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Topic Discussion: Absorption Correction
SUMMARY:
The
effect of absorption by macromolecular crystals can only be
implicitly taken into account by a purely empirical approach, the
classic interimage scaling.
A significant improvement of this approach involves the use of spherical harmonics
and is now programmed
in some data reduction programs (eg. Scalepack and Scala), which usually leads to significantly improved intensity estimations, especially in
the case of anomalous
scattering.
However, this approach needs to have enough redundancy of
measurements and should be used with care.
Dr. Lothar
Esser (NCI): In continuing the discussion about absorption correction ( of which we had
an excellent introduction by Dr. Dauter ), the following cases where it
might help come to mind:
1) Heavy atom derivatives. Data processing of crystals that contain heavy
atoms with Z >> 16 may be improved by applying an absorption correction. I know
of one case where the resulting phases of a mercury derivative were
substantially improved after an absorption correction was applied.
Similar arguments apply if the native crystal already contains atoms
heavier than sulfur ( i.e. Ca, Fe, Cu, Zn, Se, Mo etc.).
2) MAD data collection. Here the wavelength of the beam is deliberately
finetuned to maximize absorption. Despite the enormous success of SeMet
MAD experiments in that superb density maps were obtained, there is
certainly a chance that a proper absorption correction even further
improves the quality of the data.
However it seems that absorption correction overall is not critical for
obtaining useful data ( with very few exceptions ). So the question should not be if it is time to perform absorption
correction but who should do it and when. Which crystal is likely to
benefit the most from it? Dr. Dauter already pointed out that conventional macromolecular
data collection of highly redundant data, compensates for absorption
phenomena to some extend and that this again is a sink for all sorts of
physical / machine stability related phenomena that lead to the fluctuation of the intensity of symmetry related reflections.
What is the effect of not performing absorption correction of an absorbing
nonspherical crystal ? Well, usually the positions of all atoms but mostly heavier atoms are
effected by it. This leads to a distortion of bond length and angles.
Macromolecular refinement with its heavy use of geometric restraints saves
us here from disaster. Another effect of uncorrected data becomes visible in anisotropic
refinement of heavy atoms: Instead of nice round (isotropic) probability ellipsoids, one might obtain oblong (USfootballshaped) ones.
Again no problem for restrained refinement as it is common practice in
macromolecular crystallography.
So in the end, those people who benefit the most are those who are blessed
with stupendously well diffracting crystals which then allow those
fortunate people to refine atoms without restraints. But this does not mean that you should not give it a try even if you have
only 2.5A data of a highly redundant (!) SeMet MAD data set and see if it
improves your maps. However, as Dr. Dauter stressed, absorption correction in its
current form needs to be applied with good judgment. An improved Rmerge
does not necessarily mean that you improved your data quality [Is it time for an
Rfree(merge) ? ].
One practical aspect: If you use Scalepack to do a semiempirical absorption correction
and you have multiple sets of data, make sure that they all have the same reference zone.
Otherwise, the absorption correction is meaningless. If you have data
collected at Kappa=0 and merge them with data at Kappa=45, don't
try absorption correction, either. BTW Scalepack will not give you a
warning, even if you do it wrong.
Dr.
Zbigniew Dauter (NCI): The most proper way to correct for the effect of the crystal
absorption of Xrays requires the exact measurements of the crystal
dimensions and indexing of its faces as well as the knowledge of the mass absorption
coefficients of its contents. This leads to the analytical absorption correction,
which is sometimes used in small molecule crystallography. The other, empirical method of North and Phillips is based on
the so called phiscan using the fourcircle diffractometer, where the crystal is rotated around the diffraction vector of
one of the reflections and the resulting intensity function applied to rescale other reflections.
Unfortunately, these methods are not applicable to crystals of macromolecules, which have shapes difficult to measure and are
usually surrounded by some other absorbing material, either a capillary or a frozen solvent in the loop. The psiscan is
impossible on the usual singleaxis goniostat and a twodimensional detector.
In fact, the classic interimage scaling, based on a comparison of intensities of the symmetry equivalent reflections, to some
extent mimics the effect of the psiscan method, and implicitly takes into account part of the effect of absorption, as well as
some other effects (for example the varying volume of the crystal irradiated by the Xray beam). This is a purely empirical approach
involving only two parameters (the scale and Bfactor) per diffraction image.
A significant improvement of this approach, which is now programmed
in some data reduction programs (eg. Scalepack and Scala) is based
on the representation of the relative scales of all measured reflections as a threedimensional function expressed in terms
of the linear combination of spherical harmonics of various orders.
With this approach, the scales vary not only as a function of the oscillation angle, but can have more complicated shape in
reciprocal space. This method usually leads to significantly improved intensity estimations, especially in case of anomalous
scattering.
However, this approach should be used with care. The spherical harmonics can be simply treated as Lagrange
coefficients. Therefore, with increased number of such coefficients, the resulting Rmerge will
always be smaller, even if the procedure has no physical meaning. It means that it is pointless to use
spherical harmonics up to the order of 10 if only 10 degrees of data have been collected.
In principle to use this method effectively, the total rotation range should be large, preferably spanning the whole reciprocal
sphere, i.e. it needs to have enough redundancy of measurements, which also depends on the crystal symmetry.
The use of spherical harmonics is a purely empirical approach, taking into account not only absorption, but also various other
phenomena influencing intensities of diffracted reflections. These additional effects include the suboptimal calibration of
the detector, bad (not synchronized) behavior of the Xray shutter,
the uneven motion of the goniostat motors and many others. Unfortunately, it is not easy to deduce this
syndrome from the individual values of harmonic coefficients, and only
Dr. Zbyszek Otwinowski knows how to do it properly...
