[3dem] what is the ideal B factor?
Carlos Oscar Sorzano
coss at cnb.csic.es
Thu Aug 27 09:27:21 PDT 2020
I totally agree with Arjen, and I think that all the ideas thrown in are
useful in their context. Another thing to consider, SAXS curves are very
much related to the topic we are discussing, are they not?
(understanding the differences between X-ray photons and electrons
diffraction, frozen proteins and proteins in solution, possible mixtures
of various conformations, etc.). The fact that different SAXS curves are
measured for different proteins, would that show that there is not a
single Platonic curve for all proteins?
Cheers, Carlos Oscar
El 27/08/2020 a las 16:49, Arjen Jakobi - TNW escribió:
>
> Hi Alexis,
>
> You bring up an interesting point.
>
> My understanding is that Wilson statistics assumes (and is strictly
> valid only for) independent and uniformly distributed (= random)
> atoms. This is why I think that Wilson statistics as derived in the
> original paper are primarily valid in the high-resolution (better than
> 3Å) part of the Wilson/Guinier plot.
>
> At lower resolution, in particular at those spatial frequencies
> corresponding to repetitive features in real-space, i.e. the regular
> path proteins (secondary structure), and nucleic acids (base stacking)
> follow in 3D space & (ordered) solvent give rise to characteristic
> features in the pair-distribution function. This is what you typically
> see in a “Guinier plot”: this plot is in principle a (rotationally
> averaged) representation of the texture of the macromolecule and will
> contain characteristic deviations from the exponential (or linear in
> log-plot) decay expected from Wilson statistics, because at some
> resolution/spatial frequencies the arrangement of atoms is not random.
> This is true regardless of whether you consider X-ray or EM
> experiments. This is also a reason why I think B-factor estimations if
> performed including these regions are systematically off; the R^2 of
> linear regression will be poor. Once you are moving to higher
> resolution, let us say 3.0 Å and beyond, a protein structure can very
> well be considered as a collection of randomly distributed atoms and
> here Wilson statistics hold and the slope gives the B-factor. If you
> do a fit in this region of a Guinier plot, e.g. for high-resolution
> ApoF structures, the fit will be very good.
>
> Regarding Carlos Oscar’s statement that the fall-off is “arbitrary
> depending on its shape”, I do not necessarily agree but I guess the
> point he is trying to make is that the radially averaged fall-off will
> be modulated by these effect (e.g. secondary structure) and this could
> be considered a “fingerprint” of the protein in question. In practice,
> when radially averaging over the entire structure, the fall-off,
> including deviations from Wilson statistics, will be very similar for
> most proteins unless they are e.g. all-alpha, all-beta or contain
> significant amount of nucleic acids as e.g. ribosomes.
>
> This aside: Could it be that the difference you observe comes from the
> fact that in your simulated model you do not account for solvent? If
> you make a thought experiment and place your protein in a “vacuum”
> then this would lead to an overestimation of “contrast” at the
> molecule surface compared to the situation where you have solvent.
> Taking this to your simulated structure factor, the calculated
> structure factor would be expected to be systematically larger than
> the observed structure factor amplitude in regions where in the real
> situation bulk solvent is having noticeable effect (e.g. 5Å and below).
>
> If in your case you have derived your B_ideal from a fit in this
> region (e.g. 20 – 4 Å), than the calculated fall-off would probably be
> steeper than the observed amplitude fall-off. If you have fit in the
> high-resolution region then this should have no effect.
>
> Not sure if it helps.
>
> Best,
>
> Arjen
>
> *From: *3dem <3dem-bounces at ncmir.ucsd.edu> on behalf of Alexis Rohou
> <a.rohou at gmail.com>
> *Date: *Thursday, 27 August 2020 at 07:06
> *To: *Carlos Oscar Sorzano <coss at cnb.csic.es>
> *Cc: *3dem <3dem at ncmir.ucsd.edu>
> *Subject: *Re: [3dem] what is the ideal B factor?
>
> Thank you Carlos Oscar for summarizing your work so succinctly!
>
> I just would like to pick up on your concluding sentence:
>
> In conclusion, from my point of view, there is not an optimal
> decay valid for all proteins, but it depends on each specific
> protein. And the shape of the decay is not a straight line, but
> arbitrary depending on its shape.
