question. I'm wondering though why you need to compute a map in such an "exotic" resolution range? I mean, what are the motivations to compute this map using only a narrow shell of medium resolution reflections rather than the whole dataset? Thanks! Pavel. On 12/22/09 9:40 PM, Nathaniel Echols wrote:

On Dec 22, 2009, at 8:36 PM, vennila Natesan wrote:

...

How do we create anomalous map using high resolution reflections? For example, if we have 2.2A resolution data (i.e 20-2.2A), i want to know how to create the anomalous map for the same dataset but from 2.4-2.2A resolution range.

I don't think there is a single-step method for this right now. The easiest alternatives:

1) From the command-line, I think this will work: phenix.reflection_file_converter --resolution=2.2 --low-resolution=2.4 --mtz=subset.mtz data.mtz phenix.model_vs_data subset.mtz model.pdb --map-type=anomalous (which will produce map coefficients in MTZ format) If you run into problems, typing the command name alone will print out a list of options.

2) From the GUI: use the reflection file editor to output the resolution range you want, then the "Create maps" app to generate an anomalous map (in either MTZ or XPLOR format). I will add resolution limits to the latter program soon.

However, I think a map calculated for 2.4-2.2A is going to look extremely distorted.

-------------------- Nathaniel Echols Lawrence Berkeley Lab 510-486-5136 [email protected]

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Hi Kendall, I guess you need to change the default setting for group_adp_refinement_mode = *one_adp_group_per_residue \ two_adp_groups_per_residue group_selection to group_adp_refinement_mode = one_adp_group_per_residue \ two_adp_groups_per_residue *group_selection and then everything will be ok, please let me know otherwise. The parameters will look like: refinement { refine { strategy = *individual_sites individual_sites_real_space rigid_body \ individual_adp *group_adp *tls occupancies group_anomalous adp { group_adp_refinement_mode = one_adp_group_per_residue \ two_adp_groups_per_residue *group_selection group = chain X group = chain A group = chain B group = chain C group = chain D tls = chain X tls = chain A tls = chain B tls = chain C tls = chain D }} Pavel. On 12/30/09 12:29 PM, Kendall Nettles wrote:

I'm trying to refine a 3.5 anstrom structure with one TLS and one ADP per chain, but I keep getting the error message:

"Sorry: Selections for group B-factor refinement are given, but no refinement requested."

The parameters file is listed below. Any suggestions?

Best regards and happy New Year,

Kendall Nettles

refinement { refine { strategy = *individual_sites individual_sites_real_space rigid_body \ individual_adp *group_adp *tls occupancies group_anomalous adp { group = chain X group = chain A group = chain B group = chain C group = chain D

tls = chain X tls = chain A tls = chain B tls = chain C tls = chain D

}}

target_weights { mode = *every_macro_cycle optimize_wxc=true optimize_wxu=true}

}

refinement.modify_start_model.adp.set_b_iso = 80

refinement.input.xray_data.labels=IMEAN

------------------------------------------------------------------------

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Hi Kendall, when you do group isotropic ADP refinement no any ADP restrains are used. Most likely this happens for residues not fitting electron density map well. I'm not sure what is the best approach here. Imagine one residue that fits density well and its group B-factor refines to a good value and the adjacent residue is out of density and, no surprises, its refined group B is very high. In this case if we apply restrains, we will spoil the group B-factor of the good residue by the high B-factor of the bad one, and we will force the high B of the bad residue to be unrealistically smaller than supported by the density. Did you try resetting all B-factors to an average value before refinement (to make sure that what you are seeing is not a refinement artifact)? Also, you may want to try using larger groups: say, two residues per one refinable B (in this case you will need to give a long list of selections). You can try using TLS as well. Individual ADP refinement may not be supported by the data amount (data-to-parameters ratio) at this resolution, as you see by 4% increase in Rfree (although, what happens with Rwork?). Pavel. On 12/31/09 8:01 AM, Kendall Nettles wrote:

I playing around with ADP and TLS parameters for a 3.5 angstrom data set. When I use one ADP per group, the values can vary by as much as 100 between adjacent residues, which doesn't make physical sense to me. If I refine individual ADPs this doesn't occur, but R free is 4% higher. Is this a problem with my data, or something to do with the implementation of this feature? Or something else? Best, Kendall Nettles ------------------------------------------------------------------------

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Hi Kendall,

I am resetting the B factors in both cases. TLS definitely helps (does it every not help?).

The goal of using TLS model is not to reduce R-factor, but is to use more physically correct model (this is primary), and eventually reduced R-factor (this is secondary) is the reward for doing so; while increased R-factor will indicate the incorrect ADP model.

If poor fit is the problem, why do I not see the variance with individual ADPs?

Because the ADP restraints are used in case of individual ADP refinement, and no restrains are used in case of group B refinement. Did you try refining individual ADPs with two scenarios: - using "wxu_scale=true" to optimize weights; - manually setting very string ADP restraints ? I would definitely try both options.

I still don't think it makes physical sense to have one residue in a helix with an ADP of 100 and the next one with 200,

I did not say it is correct or makes physical sense. I just tried to explain why it happens (the most likely reason).

which is what I'm seeing with the one per residue setting. With respect thermal motion, the adjacent residues are attached, so it makes sense to me to have their values restrained.

Copy-paste from my previous email: """Imagine one residue that fits density well and its group B-factor refines to a good value and the adjacent residue is out of density and, no surprises, its refined group B is very high. In this case if we apply restrains, we will spoil the group B-factor of the good residue by the high B-factor of the bad one, and we will force the high B of the bad residue to be unrealistically smaller than supported by the density.""" which is saying differently : If both residues fit density well, then applying restraints will not do any harm, and this is not the case otherwise. If in your particular case both residues fit density very good and the difference between their refined group B-factors is very large, then the problem is somewhere else, and this needs more investigation (which may take a day or more). Also, I will need to be able to reproduce it myself (I need the data and model for this). I will see what it takes to add restrains for group B refinement, at least for experimenting. Pavel.