My advice is to simply cycle back-and-forth between the two data sets.
Refine against your high-resolution data until that converges, then
simply switch to the other mtz file and use F+ F-, along with "anomalous
wavelength" and probably "refine orefine no", unless you want to refine
the occupancies of Mn and other heavies. Personally, I prefer to define
occupancy refinement explicitly using the REFMAC "occupancy group" etc.
commands. Once the SAD refinement has converged (as in positions,
occupancies, and B factors stop moving), then switch back to the
high-res. Once that converges, shift back to anomalous again. Then
high-res, then anomalous, then high-res, etc.
Obviously, you want to have the same Free-R flags in both mtz files.
You might think that all this back-and-forth would be redundant. Doesn't
"refinement" of a given model and given data set always converge to the
same final coordinate set? No, it does not. All you need to do is
"jiggle" your model and re-refine and you will get something new. RMSDs
usually around 0.4 A or so, depending on resolution. What this means
pragmatically is that any given refinement has a lot of "slop" in its
final result, and is therefore quite sensitive to initial conditions.
You can therefore easily incorporate information from multiple data sets
by doing this switching back-and-forth. The information from one data
set then manifests as "bias" for the start of the next refinement. No
special software required!
I understand that it is tempting to desire software that can somehow
incorporate data from disparate crystals and give you a single "right"
model, but that can only happen if your data sets are highly
isomorphous. Think about it. What if one data set has a serine side
chain in the + rotamer and another data set has the same residue is - or
trans ? What would you expect to find in your "right" model?
Oscillating between data sets, however, allows you to have a
well-defined final refinement target, but for the things that are not
well-defined by your final data set (such as anomalous signal or
occupancy), you can "fill in" those blank spaces by creating bias in the
starting structure.
In the present case, however, both data sets are from the same crystal,
so structural differences like rotamer shifts are not expected. In
fact, why not just merge the two data sets? If you take your two sets
of FP, put them together with CAD, and scale them with SCALEIT, what is
the R-factor between them? If the R is less than 5-7% then there is
seldom any reason not to merge them, but if your two wavelengths are
10-15% different you should really ask yourself why. Always a good idea
to make an Fo-Fo map and see where the largest difference peaks are. If
they are on top of heavy atoms then you might just be seeing the f'
difference, and that is OK, but you could also be seeing
radiation-induced changes, and those are perhaps not what you are
looking for? Which data set was collected first?
-James Holton
MAD Scientist
On 1/4/2019 2:16 AM, Steiner, Roberto wrote:
Dear Piotrek
You can find some info on the use of prior phase information in
refinement with Refmac in
Acta Crystallogr D Biol Crystallogr.
<https://www.ncbi.nlm.nih.gov/pubmed/?term=steiner+pannu#> 2011
Apr;67(Pt 4):355-67. doi: 10.1107/S0907444911001314. Epub 2011 Mar 18.
REFMAC5 for the refinement of macromolecular crystal structures.
Murshudov GN
<https://www.ncbi.nlm.nih.gov/pubmed/?term=Murshudov%20GN%5BAuthor%5D&cauthor=true&cauthor_uid=21460454>1,
Skubák P
<https://www.ncbi.nlm.nih.gov/pubmed/?term=Skub%C3%A1k%20P%5BAuthor%5D&cauthor=true&cauthor_uid=21460454>,
Lebedev AA
<https://www.ncbi.nlm.nih.gov/pubmed/?term=Lebedev%20AA%5BAuthor%5D&cauthor=true&cauthor_uid=21460454>,
Pannu NS
<https://www.ncbi.nlm.nih.gov/pubmed/?term=Pannu%20NS%5BAuthor%5D&cauthor=true&cauthor_uid=21460454>,
Steiner RA
<https://www.ncbi.nlm.nih.gov/pubmed/?term=Steiner%20RA%5BAuthor%5D&cauthor=true&cauthor_uid=21460454>,
Nicholls RA
<https://www.ncbi.nlm.nih.gov/pubmed/?term=Nicholls%20RA%5BAuthor%5D&cauthor=true&cauthor_uid=21460454>,
Winn MD
<https://www.ncbi.nlm.nih.gov/pubmed/?term=Winn%20MD%5BAuthor%5D&cauthor=true&cauthor_uid=21460454>,
Long F
<https://www.ncbi.nlm.nih.gov/pubmed/?term=Long%20F%5BAuthor%5D&cauthor=true&cauthor_uid=21460454>,
Vagin AA
<https://www.ncbi.nlm.nih.gov/pubmed/?term=Vagin%20AA%5BAuthor%5D&cauthor=true&cauthor_uid=21460454>.
and references therein.
As stated in the paper (paragraph 2.2.2), the incorporation of prior
phase information by the refinement function is especially useful in
the early and middle stages of model building and at all stages of
structure solution at lower resolutions, owing to the improvement in
the observation-to-parameter ratio.
Refinement in Refmac is very fast therefore the best thing (as you
just did) is to try both options and see.
With best wishes
Roberto
On 4 Jan 2019, at 09:55, Piotr Wilk <[email protected]
<mailto:[email protected]>> wrote:
Dear Eleanor,
I have used the ACORN previously for structure solution but I will
have to read more about its functionality in structure refinement.
I have run two Refmac jobs using either native or anomalous data with
otherwise default parameters resulting in R/Rfree of 0.2013/0.2458
and 0.1862/0.2274 respectively for crystal diffracting to ~1.85A.
This seems to me, that using anomalous signal in refinement can be
useful at least in some cases.
