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Re: [ccp4bb] How small is a microbeam? |
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CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999Subject: Re: How small is a microbeam? From: Anastassis Perrakis a {- dot -} perrakis {- at -} NKI {- dot -} NL Date: 2009-04-22 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 pRbô°E2F Complex. Crystals of the pRbô°E2F(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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of >> Sanishvili, Ruslan >> Sent: 21 April 2009 22:21 >> To: 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/) >> >> 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 >> rsanishvili@anl.gov >> >> -----Original Message----- >> From: Jon Wright [mailto:wright@esrf.fr] >> Sent: Tuesday, April 21, 2009 3:36 PM >> To: Sanishvili, Ruslan >> Cc: 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 >> This e-mail and any attachments may contain confidential, copyright >> and or privileged material, and are for the use of the intended >> addressee only. If you are not the intended addressee or an >> authorised recipient of the addressee please notify us of receipt >> by returning the e-mail and do not use, copy, retain, distribute or >> disclose the information in or attached to the e-mail. >> Any opinions expressed within this e-mail are those of the >> individual and not necessarily of Diamond Light Source Ltd. >> Diamond Light Source Ltd. cannot guarantee that this e-mail or any >> attachments are free from viruses and we cannot accept liability >> for any damage which you may sustain as a result of software >> viruses which may be transmitted in or with the message. >> Diamond Light Source Limited (company no. 4375679). Registered in >> England and Wales with its registered office at Diamond House, >> Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 >> 0DE, United Kingdom >> >> 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 CCP4bb navigationCCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999 |
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