[ccp4bb] how to apply non-protein crystallographic symmetry
I have a pdb file for a non-protein having C 1 2/c symmetry. PyMol can't recognize the spacegroup. Can someone recommend software that can apply the crystallographic symmetry to give the full structure? Richard Gillilan MacCHESS Cornell University
Re: [ccp4bb] how to apply non-protein crystallographic symmetry
Thanks to all who responded so quickly! Coot worked. Richard On Jul 14, 2014, at 4:33 PM, Tim Gruene wrote: Dear Richard, Coot shouldn't have an issue with non-Sohnke space groups. I used it to build a structure in P-1. If you want to edit the molecule, you could also try ShelXle, available at http://ewald.ac.chemie.uni-goettingen.de/shelx/eingabe.php. Best, Tim On 07/14/2014 10:26 PM, Richard Gillilan wrote: I have a pdb file for a non-protein having C 1 2/c symmetry. PyMol can't recognize the spacegroup. Can someone recommend software that can apply the crystallographic symmetry to give the full structure? Richard Gillilan MacCHESS Cornell University -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] I/sigmaI or I/sigmaI
Hi Qixu, Sorry, I don't read this BB very often and missed your message. I assumed the DEC-1-2003 archives would be online somewhere, but I may be mistaken. I printed out the discussion and stored it in a notebook. So if nobody here can point to online archives, I can scan and post the pages on DropBox. These were discussions by Anthony Duff, Phil Evans, Jim Pflugrath, et al. Richard On Feb 19, 2014, at 10:21 AM, Cai Qixu wrote: Dear Folmer Fredslund, Thanks for your help. Actually, I can only find the archives until APR-30-2003 at the archives. Where is the DEC-1-2003 archives? Regards, Qixu Cai 发件人: Folmer Fredslund folm...@gmail.commailto:folm...@gmail.com 日期: 2014年2月19日 星期三 下午10:09 至: Cai Qixu caiq...@gmail.commailto:caiq...@gmail.com 抄送: CCP4BB@jiscmail.ac.ukmailto:CCP4BB@jiscmail.ac.uk CCP4BB@jiscmail.ac.ukmailto:CCP4BB@jiscmail.ac.uk 主题: Re: [ccp4bb] I/sigmaI or I/sigmaI Dear Qixu Cai, You can find information about where to find the archives here: http://www.ccp4.ac.uk/ccp4bb.php#archives Best regards, Folmer 2014-02-19 14:44 GMT+01:00 Cai Qixu caiq...@gmail.commailto:caiq...@gmail.com: Dear Richard Gillilan, Where to find the archives of Dec 2003? I can only find the archives until 2007 at jiscmail. Thanks. Regards, Qixu Cai 发件人: Richard Gillilan r...@cornell.edumailto:r...@cornell.edu 答复: Richard Gillilan r...@cornell.edumailto:r...@cornell.edu 日期: 2014年2月16日 星期日 上午12:19 至: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK 主题: Re: [ccp4bb] I/sigmaI or I/sigmaI There was an informative discussion on this very topic back in Dec 1-2, 2003 if you browse the CCP4BB archives. Richard Gillilan MacCHESS On Feb 12, 2014, at 6:43 AM, Cai Qixu wrote: Dear all, Does the I/sigmaI in “Table 1” mean for I/sigmaI or I/sigmaI ? Thanks for your answer. Best wishes, Qixu Cai -- Folmer Fredslund
Re: [ccp4bb] I/sigmaI or I/sigmaI
There was an informative discussion on this very topic back in Dec 1-2, 2003 if you browse the CCP4BB archives. Richard Gillilan MacCHESS On Feb 12, 2014, at 6:43 AM, Cai Qixu wrote: Dear all, Does the I/sigmaI in “Table 1” mean for I/sigmaI or I/sigmaI ? Thanks for your answer. Best wishes, Qixu Cai
[ccp4bb] Summary: identifying protein crystals via visible light only
My original question was: Some years ago, I remember hearing about a microscope that used *visible* light combined with some proprietary image processing algorithm to distinguish between protein crystals, salt, and background. I can't remember the company name or researchers involved. Has anyone here heard of this? None of the answers I received sound like what I remember. Nonetheless, there are two companies that apparently offer visible-light technology for recognizing crystals: (a) Tritek: proteincrystalimaging.com/index.php (b) Jan Scientific: (VISEX) see smb.slac.stanford.edu/news/Visex.pdf (their listed products seem to be UV-based, but apparently this one is visible.) Both of these are proprietary commercial products, so it is impossible to know exactly what they are doing. Richard Gillilan MacCHESS
Re: [ccp4bb] fluorescent pedal
We've used CdWO4 crystals for years. They are highly radiation resistant and colorless and give VERY bright fluorescence. Saint-Gobain has them in various forms. In principle you can mix your own CdWO4 from CdNO3 and NaWO4 ... under proper conditions you let the solution digest to form microcrystals. I tried this once, with no success, but it has been done in the literature. Another option is terbium-doped borosilicate glass. Can be bought in first-draw fiber form from a company called Collimated Holes. This material is bright, colorless, can be melted and formed, but photobleaches in bright x-ray beams over time. Of course there is always cutting thin slivers of the plastic fluorescent sheet material everyone uses at beamlines. Not sure where they get that. Richard Gillilan MacCHESS On Feb 14, 2014, at 12:18 PM, Scott Classen wrote: Hi Ronnie, We use YAG discs (Yttrium Aluminum Garnet). We buy small 10mm x 100um or 500um thick discs, break them into shards, glue them to various alignment jigs and they provide a very effective X-ray visualization tool. Our latest supplier is Star Tech Instruments (http://www.startechinstruments.com) There are other suppliers, but it does take some calling around because Google doesn't appear to be very helpful… unless you want 5000 lbs from a supplier in China. Good luck, Scott On Feb 14, 2014, at 7:54 AM, Ronnie wrote: I am trying to find out where I can get the fluorescent material (just a small flat piece) I can glue to the tip of a pin for aligning the X-ray beam of our home source. Does anyone know? Thanks in advance! Ronnie
[ccp4bb] identifying protein crystals via visible light only?
Some years ago, I remember hearing about a microscope that used *visible* light combined with some proprietary image processing algorithm to distinguish between protein crystals, salt, and background. I can't remember the company name or researchers involved. Has anyone here heard of this? Richard Gillilan MacCHESS Cornell
Re: [ccp4bb] archival memory?
This is too funny. My wife's new job is scanning Sumerian clay tablets into computer. I kid you not. On Dec 12, 2012, at 5:35 PM, Laura Spagnolo wrote: I would definitely go for babylonian clay... On Dec 12, 2012, at 10:31 PM, Adrian Goldman wrote: I say write them out onto acid-free paper: should be good for at least 300 years without active management, if there is no fire. If that doesn't work, I believe babylonian clay tablets have an even longer expected life time…. Dale, I must say I am impressed… I gave up after the exabyte to DAT transition, and decided that if I really wanted to get data sets from (my) old projects, it would be easier to regrow the crystals… Adrian On 13 Dec 2012, at 00:22, Dale Tronrud wrote: I don't believe there is a solution that does not involve active management. You can't write your data and pick up those media 25 years later and expect to get your data back -- not without some heroic effort involving the construction of your own hardware. I have data from Brian Matthews' lab going back to the mid-1970's and those data started life on 7-track mag tapes. I've moved them from there to 9-track 1600 bpi tapes, to 9-track 6250 bpi tapes, to just about every density of Exabyte tape, to DVD, and most recently to external magnetic hard drives (each with USB, Firewire, and eSATA interfaces). The hard drives are about five years old and so far are holding up. Last time I checked I could still read the 10 year old DVD's. I'm having real trouble reading Exabyte tapes. Write your data to some medium that you expect to last for at least five years but anticipate that you will then have to move them to something else. Instead of spending time working on the 100 year solution you should spend your time annotating your data so that someone other than you can figure out what it is. Lack of annotation and editing is the biggest problem with old data. Dale Tronrud P.S. If someone needs the intensities for heavy atom derivatives of Thermolysin written in VENUS format, I'm your man. On 12/12/2012 1:57 PM, Richard Gillilan wrote: Better option? Certainly not TAPE or electromechanical disk drive. CD's and DVD's don't last nearly that long and James Holton has pointed out. I suppose there might be a cloud solution where you rely upon data just floating around out there in cyberspace with a life of its own. Richard On Dec 12, 2012, at 4:41 PM, Dale Tronrud wrote: Good luck on your search in 100 years for a computer with a USB port. You will also need software that can read a FAT32 file system. Dale Glad I didn't buy a lot of disk drives with Firewire Tronrud On 12/12/2012 1:02 PM, Richard Gillilan wrote: SanDisk advertises a Memory Vault disk for archival storage of photos that they claim will last 100 years. (note: they do have a scheme for estimating lifetime of the memory, Arrhenius Equation ... interesting. Check it out: www.sandisk.com/products/usb/memory-vault/ and click the Chronolock tab.). Has anyone here looked into this or seen similar products? Richard Gillilan MacCHESS Dr Laura Spagnolo Institute of Structural Molecular Biology University of Edinburgh Room 506, Darwin Building King's Buildings Campus Edinburgh EH9 3JR United Kingdom T: +44 (0)131 650 7066 F: +44 (0)131 650 8650 http://www.biology.ed.ac.uk/research/institutes/structure/homepage.php?id=lspagnolo laura.spagn...@ed.ac.uk -- The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.
[ccp4bb] archival memory?
