Hi Everyone, just this morning I replied someone else offlist to a similar question. I replied to Joe offlist too. I guess I had to actually reply to phenixbb, so I wouldn't have to duplicate emails multiple time... This is a very typical question and honestly, and sometime I feel like people get too sensitive to "high" B-factors... The atomic displacement parameter tends to measure several physical phenomena, including: 1) dynamic disorder, that is how much an atom moves around its position defined by the atomic model, and 2) static disorder arising from (typically small) differences in equivalent atomic positions throughout the whole crystal. In addition, it measures the correctness of atomic model – a misplaced atom would have outstandingly low or high value of refined ADP compared to surrounding correctly placed atoms. Clearly this is way more than the mathematical model implemented for it in refinement is supposed to model. Within structure refinement framework the B-factors are supposed to model only the first phenomenon (dynamic disorder), while the others effects get absorbed by irrelevant refinable parameters contributing to their uncertainties. Large-scale static disorder can be modeled, for example, by alternative conformations or by using anharmonic functions for ADP (Acta Cryst. (2009). D65, 284-293) or multiple models. Having said that, the refined value of a B-factor is not necessarily directly related to mean amplitude of harmonic vibration of an atom. The matter of fact is that it has much more in it. So when you get a huge B-factor I wouldn't take it too literally and convert it into angstroms of mean amplitude of its harmonic vibration. Sometime people believe that their refined B-factors should closely match the Wilson B, and they get very excited if it not the case and start talking about "a big difference between mean B and Wilson B". But, they probably forget one "tiny" detail, that: - the Wilson B-factor is just an estimation based on assumption that the atoms are randomly distributed in the unit cell. Clearly the atoms in your model are not randomly filling the unit cell! Plus, the accuracy of this estimation drops with the resolution. - refined B-factors is what reflects your data, so I would trust these values more rather than an estimate obtained under unrealistic assumptions. Given this, can anyone seriously tell what is "big difference" (see recent Dale Tronrud's great reply on this matter)? Now, if we look at thousands of structures in PDB to see how their mean B-factors are distributed as a function of resolution, we will get something like this: Resolution, A <B> 1.25-1.5 18.96 1.5-1.75 21.99 1.75-2.0 26.51 2.0-2.25 31.75 2.25-2.5 38.53 2.5-2.75 42.49 2.75-3.0 48.01 3.0-3.25 55.8 3.25-3.5 65.25 Joe refines his structure at 1.7 and his mean B~40A**2, and the Wilson B is ~30. Yes, both refined and Wilson B are somewhat larger than observed in average over PDB structures. This fact would prompt me to triple-check if the correct refinement strategy is used, and if everything else looks ok, and if everything is good then I would not worry too much about the B-factors - there must be something in your crystal/data that they (B-factors) are trying to convey to you. All the best! Pavel. On 6/10/10 1:02 PM, Nian Huang wrote:

Hi Joseph, I had a similar situation. In the end, I ended up stopping using TLS for my structure. Do you have the same problem by just doing individual adp refinement?

Nian

On Mon, Jun 7, 2010 at 8:52 PM, Joseph Brock wrote:

...

Dear Phenix users,

I am currently using version 1.6.1-353 and I'm working on a 1.7A structure that has refined quite well using Individual_sites+individual_adp(isotropic)+occupanices+TLS: Final R-work = 0.1776, R-free = 0.2262

However, I noticed that the average B-factor reported by polygon with these settings was quite high ( < 0.1 of other structures with a similar resolution).

Discovering I could not change the value of wxu_scale when using individual_adp+TLS, I went about trying to reduce the average B using the method described here:

http://phenix-online.org/pipermail/phenixbb/2007-September/000511.html

However, I still can't seem to reduce the average B-factor. I've just done a refinement with the following non-default (extreme) target weight settings:

target_weights { wxc_scale = 1.5 fix_wxc = 2 fix_wxu = 0.1 (wxu_scale = 1)

But if anything, reducing wxu to such low values makes the avergae B-even higher while concuretnly making the gap between R-values much worse:

Final R-work = 0.1602, R-free = 0.2457

This suggests to me that I'm obviously over fitting the data but I'm at a loss on how to proceed. Should i just accept that the automatic scaling used for Phenix TLS wxu is doing a good job and that my structure actually does have an unusually high avergae B?

I'm fitting two ligands into density, which is at several places ambiguous, so I would like the most unbiased difference density possible.

Many thanks in advance,

Joe.