Regards,
Piotrek
Hmm - you can certainly generate "MAD" phases using the anom signal
from one data set, and then PARROT or some such density modification
tool to extend those phases for the higher resolution reflections..
Or ACORN can work well if he data resolution is high enough to give
good phases for all the data
Then you can use those phases in the initial refinement procedure -
the usual idea is to use them till the R factor drops below 30% or
35% then just refine against the Fobs, phasing just from the model .
But I dont think it is ever worth working at a limited resolution..
Dear Eleanor,
thank you for your comment. My crystals of interest diffract with
dmin usually between 1.4 and 1.9 A (in high energy data set) and
significant anomalous signal extends usually to approx. 4 A (in low
energy data set).
Certainly I do agree, that in "EITHER, OR" situation one can check
both approaches, compare the results and take the more convincing
one. I can easily do that in Refmac running one job against data with
Friedel pairs merged and parallel one against data with Friedel pairs
unmerged. I was considering rather an "AND" scenario in which in
addition to high resolution data (FP) I'd include information from
anomalous signal (F+ F-, DANO). I understand that this should
increase number of observations from a given sample and therefore
help to refine positions, occupancies and perhaps ADPs for at least a
fraction of atoms in a model (Mn ions and S in my case). I imagine it
as somehow analogical to adding geometrical restrains derived from
very high resolution data to refinement protocols.
I was wondering first of all if my reasoning is sensible and if there
is an existing protocol to try this?
With kind regards,
Piotrek
czw., 3 sty 2019 o 22:00 Eleanor Dodson <[email protected]
<mailto:[email protected]>> napisał(a):
Hmm - you can certainly generate "MAD" phases using the anom
signal from one data set, and then PARROT or some such density
modification tool to extend those phases for the higher
resolution reflections..
Or ACORN can work well if he data resolution is high enough to
give good phases for all the data
Then you can use those phases in the initial refinement procedure
- the usual idea is to use them till the R factor drops below 30%
or 35% then just refine against the Fobs, phasing just from the
model .
But I dont think it is ever worth working at a limited resolution..
eleanor
On Thu, 3 Jan 2019 at 20:40, Piotr Wilk <[email protected]
<mailto:[email protected]>> wrote:
Dear Eleanor,
thank you for your comment. My crystals of interest diffract
with dmin usually between 1.4 and 1.9 A (in high energy data
set) and significant anomalous signal extends usually to
approx. 4 A (in low energy data set).
Certainly I do agree, that in "EITHER, OR" situation one can
check both approaches, compare the results and take the more
convincing one. I can easily do that in Refmac running one
job against data with Friedel pairs merged and parallel one
against data with Friedel pairs unmerged. I was considering
rather an "AND" scenario in which in addition to high
resolution data (FP) I'd include information from anomalous
signal (F+ F-, DANO). I understand that this should increase
number of observations from a given sample and therefore help
to refine positions, occupancies and perhaps ADPs for at
least a fraction of atoms in a model (Mn ions and S in my
case). I imagine it as somehow analogical to adding
geometrical restrains derived from very high resolution data
to refinement protocols.
I was wondering first of all if my reasoning is sensible and
if there is an existing protocol to try this?
With kind regards,
Piotrek
czw., 3 sty 2019 o 17:02 Eleanor Dodson
<[email protected]
<mailto:[email protected]>> napisał(a):
I think any decision depends on the resolution of your
two data sets. If they are very different I would choose
the higher resolution one.
If that is the Anom data then I would use the anom signal
at least in the first cycles to improve the phases..
Eleanor
On Thu, 3 Jan 2019 at 14:59, Piotr Wilk
<[email protected] <mailto:[email protected]>>
wrote:
Dear CCP4 experts,
I'd like to ask your opinion about using anomalous
signal in refinement of crystal structures in
addition to using high resolution native data.
I am working on a series of structures for which I
have collected two data sets (from the same crystal):
1 - native with higher resolution
2 - anomalous at MN absorption edge peak.
The structures were solved with MR and preliminary
refinement using the native data only yields decent
statistics, but I also use anomalous data to verify
presence and position of manganese ions. For this I
used ANODE which lists four strong peaks (~30 sigma)
as expected for manganese ions and around 45 weaker
peaks (~9-5 sigma) for sulfur atoms in Cys and Met. I
am happy to use this information in model building
but I was also wondering if (and how) beneficial
would it be to use both high resolution structure
factors and somehow lower resolution yet highly
specific anomalous signal in the same round of
refinement?
In Refmac5 I can use either refinement with "no prior
phase information" taking FP and SIGFP or "SAD data
directly" with SIGFP F(+) SIGF(+) F(-) SIGF(-), but I
didn't find any "MAD" option to use both.
I have the following columns in my mtz files:
for native data: H K L FP SIGFP FreeRflag
for anomalous data : H K L FP SIGFP F(+) SIGF(+) F(-)
SIGF(-) FreeRflag
or : H K L FP SIGFP DANO
SIGDANO ISYM FreeRflag
I could use CAD to merge the interesting columns into
a single mtz file containing:
H K L FP SIGFP FreeRflag F(+) SIGF(+) F(-) SIGF(-)
DANO SIGDANO
I'd appreciate any comments or advise how to use both
sources of information in the refinement.
I wish you all a Happy New Year.
Kind regards,
Piotrek
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Roberto A. Steiner
Professor of Biomolecular Structure
Randall Centre of Cell and Molecular Biophysics
Faculty of Life Sciences and Medicine
King's College London
[email protected] <mailto:[email protected]>
Phone 0044 20 78488216
Fax 0044 20 78486435
Room 3.10A
New Hunt's House
Guy's Campus
SE1 1UL
London
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