At risk of lengthening an already silly, off-topic discussion ... there have been good arguments here in the past on why re-processing archived data can yield important new information. But in the very long run I think, protein structures will be all figured out and new technology will allow for true snapshots of non-periodic structures etc. ... I hope. So the value of the scientific data itself will ultimately diminish. What will not diminish is the historical and cultural information associated with scientific research: email, personal notes, powerpoint presentations, reviewer comments, proposal and paper rejections, equipment lists and prices, etc. These cannot be reproduced in the future (spouse of archaeologist talking here). So it is for this kind of data that long-term storage may be most important ... and should not be overlooked. Reminds me of a recent issue of Archaeology magazine that featured efforts to reverse engineer the 6502 microprocessor chip that many of us knew and loved in the late 70's and early 80's. Believe it or not, the designs have been lost, so a team resorted to excavating a chip using modern microscopy technology (www.archaeology.org/1107/features/mos_technology_6502_computer_chip_cpu.htmlhttp://www.archaeology.org/1107/features/mos_technology_6502_computer_chip_cpu.html). Just for interest: the sumerian tablets, some of the oldest written language, are actually packing slips. One would break open the clay wallet to see what the package was actually supposed to contain. Mundane lists of mail-order stuff back then, but valuable information today. Those which survive unbroken were probably from stolen shipments. Funny twist of fate. Richard On Dec 14, 2012, at 10:41 AM, Artem Evdokimov wrote: In terms of information density clay, paper and suchlike are not likely to be competitive with any modern storage device. However, if there really is a serious need to store relatively large amounts of information for a really long time I see no issues with laser (or afm) engraving on thin gold layers,deposited on top of e.g. fused sio2 and then counter-deposited or sandwiched between layers of some equally tough subtance e.g. the same fused silica or sapphire, etc. The assembly is mechanically stable, oxidation is not an issue and provided that gold feature size is kept reasonably large (say 50 microns per pixel) I estimate the practical read limit (noncontact optical readout) to be in the hundreds of thousands of years as long as the assembly is protected from air convection and weather by a suitable enclosure e.g. a modest size mountain. Best of all, information can be stored as words and imagess readout is simple and requires nothing more than the language to be still there at read time. As we evolve towards becoming telepathic quasicorporeal space mushrooms this may become an issue. Alternatively, given that there is no apparent issue with transmitting information across time (not a time machine but a backwards looking time telescope) all of this is unnecessary since we will be able to simply look back in time and see what we need to see. This technology naturally is immediately and profitably abused for nepharious/amusing/creepy purposes. Luckily, as a space mushroom I will have no interest in private momets of Queen Elizabeth's life, no matter how amusing they might be. -artem On Dec 14, 2012 9:20 AM, Richard Gillilan r...@cornell.edumailto:r...@cornell.edu wrote: This is too funny. My wife's new job is scanning Sumerian clay tablets into computer. I kid you not. On Dec 12, 2012, at 5:35 PM, Laura Spagnolo wrote: I would definitely go for babylonian clay... On Dec 12, 2012, at 10:31 PM, Adrian Goldman wrote: I say write them out onto acid-free paper: should be good for at least 300 years without active management, if there is no fire. If that doesn't work, I believe babylonian clay tablets have an even longer expected life time…. Dale, I must say I am impressed… I gave up after the exabyte to DAT transition, and decided that if I really wanted to get data sets from (my) old projects, it would be easier to regrow the crystals… Adrian On 13 Dec 2012, at 00:22, Dale Tronrud wrote: I don't believe there is a solution that does not involve active management. You can't write your data and pick up those media 25 years later and expect to get your data back -- not without some heroic effort involving the construction of your own hardware. I have data from Brian Matthews' lab going back to the mid-1970's and those data started life on 7-track mag tapes. I've moved them from there to 9-track 1600 bpi tapes, to 9-track 6250 bpi tapes, to just about every density of Exabyte tape, to DVD, and most recently to external magnetic hard drives (each with USB, Firewire, and eSATA interfaces). The hard drives are about five years old and so
Re: [ccp4bb] archival memory?
It's an interesting story. Even before 9/11, the tablet collection was the subject of debates on ethics in archaeological circles. Such tablets were sometimes from private collectors and probably obtained from looters on the black market. Reading them (the insides could be x-rayed ... tomography) was thought to increase the market value and so make the looting problem worse. But other scholars argued the importance of knowing the inscriptions despite lack of provenance. The collection here draws scholars from around the world. But then came 9/11. Antiquities like this are thought by some to be involved in money laundering and funding of terrorist organizations. Whether that is true or not I don't know, but the government here takes it seriously. Hence, there is movement to scan everything and repatriate the collection. It's a question of preserving access to information. Richard On Dec 14, 2012, at 10:39 AM, Boaz Shaanan wrote: But why? I thought the idea emanating from the thread was to go the other way around for long time archival, i.e. computer -- Sumerian (or Babylonian) clay. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Richard Gillilan [r...@cornell.edu] Sent: Friday, December 14, 2012 5:20 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] archival memory? This is too funny. My wife's new job is scanning Sumerian clay tablets into computer. I kid you not. On Dec 12, 2012, at 5:35 PM, Laura Spagnolo wrote: I would definitely go for babylonian clay... On Dec 12, 2012, at 10:31 PM, Adrian Goldman wrote: I say write them out onto acid-free paper: should be good for at least 300 years without active management, if there is no fire. If that doesn't work, I believe babylonian clay tablets have an even longer expected life time…. Dale, I must say I am impressed… I gave up after the exabyte to DAT transition, and decided that if I really wanted to get data sets from (my) old projects, it would be easier to regrow the crystals… Adrian On 13 Dec 2012, at 00:22, Dale Tronrud wrote: I don't believe there is a solution that does not involve active management. You can't write your data and pick up those media 25 years later and expect to get your data back -- not without some heroic effort involving the construction of your own hardware. I have data from Brian Matthews' lab going back to the mid-1970's and those data started life on 7-track mag tapes. I've moved them from there to 9-track 1600 bpi tapes, to 9-track 6250 bpi tapes, to just about every density of Exabyte tape, to DVD, and most recently to external magnetic hard drives (each with USB, Firewire, and eSATA interfaces). The hard drives are about five years old and so far are holding up. Last time I checked I could still read the 10 year old DVD's. I'm having real trouble reading Exabyte tapes. Write your data to some medium that you expect to last for at least five years but anticipate that you will then have to move them to something else. Instead of spending time working on the 100 year solution you should spend your time annotating your data so that someone other than you can figure out what it is. Lack of annotation and editing is the biggest problem with old data. Dale Tronrud P.S. If someone needs the intensities for heavy atom derivatives of Thermolysin written in VENUS format, I'm your man. On 12/12/2012 1:57 PM, Richard Gillilan wrote: Better option? Certainly not TAPE or electromechanical disk drive. CD's and DVD's don't last nearly that long and James Holton has pointed out. I suppose there might be a cloud solution where you rely upon data just floating around out there in cyberspace with a life of its own. Richard On Dec 12, 2012, at 4:41 PM, Dale Tronrud wrote: Good luck on your search in 100 years for a computer with a USB port. You will also need software that can read a FAT32 file system. Dale Glad I didn't buy a lot of disk drives with Firewire Tronrud On 12/12/2012 1:02 PM, Richard Gillilan wrote: SanDisk advertises a Memory Vault disk for archival storage of photos that they claim will last 100 years. (note: they do have a scheme for estimating lifetime of the memory, Arrhenius Equation ... interesting. Check it out: www.sandisk.com/products/usb/memory-vault/ and click the Chronolock tab.). Has anyone here looked into this or seen similar products? Richard Gillilan MacCHESS Dr Laura Spagnolo Institute of Structural Molecular Biology University of Edinburgh Room 506, Darwin Building
[ccp4bb] archival memory?
SanDisk advertises a Memory Vault disk for archival storage of photos that they claim will last 100 years. (note: they do have a scheme for estimating lifetime of the memory, Arrhenius Equation ... interesting. Check it out: www.sandisk.com/products/usb/memory-vault/ and click the Chronolock tab.). Has anyone here looked into this or seen similar products? Richard Gillilan MacCHESS
Re: [ccp4bb] archival memory?
Ha ha. Brilliant! Maybe we should just send them up to Svalbard to store with the seeds. On Dec 12, 2012, at 4:38 PM, Artem Evdokimov wrote: Or... (gasp) store a regular USB drive in a freezer, yes? If the relationship between data decay rate and temperature indeed follows the same good old Arrhenius formula then any old USB drive is virtually endless at -80C and safe for human life span at -20 (i.e. kitchen freezer, sans defrost cycles (so pack your USB in some ice packs so defrost doesn't kill it). If this works, feel free to send me money, SanDisk... Artem On Wed, Dec 12, 2012 at 3:02 PM, Richard Gillilan r...@cornell.edumailto:r...@cornell.edu wrote: SanDisk advertises a Memory Vault disk for archival storage of photos that they claim will last 100 years. (note: they do have a scheme for estimating lifetime of the memory, Arrhenius Equation ... interesting. Check it out: www.sandisk.com/products/usb/memory-vault/http://www.sandisk.com/products/usb/memory-vault/ and click the Chronolock tab.). Has anyone here looked into this or seen similar products? Richard Gillilan MacCHESS
Re: [ccp4bb] archival memory?
Better option? Certainly not TAPE or electromechanical disk drive. CD's and DVD's don't last nearly that long and James Holton has pointed out. I suppose there might be a cloud solution where you rely upon data just floating around out there in cyberspace with a life of its own. Richard On Dec 12, 2012, at 4:41 PM, Dale Tronrud wrote: Good luck on your search in 100 years for a computer with a USB port. You will also need software that can read a FAT32 file system. Dale Glad I didn't buy a lot of disk drives with Firewire Tronrud On 12/12/2012 1:02 PM, Richard Gillilan wrote: SanDisk advertises a Memory Vault disk for archival storage of photos that they claim will last 100 years. (note: they do have a scheme for estimating lifetime of the memory, Arrhenius Equation ... interesting. Check it out: www.sandisk.com/products/usb/memory-vault/ and click the Chronolock tab.). Has anyone here looked into this or seen similar products? Richard Gillilan MacCHESS
Re: [ccp4bb] archival memory?