>
> If your point of view is correct, this implies that ResLog plots and
> the resulting B factors should not be compared to each other if they
> were obtained from images of different proteins. This would be quite a
> departure from the field's consensus.
>
> Cheers,
>
> Alexis
>
> On Wed, Aug 26, 2020 at 12:32 AM Carlos Oscar Sorzano
> <coss at cnb.csic.es <mailto:coss at cnb.csic.es>> wrote:
>
> Dear Alexis and all,
>
> as a very condensed summary of what Jose Luis Vilas and us showed
> in the paper you have mentioned below is that:
>
> 1. the Fourier spectrum of a single atom is expected to decay in
> amplitude with frequency (the only way it can be flat is that it
> is infinitely thin). This is well known and comes from the
> electron atomic scattering factors.
>
> 2. the Fourier spectrum of a collection of atoms is mostly
> determined by the shape of that collection, more than on the
> specific nature of the atoms being involved (we performed
> extremely harsh modifications to the atoms and the decay did not
> change significantly).
>
> 3. the reasons normally argued to make the spectrum flat, do not
> apply to macromolecules and the reason why B-factor sharpening
> produces "nice" structures is mostly a visualization reason
> (higher amplitudes at high frequencies result in sharper edges
> whose isosurfaces are easier to track and fit with an atomic model).
>
> Because of 2, the decay of the radial average of the Fourier
> transform of a macromolecule cannot be expected to follow any
> particular shape (for instance, a straight line) a priori, that we
> can estimate its slope (also a priori) and force our 3D
> reconstruction to follow that slope. In that regard the question
> of what is the expected slope is ill-posed. From my point of view,
> the amplitude correction is much more meaningful when performed in
> the spirit of LocScale of Jakobi and Sachse. You fit an atomic
> model to the map, then convert it into a map, estimate its decay
> and force the map to follow this decay. In this way, the shape of
> the collection of atoms (and their nature) is explicitly taken
> into account. This process can be performed iteratively (with the
> corrected map, you may refine the atomic model, refine the map
> amplitudes again, ...). I also like the idea that this process is
> performed locally.
>
> If we do not want to wait for the atomic model to make the
> amplitude correction, we have devised an alternative based on the
> local resolution (E. Ramirez-Aportela, J.L.Vilas, A. Glukhova, R.
> Melero, P. Conesa, M. Martinez, D. Maluenda, J. Mota, A. Jimenez,
> J. Vargas, R. Marabini, P.M. Sexton, J.M. Carazo, C.O.S. Sorzano.
> Automatic local resolution-based sharpening of cryo-EM maps.
> Bioinformatics 36: 765-772 (2020)). There is no guarantee that it
> will follow the correct decay, but in practice we have observed
> that it normally approximates the correct decay quite closely
> (there are some examples of this in the paper).
>
> The procedure above of local correction based on local resolution
> is local and it does not require an the atomic model. If we still
> want to do a global correction without an atomic model, procedures
> like the one of phenix
> (https://urldefense.com/v3/__https://journals.iucr.org/d/issues/2018/06/00/ic5102/ic5102.pdf__;!!Mih3wA!W6nGuTLj09q-CVG7jDPGp8qSrv627xTv47mDUYkewpvi_V5XSDYBPKc1QjX6ZWKqQw$
> <https://urldefense.com/v3/__https:/journals.iucr.org/d/issues/2018/06/00/ic5102/ic5102.pdf__;!!Mih3wA!Rn8ptfdW3i-ZJAsyJRtf84MeTMX0gbKZxAVhSD_WbmT6PxiAn3RA49bpffFudf71Mg$>)
> provides some clue based on the maximum continuity of the isosurface.
>
> Finally, we found that a combination of DeepRes (E.
> Ramírez-Aportela, J. Mota, P. Conesa, J.M. Carazo, C.O.S. Sorzano.
