Dear John,
Thank you for your message. I do realise that what I wrote may
have sounded like a "categoric blanket endorsement of XDS". Perhaps I
should have slept on my draft message a little longer, but James's
long e-mail made me wake up and it seemed appropriate to say what I
wanted to say, in the form I had written it, withouth further delay.
My main point was to try and shake people out of habits that
are, in part at least, linked to the use of integration programs still
based on a 2D analysis of diffraction images, and to getting misled
into thinking that fine slicing doesn't help because it doesn't help
these programs. What I most wanted to put across is that the merits of
fine slicing and low-exposure, high-multiplicity collection protocols
will emerge faster if people are encouraged to evaluate the data they
yield through processing with XDS - hence my strong endorsement of it
in this context.
I do not consider XDS to be beyond perfectibility by any means,
but I was reluctant to make my e-mail longer by analysing its possible
improvements. Once its use has spread enough to give users of Pilatus
detectors the best data they can hope for in the current state of the
art, then its limitations will make concrete sense to enough people
for their discussion to move spontaneously to the top of the agenda.
It is indeed because I think there is so much scope for improvement on
the current diffraction image processing software, including XDS, that
I have been strongly (some would perhaps say: stridently) advocating
the deposition of raw diffraction image data, as both an incentive and
a testing ground for such developments in the future.
With best wishes,
Gerard.
On Sun, May 19, 2013 at 08:01:34PM +0100, John R Helliwell wrote:
> Dear Gerard,
> Thank you for sharing these extensive details which I feel sure everyone
> will appreciate.
> Just one aspect I wondered about namely your categorical blanket
> endorsement of XDS. Indeed a very fine program and eg most recently
> evaluated and discussed at CCP4 2011 I think it was, where it emerged 'the
> winner'. You probably guess though that I am thinking of our mutually
> emphasized point that one key reason for raw diffraction data images
> availability is to see such software improve. Is XDS already perfection? Is
> its use by users already guaranteed to yield processed data as good as it
> can get?
> Greetings,
> John
> Prof John R Helliwell DSc
>
>
> On Thu, May 16, 2013 at 6:03 PM, Gerard Bricogne
> <g...@globalphasing.com>wrote:
>
> > Dear James,
> >
> > A week ago I wrote what I thought was a perhaps excessively long and
> > overly dense message in reply to Theresa's initial query, then I thought I
> > should sleep on it before sending it, and got distracted by other things.
> >
> > I guess you may well have used that whole week composing yours ;-) and
> > reading it just now makes the temptation of sending mine irresistible. I am
> > largely in agreement with you about the need to change mental habits in
> > this
> > field, and hope that the emphasis on various matters in my message below is
> > sufficiently different from yours to make a distinct contribution to this
> > very important discussion. Your analysis of pile-up effects goes well
> > beyond
> > anything I have ever looked at. However, in line with Theresa's initial
> > question, I would say that, while I agree with you that the best strategy
> > for collecting "native data" is no strategy at all, this isn't the case
> > when
> > collecting data for phasing. In that case one needs to go back and consider
> > how to measure accurate differences of intensities, not just accurate
> > intensities on their own. That is another subject, on which I was going to
> > follow up so as to fully answer Theresa's message - but perhaps that should
> > come in another installment!
> >
> >
> > With best wishes,
> >
> > Gerard.
> >
> > --
> > On Tue, May 07, 2013 at 12:04:33AM +0100, Theresa Hsu wrote:
> > > Dear crystallographers
> > >
> > > Is there a good source/review/software to obtain tips for good data
> > collection strategy using PILATUS detectors at synchrotron? Do we need to
> > collect sweeps of high and low resolution data separately? For anomalous
> > phasing (MAD), does the order of wavelengths used affect structure solution
> > or limit radiation damage?
> > >
> > > Thank you.
> > >
> > > Theresa
> > --
> >
> > Dear Theresa,
> >
> > You have had several excellent replies to your question. Perhaps I
> > could venture to add a few more comments, remarks and suggestions, which
> > can
> > be summarised as follows: with a Pilatus, (1) use fine slicing, (2) use
> > strategies combining low exposure with high multiplicity, and (3) use XDS!
> >
> > As the use of Pilatus detectors has spread widely, it has been rather
> > puzzling to come across so many instances when these detectors are misused,
> > sometimes on the basis of explicit expert advice that is simply misguided.
> > A
> > typical example will be to see images collected on a Pilatus 6M with an
> > image width of 1 degree and an exposure time of 1 second. When you see
> > this,
> > you know that there is some erroneous thinking (or habit) behind it.
> >
> > When talking to various users who have ended up with such datasets,
> > and
> > with people who advocate this kind of strategy, it seems clear that a
> > number
> > of irrational concerns about fine-slicing and
> > low-exposure+high-multiplicity
> > strategies have tended to override published rational arguments in favour
> > of
> > those strategies: there is a fear that if the images being collected do not
> > show spots discernible by the naked eye to the resolution limit that is
> > being aimed for, the integration software will then somehow not be able to
> > find those spots in order to integrate them, and the final data resolution
> > will be lower than expected. Perhaps this may be of concern in relation
> > with
> > the use of some integration programs, but if you use XDS, which implements
> > a
> > full 3D approach to image integration, this is simply not the case: XDS
> > will
> > collect all the counts belonging to a given reflection, whether those
> > counts
> > are all from a spot on a single 1-degree image exposed for 1 second, or
> > from
> > 10 consecutive images of 0.1 degree width exposed for 0.1 second each, or
> > from 100 images obtained by grouping together the same 10 images as
> > previously collected in 10 successive passes with a 10-fold attenuated
> > beam.