I don't think memory sticks have any internal electrolytics or power supplies. Both USB and FAT32 are widely documented standards in this era, so while they might no longer be supported (FAT32 is already very old), information on how to communicate and decode data will still likely be available. RS232, for example, is now 50 years old and one can still find adapters and software. Richard On Dec 12, 2012, at 4:45 PM, Roger Rowlett wrote: Maybe the memory chips will retain their bits for 100 years, but what about the driver hardware or internal power supply? Anyone had an electrolytic capacitor last for 100 years? Just sayin... I like the image of the USB sticks in the -80 freezer, though. :) ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edumailto:rrowl...@colgate.edu On 12/12/2012 4:38 PM, Artem Evdokimov wrote: Or... (gasp) store a regular USB drive in a freezer, yes? If the relationship between data decay rate and temperature indeed follows the same good old Arrhenius formula then any old USB drive is virtually endless at -80C and safe for human life span at -20 (i.e. kitchen freezer, sans defrost cycles (so pack your USB in some ice packs so defrost doesn't kill it). If this works, feel free to send me money, SanDisk... Artem On Wed, Dec 12, 2012 at 3:02 PM, Richard Gillilan r...@cornell.edumailto:r...@cornell.edu wrote: SanDisk advertises a Memory Vault disk for archival storage of photos that they claim will last 100 years. (note: they do have a scheme for estimating lifetime of the memory, Arrhenius Equation ... interesting. Check it out: www.sandisk.com/products/usb/memory-vault/http://www.sandisk.com/products/usb/memory-vault/ and click the Chronolock tab.). Has anyone here looked into this or seen similar products? Richard Gillilan MacCHESS
Re: [ccp4bb] Izit dye stained crystal
We've found that high PEG concentration seems to compete with dye binding, so I'm not surprised your didn't see good uptake. I doubt the anomalous signal is from the dye ... if you calculate the molar dye concentration you would need to have significant occupancy in the lattice, you'll probably find that the crystals would look almost black ... unless there was a color change. I recall seeing a poster at the annual ACA meeting 3-4 years ago maybe, in which somebody was using polybrominated or polyiodinated aromatics to both dye the crystals and phase the structures at once. In fact, I think we gave the poster an award for that. I don't have my past notes handy to remember, but could look it up if you're interested. Richard Gillilan MacCHESS On Nov 30, 2012, at 4:19 PM, Sarathy Karunan Partha wrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn’t take up the dye well. But, showed nice protein diffraction (home source KCr 2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy XSCALE.INP
Re: [ccp4bb] Izit dye stained crystal
Yes, that's the one I remember! Thanks Richard On Nov 30, 2012, at 7:01 PM, Prince, D Bryan wrote: Are you referring to the I3C magic phasing triangle by any chance? Beck, et al Acta Cryst D 61(?) (2008) is the reference I think. Good luck! Bryan Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Richard Gillilan Sent: Friday, November 30, 2012 5:21 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Izit dye stained crystal We've found that high PEG concentration seems to compete with dye binding, so I'm not surprised your didn't see good uptake. I doubt the anomalous signal is from the dye ... if you calculate the molar dye concentration you would need to have significant occupancy in the lattice, you'll probably find that the crystals would look almost black ... unless there was a color change. I recall seeing a poster at the annual ACA meeting 3-4 years ago maybe, in which somebody was using polybrominated or polyiodinated aromatics to both dye the crystals and phase the structures at once. In fact, I think we gave the poster an award for that. I don't have my past notes handy to remember, but could look it up if you're interested. Richard Gillilan MacCHESS On Nov 30, 2012, at 4:19 PM, Sarathy Karunan Partha wrote: Dear all, We did some Izit dye staining to test our crystal (salt or protein) and we observed that the crystal didn’t take up the dye well. But, showed nice protein diffraction (home source KCr2.2909 A) and we collected a dataset (360 frames, 1o osc, 5 min exposure) on this dye stained crystal. The data collection statistics looks great and most interestingly we saw some anomalous signal for this data (see attached XSCALE.LP). This protein was crystallized in 0.2 M Ca acetate, 30 % PEG 400 pH 4.5. Izit dye is basically methylene blue which contains a sulfur atom (phenothiazine ring) and also has some basic dimethylamio groups. Our protein has many acidic residues that could enhance binding of this basic dye.We think the anomalous signal could be from this dye and the heavy atom search with autoSHARP and Phenix autosol is suggestive of 4 sulfur atoms. Did anyone come across similar situation with using this dye and also welcome any suggestions about using this data for S-SAD phasing. Thanks, Sarathy XSCALE.INP
[ccp4bb] BioSAXS Postdoc position at CHESS
Job opening: Postdoctoral Associate Biological Small-Angle Solution Scattering (BioSAXS) MacCHESS, Cornell High-Energy Synchrotron Source The Macromolecular Diffraction Facility of the Cornell High-Energy Synchrotron Source (MacCHESS) has an opening for a Postdoctoral Associate. Applicants should have a Ph.D. degree in a field relevant to structural biology. Preference will be given to those with experience in x-ray solution scattering on biological systems (SAXS and WAXS). Activities will include developing novel microfluidic lab-on-a-chip methods, in situ SEC, applying state-of-the art algorithms to BioSAXS data (especially as related to handling mixtures of oligomers), developing CryoSAXS technology, time-resolved SAXS using stopped flow methods. Experience in developing hardware and software for automation is desirable. Software development will be done primarily in Python. While MacCHESS postdocs are not required to do general beamline user support, they will be expected to help with the biannual BioSAXS Essentials training course. The successful postdoc will take full advantage of the rich variety of high-profile groups visiting MacCHESS by co-authoring publications and collaborating on research. MacCHESS is a heavily team-oriented environment. Good clear communication skills are a must, including fluency in the English language. This position is expected to be at least a 2-year appointment (renewed after the first year, contingent upon availability of funds and employee performance). Postdoctoral positions at Cornell are limited to no more than 5 years. The starting date is negotiable, but should be as soon as possible. Located on an Ivy League university campus in picturesque upstate New York, the Cornell High-Energy Synchrotron Source (CHESS) serves a worldwide user base of structural biologists, chemists, physicists, and engineers. MacCHESS is an NIH-supported National Resource providing support for structural biology at CHESS. Applications should be submitted at http://academicjobsonline.org/ (posting #1522) and should include a cover letter, a CV, a list of publications, and a detailed summary of research experience and interests. Applicants must arrange to have at least three letters of recommendation uploaded, as per instructions on the academicjobsonline website. For information about the position, contact Dr. Richard Gillilan at r...@cornell.edu. Cornell is an equal opportunity, affirmative action educator and employe
[ccp4bb] Fall BioSAXS beamtime available at MacCHESS
BioSAXS beamtime at CHESS is now available for Fall 2012! Dear BioSAXS users, Beamtime is still available during the Fall running period (Sept 12 - Nov 19) for BioSAXS. To apply for time, please fill out the short online express-mode form at express.chess.cornell.edu/EM_form.phphttp://express.chess.cornell.edu/EM_form.php Under Choice of experimental technique specify Other In the box provided for Special experimental and facility needs type standard BioSAXS. beamline = F2 Please also send me an email when you submit your express mode form: r...@cornell.edumailto:r...@cornell.edu To insure a successful visit, please carefully follow the sample preparation guidelines and other information given on our recently updated BioSAXS web page:www.macchess.cornell.edu/MacCHESS/bio_saxs.htmlhttp://www.macchess.cornell.edu/MacCHESS/bio_saxs.html News -- The F2 station is now dedicated to BioSAXS. You can read about our robotic sample loading system in our recent paper: High-throughput biological small-angle X-ray scattering with a robotically loaded capillary cell, Nielsen, S. S., Moller, M. Gillilan, R. E. (2012). J. Appl. Cryst. 45. We now have dual SAXS/WAXS Pilatus 100K detectors that give us automatic q-space coverage from 0.008 - 0.8 inverse Angstroms! Our AKTA Purifier size-exclusion chromatography (SEC) system is available for use at the F2 beamline. If your samples seem to aggregate rapidly after preparation, or you think you may have a mixture, you may want to investigate on-site SEC. Fractions can be collected directly into the same 96-well plates used by the BioSAXS robot. I you are interested, contact me (Richard Gillilan) for details. -- Hope you can join us this Fall! Best Richard Gillilan MacCHESS
[ccp4bb] Fwd: [ccp4bb] Do my SAXS data agree with the crystal structure?