> DeepRes: A New Deep Learning and aspect-based Local Resolution
> Method for Electron Microscopy Maps . IUCR J 6: 1054-1063 (2019))
> and BlocRes
> (https://urldefense.com/v3/__https://www.sciencedirect.com/science/article/pii/S1047847713002086__;!!Mih3wA!W6nGuTLj09q-CVG7jDPGp8qSrv627xTv47mDUYkewpvi_V5XSDYBPKc1QjVs0XdB2Q$
> <https://urldefense.com/v3/__https:/www.sciencedirect.com/science/article/pii/S1047847713002086__;!!Mih3wA!Rn8ptfdW3i-ZJAsyJRtf84MeTMX0gbKZxAVhSD_WbmT6PxiAn3RA49bpffHaOthRoQ$>)
> could help to find a B-factor that does not result in overfitting.
>
> In conclusion, from my point of view, there is not an optimal
> decay valid for all proteins, but it depends on each specific
> protein. And the shape of the decay is not a straight line, but
> arbitrary depending on its shape.
>
> I hope these reflections helped a bit.
>
> Cheers, Carlos Oscar
>
> On 8/26/20 7:05 AM, Alexis Rohou wrote:
>
> Dear colleagues,
>
> I hope you may be able to help me get my head around something.
>
> When considering the radially-averaged amplitudes of an ideal
> 3D protein structure, the expectation (as laid out in Fig1 of
> Rosenthal & Henderson, 2003 (PMID: 14568533), among others) is
> that in the Wilson-statistics regime (q > 0.1 Å^-1, let’s
> say), amplitudes will decay in a Gaussian manner, or linearly
> when plotted on a log scale against q^2, reflecting the decay
> of structure factors.
>
>
> This expectation is certainly met when simulating maps from
> PDB files, as described nicely for example by Carlos Oscar
> Sorzano and colleagues recently (Vilas et al., 2020, PMID:
> 31911170). Let’s call the rate of decay of this ideal curve
> B_ideal, the “ideal” B factor.
>
> Assuming for a moment that noise has a flat spectrum
> (reasonable so long as shot noise is dominant), one may follow
> in Rosenthal & Henderson’s footsteps and draw a horizontal
> line on our plot to represent the noise floor. As more
> averaging is carried out, the noise floor is lowered relative
> to our protein’s amplitude profile. As more particles are
> averaged (without error, let’s say) the intersection between
> the protein’s ideal radial amplitude profile and the noise
> floor moves to higher and higher frequencies.
>
> This is the basis for the so-called ResLog plots, where one
> charts the resolution as a function of the number of averaged
> particles. The slope of the ResLog plot is related to the
> slope of the radial amplitude profile of the protein. Assuming
> no additional sources of errors (i.e. ideal instrument and no
> processing errors), B_ideal (the slope of the ideal protein
> amplitude profile) can be computed from the slope of the
> ResLog plot via B_ideal = 2.0/slope.
>
> Now, to my question. By looking at the slope of a schematic
> Guinier plot generated using Wilson statistics and atomic
> scattering factors for electrons, I estimated a B_ideal of
> approximately 50 Å^2 (decay of ~ 1.37 natural log in amplitude
> over 0.1 Å^-2). The problem is that recent high-resolution
> studies have reported ResLog-estimated B factors of 32.5 Å^2
> (Nakane et al., 2020) and 36 Å^2 (Yip et al., 2020), leading
> me to wonder what is wrong in the above model.
>
>
> I see several possibilities:
>
> (1) B_ideal is actually significantly less than 50 Å^2. This
> would be consistent with the empirical observation that
> “flattening” maps’ amplitude spectrum (i.e. assuming B-ideal =
> 0 Å^2) gives very nice maps. Either:
>
> a. I mis-estimated B_ideal when reading the simulated
> amplitude spectrum plot. Has anyone done this (i.e. fit a
> B factor to a simulated map’s amplitude spectrum, or to a
> simulated spectrum)? What did you find?
>
> b. The simulations using atomic scattering factors and
> Wilson statistics do not correctly capture the actual
> amplitude profile of proteins, which is actually much
> flatter than the atomic scattering factors suggest.
>
> (2) B_ideal actually is ~ 50 Å^2, but the assumption of a
> flat noise spectrum is wrong. I guess that if the true noise
> spectrum were also decaying at a function of q^2, this would
> cause the ResLog plot to report “too small” a B factor
>
>
> What do you think?
>
> Cheers,
> Alexis
>
>
>
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