Hello All,
and sorry for the stream of non-software-related posts, but does anybody
have a recipe for how much 1M or concentrated HCl is needed to pH Jeffamine
M-600 down to ~7? It seems that once the amines are protonated, there should
be little buffering strength, so a rough recipe for pH 5-8 should work, I
think.
Jacob
*******************************************
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: j-kell...@northwestern.edu
*******************************************
----- Original Message -----
From: "Thomas Earnest" <tnearn...@lbl.gov>
To: <CCP4BB@JISCMAIL.AC.UK>
Sent: Wednesday, April 22, 2009 6:13 PM
Subject: Re: [ccp4bb] How small is a microbeam?
Nukri, Tassos, et al.,
The microdiffraction work that began and has been ongoing at ESRF, and
continued elsewhere such as SLS, is truly
impressive. More importantly it has provided high-impact structural
biology information. The GMCA-CAT
work is really raising the bar, and it is also great to see that the
team is continuing to develop this and making further improvements
that will benefit the community. With the ESRF upgrade plan to go to
even smaller beams this will certainly be
an area of further development there as well.
So, Nukri, please accept some well-deserved flattery.
- Th
Anastassis Perrakis wrote:
Dear Nukri -
I see, and you are perfectly right. I can also clearly say that we have
never done what you describe at the time, and indeed I first heard of it
last summer by you ;-) What we had found but not in a systematic manner,
was what I quoted, that some small areas have indeed much lower mosaicity
than others.
We can thus conclude, that the poor old field of crystallography methods
still has enough space for innovation and pioneers - I hope a few NIH and
EU people
are reading the bb. ;-)
Best -
A.
On Apr 22, 2009, at 23:54, Sanishvili, Ruslan wrote:
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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*P** **please don't print this e-mail unless you really need to*
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The
Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
*P** **please don't print this e-mail unless you really need to*
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