In theory, there should be a simple way to calculate P(r) directly from the crystal structure rather than indirectly from the expected scattering curve. Distribution of pair distances, r^2 weighted. This would remove any ambiguity about choice of Dmax. ... but I can't think of any of the common SAXS programs that do it that way. Clearly, since you have the crystal structure, you know the exact Dmax (the maximum diameter of the object). I would use the maximum atom pair distance from the crystal structure, then add a little bit to account for the width of the atoms and solvation layer. Do this only for the theoretical curve. The Dmax for the solution structure may be different due to conformational fluctuations. Dmax is not a well-defined quantity in reality and has a large error range. I would describe the feature you see in the theoretical P(r) at 70A as a shoulder. Such a feature in a dimer is not surprising because you have two large domains separated by a distance. Do you see it in the monomer P(r)? The fact that it is smoother in the solution data is also not surprising, since you can expect domains to move around on average. Some programs actually attempt to model this kind of disorder by sampling conformation space to see which various conformations best fit the curve. Probably that's more analysis than would be useful to you. Richard On Jun 17, 2012, at 1:11 AM, Xun Lu wrote: Drs.Caldwell, Briggs, and Gupta, Thank you very much for the advices. I regret that I didn't show any figure in the earlier post. Here I've attached a figure showing the data quality and some fittings. Data look OK, right? This question may sound silly, but I just want to make sure. As I said in the earlier post, I tried Crysol. I used the crystal structure (dimer+DNA) as the model, and the fitting was OK, right? In fact, I also tried monomer+DNA as the model (I simply deleted one monomer from the PDB file). This kind of comparison may be meaningless, but I was just curious. I am wondering how people judge whether the fit is good or not. Another question, I tried to generate an envelope from SAXS data using Gasbor and Dammin (people say Dammin is better at protein-DNA complex, although it still uses the same bead for both DNA and protein?). The generated envelope was nothing like my crystal structure. As people have pointed out, protein and DNA scatter differently. SANS is the way to go. So I should give up on modeling SAXS data? I've almost given up, because anyways I have the crystal structure, and SAXS is only a small part of this paper. Thanks, Xun
[ccp4bb] BioSAXS and other postdoc positions open at CHESS
Note: CHESS has multiple postdoc positions open in various areas of x-ray science. I am posting the BioSAXS ad here, but applicants will automatically be considered for all the positions. Job opening: Postdoctoral Associate Biological Small-Angle Solution Scattering (BioSAXS) MacCHESS, Cornell High-Energy Synchrotron Source The Macromolecular Diffraction Facility of the Cornell High-Energy Synchrotron Source (MacCHESS) has an opening for a Postdoctoral Associate. Applicants should have a Ph.D. degree in a field relevant to structural biology. Preference will be given to those with experience in x-ray solution scattering on biological systems (SAXS and WAXS). Activities will include automating BioSAXS data processing at the beamline, developing novel microfluidic lab-on-a-chip methods, applying state-of-the art algorithms to BioSAXS data (especially as related to handling mixtures of oligomers), developing CryoSAXS technology, and modeling macromolecular complexes. Experience in developing hardware and software for automation is desirable. Software development will be done primarily in Python. While MacCHESS postdocs are not required to do general beamline user support, they will be expected to help with the biannual BioSAXS Essentials training course. The successful postdoc will take full advantage of the rich variety of high-profile groups visiting MacCHESS by co-authoring publications and collaborating on research. MacCHESS is a heavily team-oriented environment. Good clear communication skills are a must, including fluency in the English language. This position is a 1-year appointment, renewable for up to 3 years total, contingent upon availability of funds and employee performance. The starting date is negotiable. Located on an Ivy League university campus in picturesque upstate New York, the Cornell High-Energy Synchrotron Source (CHESS) serves a worldwide user base of structural biologists, chemists, physicists, and engineers. MacCHESS is an NIH-supported National Resource providing support for structural biology at CHESS. Applications should be submitted at http://academicjobsonline.org/ (posting #1522) and should include a cover letter, a CV, a list of publications, and a detailed summary of research experience and interests. Applicants must arrange to have at least three letters of recommendation uploaded, as per instructions on the academicjobsonline website. For information about the position, contact Dr. Marian Szebenyi at dm...@cornell.edumailto:dm...@cornell.edu. Cornell is an equal opportunity, affirmative action educator and employer.
Re: [ccp4bb] very informative - Trends in Data Fabrication
On Apr 9, 2012, at 11:47 AM, aaleshin wrote: Thank you Phil, for clarification of my point, but it appears as cheating in a current situation, when an author has to fit a three dimensional statistics into a one-dimentional table. Moreover, many of journal reviewers may never worked with the low-resolution data and understand importance of every A^3 counts. It is not clear to me how to report the resolution of data when it is 3A in one direction, 3.5A in another and 5A in the third. Alex In the very low resolution world of SAXS, the whole idea of resolution is problematic. One can quote the minimum d-spacing (maximum angle) measured, but it is not a useful number to report. People are much more concerned about the quality of the data at maximum d-spacing (lowest angle). Perhaps very low-resolution crystallography is starting to enter this regime as well in which resolution concerns are turned upside down. Granted, SAXS is a heavily averaged experiment which can densely sample q space, but which does not even attempt to produce density. But the point I think that is appreciated in the SAXS community, is that the connection between extent of data in reciprocal space and model features is not simple. Richard Gillilan MacCHESS
Re: [ccp4bb] SAXS workshop
The MacCHESS BioSAXS Essentials minicourse filled up just a few days after we made the announcement over a week ago. Hopefully the overflow will find their way to Brookhaven and other places. To date, there has NOT been a lot of interest among potential organizers in holding such a course at the annual American Crystallographic Association meeting. Given the intense interest in the subject, I would like to know if any of you CCP4 readers would be interested in attending a lecture-only BioSAXS HOWTO workshop at the ACA meeting in Hawaii 2013. Please be realistic, I know many would LIKE to go ... but would you actually be able to go? I would also be interested in hearing from any interested potential organizers. (Please also keep in mind that SAS2012 is in Australia Nov 2012, so that may make a difference in your travel plans). Richard Gillilan chair-elect ACA SAXS SIG MacCHESS Cornell University Ithaca, NY On Feb 6, 2012, at 1:06 PM, Robert Sweet wrote: And here's one from Brookhaven -- app. deadline is three weeks away: http://workshops.ps.bnl.gov/default.aspx?w=SAXSMar2012 http://www.chess.cornell.edu/BioSAXS%20course/index.htm Getting Started in Biological Small-Angle X-ray Solution Scattering Feb 24-26, 2012 Students will have the opportunity to collect data on CHESS beamlines using protein standards and/or their own samples. -- = Robert M. Sweet E-Dress: sw...@bnl.gov Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Biology Dept Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) =
[ccp4bb] BioSAXS beamtime available
Dear BioSAXS users, Beamtime at MacCHESS will be available during the Spring running period (March 7-March 27) for BioSAXS. To apply for time, please submit an online express-mode proposal at http://express.chess.cornell.edu/EM_form.php Under Choice of experimental technique specify Other In the box provided for Special experimental and facility needs type standard BioSAXS. Please also send me an email when you submit your express mode form: r...@cornell.edumailto:r...@cornell.edu To insure a successful visit, please carefully follow the sample preparation guidelines and other information given on our BioSAXS web link: http://www.macchess.cornell.edu/MacCHESS/bio_saxs.html News -- BioSAXS Essentials III, the introductory SAXS minicourse for biologists is open for registration! Have your students apply soon, the course is already rapidly filling up ... only 4 more slots left: http://www.chess.cornell.edu/BioSAXS%20course/Registration/register02242012.html MacCHESS has purchased two Pilatus 100K-S detectors which are dedicated exclusively to BioSAXS. The first detector is now in full-time operation for users at F2 station. We have also purchased an AKTA Purifier size-exclusion chromatography (SEC) system for use at the F2 beamline. If your samples seem to aggregate rapidly after preparation, or you think you may have a mixture, you may want to investigate on-site SEC. If you are interested, contact me (Richard Gillilan) directly for details. -- We look forward to an exciting Spring running period and hope you can join us! Best Richard Gillilan MacCHESS
Re: [ccp4bb] IUCr committees, depositing images
For the record, the amount of disk storage space per unit cost has doubled every 14 months for the last 30 years. It's an exponential relationship: www.mkomo.com/cost-per-gigabyte So data generated at a very high rate today, will be trivial to store in the near future. That's not to say it is cost free, of course ... but exponentially approaching free. I worked at a Supercomputing facility for 7 years. At that time whole rooms were filled with state-of-the-art tape archive robots that could hold an unimaginable amount of data: a whole terabyte. Today, of course, that same volume costs under 100 USD with much faster I/O ... and I have personal copies of everything I generated (even digitized, uncompressed analog video). To keep data backed up and online, of course costs something, but distributed/cloud computing is also changing that picture dramatically. I am curious to know: those who have Pilatus 6M, for example. How much data do you generate in a year? I suspect this is limited by beam intensity ... at the moment. Richard On Oct 18, 2011, at 6:52 AM, Chris Morris wrote: Some crystals are hard to make, so storing all the data the best way to get reproducibility. On the other hand, no one needs more images of lysozyme. So using the same standard for every deposition doesn't sound right. The discussion should be held on the basis of overall cost to the research budget - not on the assumption that some costs can be externalised. It is too easy to say you should store the images, in case I want to reprocess them sometime. IT isn't free, nor is it always cheaper than the associated experimental work. The key comparison is: Cost of growing new crystals + cost of beam line time With: Cost of storing images * probability of processing them again At present, detectors are improving more quickly than processing software. Sample preparation methods are also improving. These forces both press downward the probability that a particular image will ever be reprocessed. regards, Chris
Re: [ccp4bb] not so good news (Steve Jobs RIP)
Jobs and Apple not only deeply shaped my career, but also my son's path as well. I bought an Apple II+ with my first Summer's earnings as an undergraduate lab assistant in organic chemistry in the very early 80's. Spent winter break writing a printer driver in Apple 6502 code so I could plot molecular orbitals. Skipped the first Macs when they came out, but returned to Apple when the Performa was popular. I may have been the first to announce on this list that OS X would be based on unix ... and I enthusiastically got a copy as soon as I could get my hands on it. It's true there will never be another Jobs, but I fully expect more great surprises ... the universe is only just getting started. Richard
Re: [ccp4bb] Bicarb at low pH
Hi Jacob, high-pressure cryocrystallography methods may be useful in this case. I copied your question to Chae Un Kim here at MacCHESS and he forwards this suggestion: -- Hi Richard, I think pressure cryocooling might be useful. They may want to check the following reference. JF Domsic et al (2008), Entrapment of carbon dioxide in the active site of carbonic anhydrase II, J. Biol. Chem. 283, 30766-30771 Best regards, chaeun -- On Oct 2, 2011, at 6:22 PM, Roger Rowlett wrote: You can't change the pKa of CO2, which is 6.3. Any attempt to grow bicarb complexes below pH 7.5 will be problematic due to CO2 bubble formation, which may crack the crystal. What we do in these situations is to soak crystals in a cryo solution at a higher pH for as long as practical, then transfer to another, identical solution with bicarbonate fo li and soak. This can be tricky, because crystals may take a long time to pH equilibrate, crack, or dissolve. At pH 7.5 or above, CO2 formation at chemical equilibrium is minimal. At low pH, you can also consider using acetate as a bicarbonate analog. Roger Rowlett On Oct 2, 2011 4:56 PM, Jacob Keller j-kell...@fsm.northwestern.edumailto:j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, I would like to soak my crystals in bicarbonate (a possible substrate), but the crystals have grown--and only grow--in pH 5.2-6.0, so the bicarb/CO2 will just keep evolving out of the solution and reliquishing its hydroxyls until the pH is elevated sufficiently out of range. Does anyone have a clever way of getting bicarb into these crystals? Grow them under CO2? Transfer them to higher pH, and hope for the best? Jacob Keller
Re: [ccp4bb] Mac OSX 10.7 Lion
Listening to Jobs speak recently, I got the distinct impression that the end of the era of general desktop computers PC's may be on the horizon. Of course that's iPad sales rhetoric, but it may be that the public moves away from general computers and that surely will have implications for scientific computing. Richard On Sep 19, 2011, at 5:16 AM, Andreas Förster wrote: I've bitched enough about all things Mac, but this one's just too good to pass on (from the article that Peter linked to): The fundamental issue here is Lion's assumption that you don't know what you're doing, and it's going to ensure you're protected from cock-ups that, in your ignorance, you may make. The first half of that sentence has always been my major gripe with Mac OS. If there just were a Pro version for those that know what they're doing, but the iPadification of computing goes exactly the opposite way. Andreas
Re: [ccp4bb] Off topic_protein degradation.