> > The hallmark of the Pilatus detector is to lead to equivalent signal/noise
> > ratios for the last two ways of measuring that reflection, because it is a
> > photon counter and has zero readout noise: therefore the combination
> > Pilatus+XDS is a powerful one.
> >
> > What is different between these three strategies, however, is the
> > quality of the overall dataset they will produce. There is nothing new in
> > what I am describing below: it is all in the references that Bob Sweet gave
> > you in his reply, or is an obvious consequence of what is found in these
> > references.
> >
> > In case 1 (1-degree, 1 second - "coarse slicing") you would presumably
> > also be (mis-)advised to use a strategy aiming at collecting a complete
> > dataset in the minimum number of images. These strategies used to make
> > sense
> > in the days of films, of image plates, and even of CCDs because of the
> > image
> > readout noise, but they have no place any longer in the context of Pilatus
> > detectors. First of all, using 1-degree image widths can only degrade the
> > precision with which 2D spots on images are lifted to 3D reciprocal space
> > for indexing, and hence worsen the quality of that indexing and therefore
> > the accuracy with which the spot locations will be predicted (unless you
> > carefully "post-refine") - then the integration step perhaps does need to
> > "hunt" for those spots locally, and needs them to be somewhat visible.
> > Secondly, 1 degree is usually greater than the angular width of a typical
> > reflection: the integration process will therefore pick up more background
> > noise (variance) than it would have done with a smaller image width.
> > Thirdly, by collecting only enough images to reach completeness you will
> > have substantial radiation damage in your late images compared to the early
> > ones (if you don't, it means you have under-exposed your crystal) and will
> > therefore end up with internal inconsistencies in your dataset, as well as
> > perhaps some extra, spurious anisotropy of diffraction limits as a result
> > of
> > having to impose increasingly stringent resolution cut-offs in the later
> > images. This will affect the internal scaling of that dataset and the final
> > quality of the merged data.
> >
> > In case 2 (0.1 degree, 0.1 second - "fine slicing") you will have a
> > more precise sampling of the 3D shape of each spot, hence more accurate
> > indexing and prediction of spot positions if you use a genuinely 3D
> > integration program like XDS. Thanks to that increased precision, spots can
> > be integrated "blind", even if they are not terribly visible in the images,
> > and the same number of photons will be collected with no penalty in terms
> > of
> > noise level, thanks to the photon-counting noiseless-readout nature of the
> > Pilatus detector. An improvement will be that the finely sampled 3D shape
> > of
> > the spots will be used by XDS to minimise the impact of background variance
> > on the integrated intensities. On the other hand, the differential
> > radiation
> > damage between early and late images will still be the same as in case 1 if
> > you have chosen one of those old-style strategies (and associated beam
> > intensity setting) that aim at just about exhausting the useful lifetime of
> > the crystal by the time you reach completeness.
> >
> > In case 3 (like case 2, but collecting n times more images with an
> > n-fold attenuated beam once you have collected a few "characterisation
> > images" without that attenuation to carry out the initial indexing) you
> > still have the two advantages of case 2 (the same total number of photons
> > will be picked up by XDS, even if the individual images are now so weak
> > that
> > you can't see anything) but you are spreading the radiation damage so
> > thinly
> > over multiple successive complete datasets that you can choose to later
> > apply a cut-off on image number at the processing stage, when the
> > statistics
> > tell you that diffraction quality has become degraded beyond some critical
> > level. This is much preferable to having to apply different resolution
> > cut-offs to different images towards the end of a barely complete dataset,
> > as in cases 1 and 2. The impact of radiation damage will be quite smoothly
> > and uniformly distributed across the final unique reflections, and your
> > scaling problems (as well as any spurious anisotropy in your diffraction
> > limits) will be minimised.
> >
> >
> > This is becoming quite a long message: you can see why I included a
> > summary of it at the beginning! Returning to it for a conclusion: Pilatus
> > detectors, fine-slicing with low-exposure and high-multiplicity strategies,
> > and XDS are a unique winning combination. If fears that another integration
> > program may not perform as well as XDS on fine-sliced data make you feel
> > tempted to revert to old-fashioned strategies (case 1) because it
> > supposedly
> > makes no difference: resist the temptation! Switch to those Pilatus-adapted
> > strategies and to XDS, and enjoy the very real difference in the results!
> >
> >
> > With best wishes,
> >
> > Gerard
> >
> > and colleagues at Global Phasing.
> >
> > --
> > On Tue, May 07, 2013 at 12:04:33AM +0100, Theresa Hsu wrote:
> > > Dear crystallographers
> > >
> > > Is there a good source/review/software to obtain tips for good data
> > collection strategy using PILATUS detectors at synchrotron? Do we need to
> > collect sweeps of high and low resolution data separately? For anomalous
> > phasing (MAD), does the order of wavelengths used affect structure solution
> > or limit radiation damage?
> > >
> > > Thank you.
> > >
> > > Theresa
> >
> > --
> >
> > ===============================================================
> > * *
> > * Gerard Bricogne g...@globalphasing.com *
> > * *
> > * Global Phasing Ltd. *
> > * Sheraton House, Castle Park Tel: +44-(0)1223-353033 *
> > * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 *
> > * *
> > ===============================================================
> >
>
>
>
> --
> Professor John R Helliwell DSc
--
===============================================================
* *
* Gerard Bricogne g...@globalphasing.com *
* *
* Global Phasing Ltd. *
* Sheraton House, Castle Park Tel: +44-(0)1223-353033 *
* Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 *
* *
===============================================================
