Hi Tassos,
Your, Christian Riekel’s and others’ work on introducing practical
microdiffraction in PX was undoubtedly remarkable and there have been
number of publications attesting that. You may be also pleased to
know that Microdiff made its way to APS as well – couple of groups
here have them on their beamlines.
Thomas can correct me if I am mistaken but what I think he meant by
such flattering description as “pioneering”, was not introduction of
microbeams or collecting data with small beam when it could not be
done with larger one. The team I am part of has discussed one very
specific application: Say you have 200 micron crystal. If you scan it
(by collecting diffraction images) with low dose and small (e.g. 5
micron) beam, chances are great that you will find the crystal to
have better ordered region which extends, say, 40 microns. Then you
center this region and collect data not with 5 micron but with 40
micron beam. BTW, turns out such inhomogeniety can be the case not
only in poor crystals but in seemingly excellent ones as well. I know
this approach is not earth-shattering and probably not pioneering
either but I wanted to make sure that it was not confused with the
implementation of the microbeam itself for which all structural
biologists should be grateful to truly pioneering work done at ESRF
little over decade ago.
Cheers,
N.
Ruslan Sanishvili (Nukri), Ph.D.
GM/CA-CAT
Biosciences Division, ANL
9700 S. Cass Ave.
Argonne, IL 60439
Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov <mailto:rsanishv...@anl.gov>
------------------------------------------------------------------------
*From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf
Of *Anastassis Perrakis
*Sent:* Wednesday, April 22, 2009 4:39 AM
*To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
*Subject:* Re: [ccp4bb] How small is a microbeam?
On Apr 22, 2009, at 1:03, Thomas Earnest wrote:
The other use for these ultra-small beams is to illuminate part of a
larger xtal to find the best diffracting (or leat mosaic) regions
and/or to
raster out of the radiation damaged areas.
Indeed. Copying from PNAS, 2003, Steve Gamblin's team reported:
Crystal structure of the retinoblastoma tumor suppressor protein
bound to E2F and the molecular basis of its regulation.
Xiao B, Spencer J, Clements A, Ali-Khan N, Mittnacht S, Broceño C,
Burghammer M, Perrakis A, Marmorstein R, Gamblin SJ.
Structure of the pRbE2F Complex. Crystals of the pRbE2F(409 – 426)
complex grew in a plate-like habit with t ypical dimensions
200x200x10 um3. Repeated attempts at dat a collection f rom
f lash-c ooled cr yst als using synchrotron x-ray sources
were thwarted by very high crystal mosaicity and the resulting data
could not be adequately scaled. Using the same crystals, a data set
was collected by using the microfocus diffractometer on
station ID13 at ESRF (20), currently the only such device installed
at a synchrotron source, utilizing a 10x10 aperture. The diffraction
images from the microdiffractometer displayed much lower mosaicity
and produced a good quality data set on data scaling and reduction.
The structure was solved by molecular replacement and produced
initial electron density maps in which the E2F peptide could be
readily located.
The paper was published in 2003 (PNAS) but the experiment was done in
the spring of 2000, if my memory serves me well, and took about an
hour to complete.
I admit I chose to use this paper not only because I am co-author,
but also because of the fact that this was casually "buried" in that
paper and not much advertised, since it was pretty easy to do.
Gerbhard Shertler's work at ID13 has been surely described very well,
and there are many other examples from over a decade ago, using
micro-beams (5-10 micron sizes)
This way even "large" xtals
can benefit from this.
Nukri should chime in on this point as well since GMCA-CAT is
pioneering
this approach.
I have been out of that business for a decade now and lots has
happened, the microdiffs are at ESRF, SLS and Stanford (or so I think
for the latter) and are in use. Nukri's team has made a fantastic
contribution to this field, as I could witness a year ago at APS and
Id23-2 at ESRF or PX-I at SLS are routinely accessed. I am sure that
this is not all.
Still, to my experience as a user lately, there is loads to be done
with micro-beams, or nano-beams, of the "usual size".