BioSAXS can also tell you if the protein is folded or not, but in either case, you may want to purify on site since degradation is so fast. Many BioSAXS beamlines (including ours at MacCHESS) now have SEC systems on site. Richard Gillilan MacCHESS Cornell University On Aug 19, 2011, at 3:55 AM, herman.schreu...@sanofi-aventis.commailto:herman.schreu...@sanofi-aventis.com herman.schreu...@sanofi-aventis.commailto:herman.schreu...@sanofi-aventis.com wrote: Dear Bei, The first question to ask is not whether there is spontaneous disulfide bond breakage, but whether the correct disulfide bonds have been made in the first place. Extreme protease sensitivity could point to an unfolded/misfolded protein. If you know some protein NMR people, you could ask them to check. Even a 1-D NMR spectrum could give some information whether the protein is correctly folded or not. Another way to check is to see if your protein has proper enzymatic/biological activity. If this activity is ok, the folding is probably ok as well. You may have a protease contaminant, so you may want to check the purification protocol. The least you could do is to add some protease inhibitors to your crystallization setups. I once added PMSF for this purpose. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of joybeiyang Sent: Friday, August 19, 2011 6:28 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off topic_protein degradation. Dear all, I am trying to crystallize a protein for which the yield and solubility were both fine. However, this protein has a severe problem of degradation. When stored at RT, the protein will degrade madly into pieces, while stored at 4 degree, the degradation is much slower and a relatively stable truncate form can be get. I am going to try to crystallize the protein at 4 degree, however I still want to understand what's going on there at RT because this protein was supposed to be very stable, it is Cys rich, and the 6 Cys were predicted to form 3 disulfide bonds which hold the protein as a globule, how can a protein with 3 stabilizing disulfide bond be fully degraded like this? Is there a possibility of spontaneous disulfide bond breakage at pH 8 ? Another question is I tried limited proteolysis with this protein, however even at 1:1000(w/w, chymotrypsin), the protein is degraded into pieces in about 2 hrs (again, a relatively stable truncated form can be get between 10 min and 30 min). I am wondering how is the crystallization probability correlated with proteolysis stability? Does this phenomenon indicate that the crystallization probability of my protein is pretty low? Any comments would be greatly appreciated. Bei 2011-08-15
[ccp4bb] Fwd: [ccp4bb] Blue color upon X-ray exposure?
I have personally noticed that the blue color only appears when the pH of the hyperquenched solution is higher than 7 or so. I assume this is because solvated electrons react with protons to form their conjugate base: the hydrogen atom. The latter species is highly reactive as well, but it is not colored. ... We see a very strong dark blue in cryoSAXS experiments on lysozyme buffer at pH 4.5 (acetate) containing high glycerol content. We've also seen that color fade to light yellow/brown over time while in the cryostream once irradiation has stopped (I don't recall if that particular solution had protein or not). The blue color appears long before the Henderson limit and does not seem to affect the scattering profile. I believe a similar effect is at work in bottle glass that has been exposed to sunlight for a long time - color centers. Blue ice seen in the far north however, appears to be purely a light-scattering phenomenon and not a result of trapped electrons. Richard Gillilan MacCHESS -James Holton MAD Scientist Todd Geders wrote:
Re: [ccp4bb] decrease of background with distance?
David had developed an empirical theory to model the air, solvent, Compton acoustic contributions and correct the integrated data for these, without background correction of course since the optic DS background was ultimately to be our data! ... Hi Ian, did David publish this theory somewhere? I'd love to get a reference. I'm genuinely confused by this because I thought the whole point of modern focusing optics (or at least the confocal mirror design) is to focus the beam onto (or close to) the sample, in which case wouldn't the photons diverge from the 'virtual source' (actually a real image of the real source) at the crystal, instead of from the real source? So then Bragg spots (and therefore also the acoustic DS) should diverge from the position of this virtual source? Cheers -- Ian This does seem confusing now. Here at CHESS, focus is either at the collimator (just upstream of the sample), or, in the case of capillaries, on the sample. In rare cases, on the detector. True, the focal spot would be a virtual source and, if the spot is reflected by an ideal crystal, that wouldn't change anything. But it is a very well collimated source even though it is slightly divergent (by a few mrad at most) ... this is in contrast to background scatter where photons are emitted in the whole q range (assuming it is that kind of scatter). So maybe it is more accurate to say Bragg reflections and background scatter are two rather different kinds of sources located at the crystal. Pardon my ignorance, but how can lattice phonons be a significant effect at low temperature? I presume the correlated displacements you refer to must be phonon modes frozen in place to create static disorder or something like that ... or perhaps stuff is moving more than I think at 100 K. Richard
Re: [ccp4bb] decrease of background with distance?
Thank you all for your informative responses! While examining the effects of unusual beam profiles on data collection due to capillary optics, I had collected a wedge of data on a large, high-quality lysozyme crystal at 8 different sample to detector distances. I restricted the analysis of all data to the resolution range seen at the largest distance, so each data set contained nearly the same reflections with the same redundancy. I/ sig does indeed improve with distance: starting at 7.89 at 135 mm and reaching its maximum of 10.35 at 614 mm. The total linear R factor for that range also falls from 0.09 down to 0.059. So, even with a 1.8 mrad divergent beam (about as divergent as practical at a beamline), the effect is significant. Surely someone has done this experiment before long ago. I like Jim Plufgrath's way of looking at the problem: reflections and background scatter have different effective source sizes and distances. For a reflection, the source is the same as the beamline source (some place way upstream). For solvent the source is the sample itself, for air, the source is spread out over a range of distances ... thus background is very divergent. Richard
Re: [ccp4bb] Mosaicity beam divergence
Sorry, I meant to say does divergence add to the reported mosaicity value. If so, do actual mosaicity and divergence add in quadrature to give the reported value? On Aug 6, 2009, at 6:14 PM, Richard Gillilan wrote: Does anyone know if beam divergence gets included in the mosaicity values reported by HKL2000? (i.e. does it add to the measured divergence (in quadrature)?) Richard Gillilan MacCHESS
[ccp4bb] Foils for energy calibration
In the past we've used elemental foils from exafsmaterials.com for energy calibration of our MAD beamline. These standards are for EXAFS and XANES. Most are thin (5 micron) metal foils. Has anyone had experience with other sources of standards or other forms (such as compounds rather than pure elements)? I notice that a number of companies offer XRF standard kits. Richard Gillilan MacCHESS
Re: [ccp4bb] Scalepack error model?
Hi Zbyszek, thanks for your reply! The scalepack log file gives the formula: Chi**2 = SUM ( (I - I) ** 2) / (Error ** 2 * N / (N-1) ) ) which equivalent to the Jay Ponder's formula, with the important addition, that sigma_avg and Iavg represent the average of all _other_ measurements with the same reduced hkl index. All sigmas are calculated from the error model described in the publications. So sigma_avg and I_avg in Ponder's formula are values that exclude the I_meas(hkl). That makes sense since we want the distributions of I_avg and I_meas to be independent. I'm still confused about how the Error value is calculated. What we want is the sigma that corresponds to the distribution of I_avg - I_meas values. As Ponder says this is sigma_avg^2 + sigma_meas^2. So I assume that sigma_avg is simply the calculated sigma of ALL the measured I's with the same reduced hkl index (no error model used for this term). The error model comes in with the sigma_meas term. That is calculated by scaling the measured sigma's from the integration with a factor derived from the error model. The error model as given in the International Tables F Eqs. (11.4.8.5-7) is W = 1/[(sig*E1)^2+( I_corr*E2)^2](5) I_corr = SUM (I_corr*W)/ SUM W (6) sig(I) = I/sqrt[SUM W] (7) So, to get sigma_meas above, presumably would use (7) for some I value (which one?). How does one compute the values of I_corr and W ? - Thanks Richard Gillilan MacCHESS Some of the error model parameters are defined at the moment by user, they can be refined iteratively by experimenter by adjusting parameters in subsequent runs of scalepack, but most of the time it is not required. New version will adjust all these parameters automatically. Zbyszek Otwinowski Richard Gillilan wrote: chi-2 = 1/N sum (I_avg - I_meas)^2/(sigma_avg^2 + sigma_meas^2)
[ccp4bb] Scalepack error model?
