Dear all,

     There have been warnings circulating for quite some time now about the
possible impact of SARS-CoV-2 on the cognitive abilities of patients who
have recovered from Covid-19, and I fear that I have reasons to worry about
possibly having fallen victim to these ... .

     I came across a remarkable piece of work published in 

                https://doi.org/10.1016/j.str.2020.01.008

entitled "Experimental Phasing of MicroED Data Using Radiation Damage". I
will follow my notes, in order to ensure that I do not stray even further
from the logic of this paper than I may have done already in writing them
down.

     The subject matter of this article is a famous seven-residue peptide of
exceptional structural stability (which is why it is associated with severe
pathologies), giving radiation-hard microcrystals capable of yielding 1.0
Angs resolution electron diffraction data enabling the solution of this (in
effect, small-molecule) structure by direct methods (PDB code 6CLI) from
such data. A thorough study of radiation damage had been conducted on these
crystals in a previous paper (https://doi.org/10.1016/j.str.2018.03.021),
revealing a detailed picture of site-specific radiation damage affecting in
particular the fully-occupied Zn atom co-crystallised with the peptide.

     The material presented in this new paper is a by-product of the earlier
analysis at 1.0 Angs resolution, whereby two datasets were assembled to
correspond to two substantially different stages of radiation damage (PDB
codes 6CLI and 6CLJ) and were truncated to 1.4 Angs to make direct methods
ineffective. The question is then whether this differential radiation damage
can be exploited as a source of experimental phase information, as has been
done successfully with the so-called RIP method in X-ray crystallography,
the implication being that this could then constitute a generally applicable
method for experimentally phasing electron diffraction data. This ambition
is clearly articulated in the last paragraph of the Introduction: "Here, we
demonstrate that radiation damage from exposure to the electron beam can be
used to solve the phase problem in Micro-ED experiments".

     Enough to get you sitting on the edge of your seat ... .

     The delicate scaling between the two datasets (the first called "low
dose" and the second called "damaged" was accomplished by the DSCA method in
SHELXC so that the difference Patterson showed a single peak, interpreted as
being due to radiation damage on the Zn atom between the two datasets. As we
are in P1 this Zn atom could have been placed at the origin, but for the
sake of comparability, a difference Fourier map was computed by combining
the amplitude differences corresponding to the scaled intensities with the
phases from 6CLI, in which the highest peak corresponded to the position of
the Zn atom in 6CLI. 

     Now the heart of the matter. The maps produced from this single Zn as
sole source of phase information were uninterpretable. Density modification
did not help, presumably because these crystals are close-packed so that
there is no solvent to flatten. The method used instead in order to "solve
the phase problem" (sic) consists in placing atoms in trial positions and
applying some selection criteria to prune out the worst choices. None of
these criteria, however, can dispense with using the wMPE (weighted mean
phase error) from the known structure (6CLI) as a guide. In whatever way it
may be described, therefore, the procedure used seems depends on already
knowing the perfect structure from the previous work at 1.0 Angs resolution.

     This is where I began to question my own sanity, and to wonder whether,
like Rip van Winkle (https://en.wikipedia.org/wiki/Rip_Van_Winkle), I had
somehow fallen into a deep sleep and completely missed a revolution: I was
still clinging to the old-fashioned conception of experimental phasing as
the technique that has the power to produce three-dimensional electron
density maps for macromolecules with total objectivity; now, however, the
new paradigm is clearly that the "experimental" component in experimental
phasing consists in experimenting with the trial placement of atoms while
keeping an eye on the agreement between the corresponding phases and those
for ... the already known structure!

     The problem must obviously lie on my side, since this work has been
published not only in the highly-respected journal "Structure", but even
under the prestigious "Resource" label of this journal, reserved for
articles that are expected "to highlight significant technical advances,
exciting new methods [...] that are of value and interest to the broad
Structure readership and have high impact on the field of structural
biology". 

     I can see that it should indeed have some impact, in the sense of
reassuring Structure readers that if they too happen to have pathologically
stable crystals capable of diffracting to 1.0 Angs resolution (so that, when
truncated to 1.4 Angs resolution, the CC1/2 statistic still has a value of
about 95% in the outer shell), with one fully occupied Zn atom for every 7
amino-acid residues, the light atoms having an average B-factor of about 1.8
Angs^2, then they too can look forward to being able to solve that crystal
structure - on condition of knowing it already, of course.


     Stepping back, though, this makes so little sense to me that I will be
extremely grateful for guidance while I recover from what must have been a
totally asymptomatic Covid-19 episode with, clearly, very serious cognitive
after-effects.


     With many thanks in advance,

                Gerard

########################################################################

To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1