Think of this very simple scenario:
A 50x50x1 micron plate. When the plate in 'edge on' a 1 micron beam
(perfectly centered, for which you would need X-ray based images and
not optical centering) you irradiate a 50x1x1=50 micron^3 volume,
right? If that plate rotates face-on the same bean will irradiate a
1x1x1=1 micron^3 volume.
A 7x7 micron beam at the same time would irradiate 7x7x1=49 micron^3
volume. If you would need a nice uniform exposure the size of the
beam should adapt while rotating the crystal, some sort of accurate
volume reconstruction would be necessary, and all these would make a
huge difference. And I did not
say anything about anisotropy in anomalous scattering (Schiltz M,
Bricogne G. 2008) or kappa's for centering etc etc.
For me the challenge in micro-beams remains to target micro-beams in
needle and plate crystals and adapt size and shape in the rotating
crystal which should be perfectly centered and oriented for the
experiment.
If one can collect data from 1x1x1 micron crystals leaves me
indifferent, since I cannot harvest them and mount them in a loop,
but Jan-Pieter Abrahams and team will be thrilled to hear about it
(see ActaD 63, 656-70, 2007)
Having said that, X-ray data collection from tenths of 1x1x1 in a
"loop" (why do they have to be in a loop again?) can be exciting, but
would be
a 'niche' compared to the bigger needles and plates that are out
there starving for appropriate X-ray attention.
A.
Nave, C (Colin) wrote:
Hi
Yes good data with a micron size beam but, in this case, the path length
was 20- 30 micron.
I presume one would like a complete data set rather than a single or a
few processable images. If the latter, then in principle anything is
possible provided background is minimised and a low dose approach is
taken - as for single particle cryo electron microscopy.
I presume how to do all this will be one of the issues to be discussed
at the workshop (which I am looking forward to).
Regards
Colin
-----Original Message-----
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Sanishvili, Ruslan
Sent: 21 April 2009 22:21
To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] How small is a microbeam?
Hi Jon,
You can indeed get data with 1 micron(ish) beam. See for example
http://journals.iucr.org/d/issues/2008/02/00/wd5082/index.html
Different question is whether there is any benefit in using micron size
beam. It is subject of much work and discussion (e.g.
http://www.nsls.bnl.gov/newsroom/events/workshops/2009/mx/)
<http://www.nsls.bnl.gov/newsroom/events/workshops/2009/mx/%29>
Regards,
Nukri
Ruslan Sanishvili (Nukri), Ph.D.
GM/CA-CAT
Biosciences Division, ANL
9700 S. Cass Ave.
Argonne, IL 60439
Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov <mailto:rsanishv...@anl.gov>
-----Original Message-----
From: Jon Wright [mailto:wri...@esrf.fr]
Sent: Tuesday, April 21, 2009 3:36 PM
To: Sanishvili, Ruslan
Cc: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
Subject: Re: [ccp4bb] How small is a microbeam?
Sanishvili, Ruslan wrote:
.......... Reasons for discriminating
5-10 micron beams (minibeam) from ca 1 micron (microbeam) might have
been not so much their size but what it involved to achieve these
sizes.
Might I ask - do you really get data from 1 micron protein crystals? The
reduction in scattering power (==crystal volume) from 5x5x5 microns to
1x1x1 is 125 and so it seems to present a grand challenge. I had
understood there to be a more fundamental size limit, coming from
radiation damage, which is still several microns for typical proteins.
Do you suggest that ~1 micron sized crystals are no longer exclusively
in the domain of powder diffraction? Millions of crystals working
together to increase the signal does help a lot for such tiny ones :-)
Going back to the original question, with 'nano' instead of 'micro', the
FDA has defined [1] a "100 nm size-range limit of nanotechnology".
Name suggetions for 100nm - 999 nm are most welcome. Are they
"submicron"?
Cheers,
Jon
[1] http://www.fda.gov/nanotechnology/regulation.html
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