Does anyone know of a detailed rigorous discussion of how the scalepack error model/Bayesian reasoning works? The scalepack manual has no equations for this. Richard Gillilan MacCHESS
Re: [ccp4bb] Scalepack error model?
Thanks Joe and others. Bits and pieces of this story appear in 11.4.8 of International Tables volume F, Borek et. al. Acta Cryst D59 (2003) and the Scalepack manual, but none are complete or have enough detail to follow easily. None of them give the expression for Chi-square for this problem. I found a presentation by Jay Ponder online (for his Bio5325 course) that gives: chi-2 = 1/N sum (I_avg - I_meas)^2/(sigma_avg^2 + sigma_meas^2) where the sum probably runs over all reflections and the I_avg is the average of the appropriate group of symmetry-related reflections. Sigma_avg^2 should be the sigma computed from the error model below (not given in the presentation) I think and sigma_meas is the sigma^2 from the actual symmetry-related reflections. One would then adjust the error parameters below to give chi-square approx unity and this leads to the proper scaling factors for intensities and sigmas. One confusing hitch seems to be that (according to the International Tables F Eqs.(11.4.8.5) and (11.4.8.6)), the error model is also implicitly defined and must be solved iteratively ... though it's hard to see that from the text. Does this sound right? Richard On Jul 17, 2009, at 4:12 PM, cockb...@pasteur.fr wrote: Dear Richard, I *think* it works like this, don't know if it's detailed or rigourous enough for you! If I(l,h,i) is the intensity of the ith observation of reflection h on frame l, with error sig(l,h,i), and S(l) is the (inverse) scale factor to be applied to frame l, then the error in the scaled intensity I(l,h,l)/S(l) is parameterised in terms of the error scale factor (E1) and estimated error (E2) as sqrt( (E1*sig(l,h,i))**2 + (E2*I(h)*S(l))**2 ) where I(h) is the weighted mean of the scaled intensity values for index h (i.e. the merged, scaled intensity). The reasoning behind this that the errors in the intensities of strong and weak reflections generally arise from different sources. Weak data are noisy, whilst very strong data can often be systematically badly measured, especially in DENZO, which assumes that all your spots are the same size and shape, or overloaded. E1 and E2 thus tend to dominate the error model at high and low resolutions, respectively. Hope that helps, Joe Does anyone know of a detailed rigorous discussion of how the scalepack error model/Bayesian reasoning works? The scalepack manual has no equations for this. Richard Gillilan MacCHESS
[ccp4bb] Fwd: [ccp4bb] structure - function
Begin forwarded message: From: Richard Gillilan r...@cornell.edu Date: June 23, 2009 9:43:20 AM EDT To: Nadir T. Mrabet nadir.mra...@medecine.uhp-nancy.fr Subject: Re: [ccp4bb] structure - function A very interesting question. Stephan Jay Gould was well known for his argument that evolution is contingent on all kinds of factors (like meteor impacts etc.). He argued that if we replayed geologic history with minor tweaks, life might be totally different or not exist at all. Based on these ideas, I think a lot of people will say evolution has no direction. It meanders aimlessly. This is in contrast to idea that life is somehow always improving or becoming more complex or more advanced (some kind of progress). But I think anyone who's used evolutionary programming to solve numerical problems realizes that it can be an optimization process under some conditions that finds best solutions. I think there are certainly situations in which external contingencies are limited and so evolution can be viewed as a process that sorts out best solutions to certain physical problems. If a certain reaction needs to be catalyzed in order for a species to live and reproduce, there may well be only one, or a few good ways to do this ... limitations of fundamental physics. The fold and sequence of the proteins may be different, but they still have to reduce the activation barrier for the reaction, and that involves binding a certain fixed intermediate geometry of a species that has donors and acceptors in fixed locations. Similarly, there are a fixed number of ways objects can achieve locomotion. Creatures evolved to move efficiently in their element to compete for resources have to solve the same basic physical problems. When those problems are simple and have only a few solutions, you have convergence. ps, if you haven't seen some of Karl Sims' evolved virtual creatures videos, they are really worth watching. Astounding in fact: www.karlsims.com/evolved-virtual-creatures.html Richard Gillilan MacCHESS On Jun 22, 2009, at 11:16 AM, Nadir T. Mrabet wrote: Ok, now we can perhaps debate of another problem. With a multiple choice question that has more than one acceptably good answer, is it convergent, or rather independent, evolution? This multiple choice question is open for discussion. Greetings, Nadir Pr. Nadir T. Mrabet Cellular Molecular Biochemistry INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: nadir.mra...@medecine.uhp-nancy.fr
Re: [ccp4bb] Phantom Crystals - a recap
If I understand the idea correctly, I would still expect to see good Bragg spots, but the amplitudes would represent the rotationally averaged protein. This is like the hexagonal water lattice (Ih): there is disorder in how the water molecules are oriented at each lattice point (not really disorder, but more than one choice for orientation), but the structure is solvable and the resulting density is a spatial average where hydrogens appear to be nearly overlapping. I agree that the lattice itself has to be distorted or imperfect for the Bragg spots to go away. It would be interesting to see how much lattice distortion can occur before the spots are gone. Actually I'd like to be able to simulate stuff like this for several reasons. Not sure how to do it other than brute-force building a massive lattice of proteins and applying FFT directly. Maybe separate treatment of structure factor and form factor would be easier. Surely this has been done in the solid state/ small molecule/diffuse scattering literature ... Ideally a system where you can tweak a parameter to go from crystal lattice to solution scattering continuously. Richard I would think that a perfect HCP lattice, no matter the disorder in the organization of the molecules, would lead to Bragg diffraction, albeit of low resolution. The ghost crystals probably consist of very imperfect lattice(s) which fluctuate in their dimensions and kind over space and time. Jacob Keller *** 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: George DeTitta To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, June 23, 2009 12:37 PM Subject: [ccp4bb] Phantom Crystals - a recap Thanks to all who replied regarding experiences with phantom crystals (objects with crystal-like morphologies but NO diffraction). The answers were more fascinating than the original poorly worded inquiry deserved. Here is a recap. The observation of phantoms may be rare but not so rare: a number of people replied with first hand experience. Classes of compounds that may lead to these bad actors: membrane-associated proteins and RNAs. NO diffraction may be interpreted as no OBSERVABLE Bragg diffraction, but beware of behind-the-beamstop diffraction; i.e. a few Bragg peaks that are not typically observed unless care is taken to insure a small beamstop. I think of a mental image as follows. Say proteins are spherically shaped and present as cats’ eyes marbles. You might be able to lay them down in a perfect HCP lattice but rotationally the eyes might point in all directions. The object at macroscopic dimensions would look like a crystal but at atomic dimensions there would be no buildup of scattering from cooperative effect of many atoms at the same lattice spacing. Thanks to all. George George T. DeTitta, Ph.D. Principal Research Scientist Hauptman-Woodward Institute Professor and Chairman Department of Structural Biology SUNY at Buffalo 700 Ellicott Street Buffalo NY 14203-1102 USA (716) 898-8600 (voice) (716) 898-8660 (fax) www.hwi.buffalo.edu
Re: [ccp4bb] structure - function
Some years ago, I produced a short computer-animated video (complete with music and narration) for Jon Clardy's group on chorismate mutase. The e. coli and b. subtilis forms of chorismate mutase catalyze the same claisen rearrangement but have completely different folds and hydrogen bond networks ... convergent evolution I presume. I have a HUGE Quicktime version of the movie digitized from the original master tape, but have not taken the time to burn it to DVD yet. I suppose I should put all my videos on youtube someday. (Also available as VHS tape). Let me know if you would like a version for teaching purposes and I'll see if I can burn a copy. Richard On Jun 22, 2009, at 6:55 AM, Justin Lecher wrote: Hello everxone, I am looking for an example of two proteins where the primary sequence does not show any significant similarities, but which have the same function due their structure? I want to use it to demonstrate that function could not always deduced from sequence alignments, but from structure alignments. Does anyone could give me some good examples? Thanks Justin -- Justin Lecher Institute for Neuroscience and Biophysics ISB3 - structural biochemistry Research Centre Juelich GmbH, 52425 Juelich,Germany phone: +49 2461 61 5385
Re: [ccp4bb] Phantom Crystals
We do see these from time to time with users, but nobody pays attention to them. It once happened to us a number of years ago. They were perfectly good looking lysozyme crystals treated with heavy metal soak. They diffracted fine when fresh, but failed to diffract at all after a couple months of storage. I think Art Weaver studied these kinds of cases years ago using electron microscopy to see if there was any visible problem with the lattice. Check this out: A.J. Weaver, A.W. McDowall, D.B. Oliver, and J. Deisenhofer, J. Struct. Biol., 87 (1992). In Art's case, he was able to extract lattice and packing info from the FFT of the EM images (at 40 A). Perhaps if he had access to a SAXS line, he would have seen Bragg spots. Hard to know. It seems to me that there should always be some anisotropic scattering at very low angles no matter how badly the lattice is distorted. Richard Gillilan MacCHESS BTW, I've also seen and harvested ghost crystals that were bubbles. Folds of skin can look like crystal edges sometimes. Speaking of ghosts, I had a dried out drop that looked just like a statue of Buddha I thought. One of my students came upon one that looked just like a couple of monkeys kissing. The ACA should host a pareidolia contest someday. I’d appreciate it if people could tell me their experiences with what I would call “phantom crystals”, or “ghost crystals”. These are objects that display the seeming morphology of crystals (clear facets, sharp edges) but do not diffract X-rays AT ALL. I would not count objects that diffract to 30 A in this category. I mean objects that don’t show a single Bragg spot.
[ccp4bb] sharp beam focus and radiation damage?
I know that sometimes people like to defocus the x-ray beam at the sample so that the intensity profile is not sharply peaked. I think the rationale is that the sharp peak will cause damage, but contribute few photons to the overall diffraction pattern. Does anyone know of a reference where this phenomenon has actually been tested (or even just used)? Richard
[ccp4bb] How small is a microbeam?
Just an interesting question of semantics that annoyingly comes up from time to time when people are comparing x-ray beam diameters. What counts as microbeam? Of course micro has the precise meaning in SI as being a factor of 10^-6. The problem is that the prefix micro just means extremely small in common usage. The term is used very confusingly everywhere. Take microwaves. Microwaves have wavelengths from 1 millimeter to 1 meter. Go figure. They're just extremely small radio waves. Now I believe that it is more widely accepted that nanofabrication is making objects that are measured in nanometers. So shouldn't microbeams rightly be x-ray beams with diameters measured in microns (i.e. 1 mm and = 1 micron). Of course this makes all crystallography beams microbeams and everything smaller than 1 micron a nanobeam. That won't be popular. I've always called anything smaller than 50 microns microbeam because that's about as small of an aperture-based collimator as we could make. So a user should ask for microbeam if regular collimator is too large. I was always puzzled at the APS habit of calling this minibeam, but it's starting to sound better all the time. But in practice, I think microbeam sometimes means smaller beam than yours. So microbeam used to be 30 microns, 10 or 5, now maybe 1 micron. Pretty soon no microbeam at all. I think maybe I'll stick with small, smaller than usual, and someday extremely small. I'd love to hear people's opinion on the topic. Richard Gillilan MacCHESS
Re: [ccp4bb] long term data backup
$3M/TB clay tablets Probably the only medium that will still be practically readable a few decades from now! Having seen a few up close here on Cornell campus, I recommend you save the archeologists some headache and bake them. At least you can write on both sides. The unfortunate fact is that once there is a black market for them, some journals will refuse to publish the data. ;-) Richard
[ccp4bb] Fwd: more about low order reflections
Since Sacha is having trouble posting directly the list I will forward his latest message since It addresses my second set of questions: Begin forwarded message: From: Alexandre OURJOUMTSEV sa...@igbmc.fr Thank you, Richard, for your questions ! Unfortunately, I failed to pass my mail to the CCP4bb - I discovered that I changed my e-mail address and completely forgot to update my CCP4 subscription ! I asked Pavel Afonine re-send my mail from his address. Coming to your last questions : - yes, eventually the sharper the map signal, the larger amount of low-resolution 'long and low' waves you can take off keeping yet the signal in the map; however, I do not have particular numbers . - yes, in the 1991-year paper there is an example how low- resolution data can be restored from the higher-resolution ones (but still having experimental data is much better that go to a risky business of data recovering) - I am not completely sure if the strongest data are always the lowest resolution one; I think due to the bulk-solvent contribution this is not always the case (I recall the paper by Phillips, 1980 - I think at his plot Fobs goes down near the origin). - yes, having the magnitudes for the whole CONTINUOUS Fourier transform (that means NOT from a crystal but from an aperiodic object) allows one (in theory) to recover unambiguously the phases, that people like Millane, Saxton et all try to do. I hope this helps. Be well ! Sacha
[ccp4bb] Importance of low order reflections?
Several times I have heard that low order (small angle) reflections are more important when solving low-resolution structures. I presume it is more than just a question of obtaining greater number of reflections. Does anyone know why low-order reflections are so important in these cases? Richard Gillilan MacCHESS
Re: [ccp4bb] Importance of low order reflections?
On Feb 20, 2009, at 2:58 PM, James Holton wrote: I made a movie demonstrating the effect of leaving out low-res data on an electron density map once: http://bl831.als.lbl.gov/~jamesh/movies/index.html#lores Very cool movie. Many thanks to those who answered my original question by private email. After reflecting for a while, I realize I should have asked a slightly different question. James, your movie shows nicely that one can still have pretty good density even with data below 6A omitted. I presume there is still a reasonable-looking envelope at that point, though I can't tell from looking. The resolution limit for that set is 1.5A. Now here's my question: suppose you increase the resolution to 1.0A. Can you then cut even more low resolution (say down to 5A) before the density goes bad? I am used to thinking that the envelope shape comes entirely from the low-resolution data. Indeed, a SAXS pattern with data ranging from say 200A to 15A will generate a nice envelope. It occurs to me, however, that this is not the whole story. If you have the molecular coordinates, you have the envelope; and there is lots of high-precision molecular coordinate information encoded in the higher resolution shells. Conventional processing techniques may not work in that regime, but it seems that the information is there. Perhaps someone with expertise in ab initio methods can comment, but I recall hearing that the molecular structure is actually over- determined if one had access to the full molecular transform, so we are always only using bits and pieces of it in crystallography. But to what extent that can, in principle, be abused by leaving out low resolution I would like to know. Richard Gillilan MacCHESS
Re: [ccp4bb] definition of I Sigma I
There was a detailed and useful discussion of this on this list back around Dec 1, 2003. If you search in the archives for I on sig I you will find it. Best Richard On Dec 10, 2008, at 5:14 PM, ANDY DODDS wrote: Hi, does anyone have a definition of I Sigma I please. Any definitions that i have found are not very informative for novices. thanks andy
Re: [ccp4bb] Crystallogrphy today
6.cryo crystallography This is routine. Well maybe there are still some surprises. Consider the case of high- pressure cooling. 8.radiation damage see cryocrystallography, and take lots of vitamin C There may be some surprises here too as one looks at smaller crystals with smaller beams. Just a few other comments. Students starting out now in this field may have the opportunity to work with the next-generation x-ray sources such as FEL's and ERL's. These sources are not just brighter than the current synchrotrons. They will provide fully-coherent, high spectral brilliance x-rays in shorter pulses than are currently available. A number of novel techniques are being proposed that will take advantage of these kinds of beams, but who knows what opportunities wait in the future for the next highly-motivated student. One place to start looking is coherent diffraction imaging ... I think C. Riekel et. al. just published a Phys Rev Letter on this subject. Richard Gillilan MacCHESS
[ccp4bb] Fwd: [ccp4bb] Spooky, moving crystals
I recently had exactly this problem only I caught the crystal frozen in the act of being catapulted out of the loop. I was using a thicker- than usual oil for cryoprotectant and kept seeing empty loops with what looked like long clear hairs attached. Finally, one loop had a graceful arc of frozen oil and on the very tip was the crystal! I wish I have taken a picture of it. There is an optimum magnet strength. To strong and you have problems like this, too weak and you find that users are knocking their base off the mount when they disengage the cryotongs. Especially in confined spaces, weak magnets are a problem. Richard Gillilan MacCHESS On Jul 21, 2008, at 2:39 PM, Edward Snell wrote: This reminded me of a haunted beamline that removed crystals from the loop. You'd loop the crystals up nicely, block the stream, transfer the crystal fast to the goniometer head, unblock the stream then look in the microscope - no crystal! After a few tries (and head scratching) the culprit was discovered to be a Hampton strong Magnetic Base that flipped the crystals out of the loops when you were mounting the pin. It was so strong that the pin 'clicked' onto the magnet. Interestingly, looking below the goniometer head, there was a whole graveyard of dead crystals lying there - many other users had the same problem. The magnet was replaced with a Hampton light magnetic base and the problem went away completely. With the concurrence of the beamline scientist the light base remained with the beamline for many years until a recent upgrade when they replaced the user with a guy called Sam. Of course, no names and no synchrotrons will be revealed ;) Seriously though, 10 to 20 minutes until the sample stops moving sounds very ominous. I may have misunderstood, but it sounds like you are warming the crystals for data collection after cryomounting them? Once cooled they should remain that way. Cheers, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Email: [EMAIL PROTECTED] Telepathy: 42.2 GHz Heisenberg was probably here! -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Kevin Jude Sent: Monday, July 21, 2008 2:20 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Spooky, moving crystals I've seen haunted crystals before - the culprit was indeed with the mounting of the pins in their bases (I was re-using some pins and apparently the adhesive had cracked or otherwise failed). Fortunately I never leave home without a tube of Duco cement and was able to correct the problem in situ. kmj Mark J. van Raaij wrote: Dear all, in a recent synchrotron trip we had a problem with our crystals moving after mounting them onto the goniometer, in some cases they moved out of the beam and even out of the zoomed camera picture - it seemed the pins, upon equilibrating to room temperature, extended. It happened with pre-mounted litho-loops only, not with pre-mounted mitegen loops on the same trip, so one possible cause is different metal allows used in the pins, somehow the mitegen ones being more suitable. We used two-component glue to stick the pins into the metal bases (Spine), so that might be another possible culprit. Perhaps we did not allow sufficient time for the glue to react before freezing into liquid N2 and it continued its reaction upon thawing, somehow pushing the pin a bit out of the base. In this case the difference between litholoops and mitegen loops may have been the thickness of the pins, the latter somehow allowing expansion of the glue along the sides, the former not. In any case, I am wondering if any of you has seen this before, so we know how to avoid it in the future. In some cases, it took 10-20 min. for the crystal to stop moving, which, with the current data collection speed and robotic mounting, is significant. Fortunately, it did not affect our trip too much, as we has sufficient time in the end. Greetings, Mark Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/
[ccp4bb] Structural importance of ordered water?
Direct hydrogen bonds between sidechains are obviously important to structural stability in proteins. From time to time I see cases of water-mediated bonds in which a single water molecule seems to play an important role (sometimes taking the place of a missing ligand atom in an apo structure, for example). But what about larger chains and networks of water? Assuming a structure is high enough in resolution and well-ordered enough to observe such things, has anyone systematically studied the structural importance of multiple water interactions (I do know of a paper by Faerman and Karplus back in 94, but perhaps there is more recent work). Has anyone here ever seen a plausible argument that a chain of several hydrogen-bonded waters enables residue A to interact with residue B, some considerable distance away? I have to say, I am skeptical of arguments based on water positions. Thanks Richard Gillilan MacCHESS
Re: [ccp4bb] alternatives to IZIT
Several people have asked about the concentrations we use for various dyes. In the case of fluorescein, we started with 45mg/ml as a stock solution. This is quite arbitrary and we often found that our initial stock solutions needed to be diluted 1:10 or 1:100 before use. It is more art than science. We also did most of our work actually growing crystals in the dye rather than using as we would IZIT. So the short answer is: you'll have to experiment. You want to inject just enough dye into the crystallization drop to give a mild coloration. It is probably wise to dilute your stock with well solution, then inject the smallest volume possible so as not to dissolve your crystal. Then, over time the drop becomes clear as the protein crystal absorbs and concentrates all the dye. If you put too much dye in the drop, the drop is not as clear and it is harder to distinguish crystals from colored precipitate. We have also seen crystals of dye form under higher concentrations. I am not aware of any salt crystals that are known to take up dye. It is presumed that the dye is absorbed by the protein crystals because of solvent channels (of which salts have none). We observed even very low solvent content crystals to take up dye. A negative result, however, does not rule out protein. We did see a case in which dye prefers to remain in solution rather than in crystal. I believe this was high PEG conc. Under these typical IZIT-like staining conditions, we have not observed any anomalous signal due to heavy atoms. This is presumably because the concentration of dye in crystal is too low. At least, that argues against any dramatic changes in crystal quality as a result of dye. Richard On Jun 16, 2008, at 11:18 AM, Jim Pflugrath wrote: Whatever dye(s) you use, be sure to run some positive and negative controls to see how the dye really works. Jim On Sat, 14 Jun 2008, Mark Del Campo wrote: Before I place an order for some Izit, are there some other dyes I can use to check if I've got a protein crystal? Thanks, Mark
[ccp4bb] MacCHESS on F2 station available
We still have openings available for the MAD station F2 during the month of April. There have been a number of recent improvements to F2, so if you haven't used the station in a while, you might want to check it out. Microbeam optics are available for this line on request. Interested users should contact Kathy Dedrick [EMAIL PROTECTED] Best Richard Gillilan MacCHESS, Cornell Ithaca NY
[ccp4bb] Fit2D?
I've been trying to get a copy of Fit2D for MacOSX, but the esrf web site with the executables has been unavailable for some time. The actual documentation is still online, including mirrors, but not the link to the executable. The author has also not responded. Anyone know what's up with Fit2D? Is it still being supported? Richard Gillilan MacCHESS Cornell
[ccp4bb] ACA Microcrystals Session: deadline approaching
Just a final reminder. The deadline for submitting abstracts (posters and talks) has been extended to January 12 (this Saturday). Students are strongly encouraged to submit abstracts and will be considered for the Etter Student Lecturer Award if invited to speak. There is still room in the schedule for new contributions. Submit your abstract online at this link: www.amercrystalassn.org/ AbsSubmit/ Microcrystallography Session ACA 2008, Knoxville TN (Session 13.14, Thursday June 5) Organizers: Richard Gillilan, Ruslan Sanishvili Frontiers of structural biology are continuously expanding. Membrane proteins, hetero-molecular assemblies, multi-domain proteins, and many other important biological systems only produce microcrystals. Increasingly, synchrotron beamlines are evolving to meet the challenges of obtaining good diffraction data from samples of ever decreasing size. While x-ray optics, background scattering reduction, beam stability, and mechanical design innovations continue to improve, this session will focus on supporting technologies, data collection strategies, and important biophysical questions surrounding the occurrence of microcrystals. What causes crystals to be small? Within larger crystals, how much does diffraction quality vary from spot to spot and how can x-ray microbeam be effectively used to collect the best data? How can we avoid or mitigate radiation damage as a consequence of higher flux densities used with smaller diffracting volumes? The session will also touch upon recent advances in microcrystal recognition, manipulation, and harvesting, especially within the context of high-throughput screening.
Re: [ccp4bb] To bathe or not to bathe.
Oops, sorry. The x axis of the previous plot is actually not resolution, but Q. My bad. Richard On Nov 27, 2007, at 11:26 AM, Richard Gillilan wrote: Just a couple small images that may be of interest. The x scale is resolution in Angstroms, the y scale is intensity (arbitrary units). I don't recall if the plot is corrected for CCD pedestal values, so the difference is not quite as dramatic as x49, still very nice clean background if you are looking for a faint signal. The CCD images below are equal exposures shown with equal contrast settings: HeN2.tiff HeN2imgs.tiff
Re: [ccp4bb] To bathe or not to bathe.
I just noticed this thread. I should make a few comments. We regularly provide microbeam with and without Helium here at MacCHESS. Yes, there are cases in which microbeam can give you good diffraction on large crystals when a larger beam cannot. Just last week we had a user group collecting on column-shaped crystals about 40 microns wide by 200-300 microns long. When exposed using a 150 micron diameter beam, the diffraction patterns were a mess, producing multiple lattices. When they exposed the crystals using a 20 micron beam, they were able to find enough sweet spots with single lattice to give good results. I can only offer anecdotes at this time, but, based on user experiences so far, this is not uncommon at all. I don't usually think of microbeam alone as a significant reduction in background except that one avoids hitting solvent (which is a major source of scatter!). While the beam scatters less air due to its small size, microbeams also can have much increased flux which compensates for the smaller size ... so I don't think there will be less air scatter unless you are just using an aperture with no flux gain. Regarding helium: we regularly use helium at 95K combined with a helium enclosure. The setup is awkward for manual sample mounting, but quite convenient for automounting. The main advantage here is that direct beam scattering with air is nearly eliminated. How important is this effect? It depends upon how much free path there is between the end of the optic (slits, etc) and the beamstop. It also depends upon how small your sample is and how strongly it diffracts. In a carefully-controlled experiment using the identical crystal and orientation range with both nitrogen and helium, I saw a signal-to-noise improvement by over a factor of 3 in the 2.5 Angstrom range and lower. At higher resolution the benefit decreases, but still looks no worse than a factor of 2. This is with a 50 micron crystal illuminated with an 18 micron beam. I am currently working on guidelines for when helium and microbeam are necessary (based on both simulations and explicit measurements). At the present time, my feeling is that crystals below 50 micron can certainly make the extra hassle worthwhile. It really depends upon how badly you want that extra resolution. In the case above, it pushed the resolution from above to below the 2.0 Angstrom mark based on I/SIG. As for radiation damage, I do think it is important that a beam intensity profile be as flat at the top as possible (not a sharp hotspot) .. so defocusing a little may be useful. This is not usually a factor that regular users have any control over. I don't have any hard data on this. Richard Gillilan MacCHESS
Re: [ccp4bb] To bathe or not to bathe.
Hi Juergen, the original calculation was done with I/SIG's from scala. Yes, I am aware of the problems obtaining reliable and meaningful I/SIG with CCD data. I have gone through the exercise of trying to get agreement between scala and scalepack by optimizing error model parameters ... though not yet with this particular dataset. Keep in mind that these are nearly identical datasets (actually the same Bragg reflections), so it is a relative improvement figure and not absolute I/SIG that are relevant here. I had not thought to go as far as density comparison. That's a good idea. Unfortunately the datasets were kept incomplete on purpose to reduce possible radiation damage effects. Richard On Nov 26, 2007, at 11:38 AM, Juergen Bosch wrote: Richard Gillilan wrote: I am currently working on guidelines for when helium and microbeam are necessary (based on both simulations and explicit measurements). At the present time, my feeling is that crystals below 50 micron can certainly make the extra hassle worthwhile. It really depends upon how badly you want that extra resolution. In the case above, it pushed the resolution from above to below the 2.0 Angstrom mark based on I/SIG. Hi Richard, I/SigI based on which program ? Default modes or tweaked by expert ? I would give this particular dataset a chance to be processed by all available programs and then do the comparison, or actualy all the datasets you have with various setups. Should be a nice table comparing program X versus Y and Z with the given data. And if it's SeMet data that would even be better - runnig e.g. Shelx and demonstrating which setup leads to an interpretable electron density. Juergen -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] To bathe or not to bathe.
In our current helium box, there is a total of about 28 mm of beam exposed. 10 mm from the aperture of the optic and 18 mm from sample to beamstop. The 10 mm side working distance is very tight for hand mounting (little room for tongs) and falls just outside the shield stream for cryo. Could probably decrease the 28 mm some, but not below 10 mm I think. I think our standard setup places the collimator ion chamber about 20 mm from sample ... so 30-50 mm may not be far off the mark for typical stations. Usually, it is the virus crystallographers who are fussy about getting the smallest beamstop. Next time, I'll have to whip out a ruler and see what distance makes them happy. It would be interesting to know typical distances for other facilities. Anyone else made these measurements? Richard Gillilan MacCHESS Note the density of air is approximately 1000 times less than a protein crystal. The total scatter for a beam going through a 50 micron thick crystal will be similar to that from 50mm air. Most beamlines will have a path length less than this but nevertheless the air scatter will be significant with small crystals. In principle, with smaller beams one can have smaller beamstops nearer the sample thus reducing the path length through the air. Colin
[ccp4bb] Microcrystallography: Call for Papers
Just a reminder that we are organizing an ACA session this year on microcrystallography in Knoxville TN. Please check out the session description below and consider submitting an abstract if you work in any of the topic areas. Students are strongly encouraged to submit abstracts and will be considered for the Etter Student Lecturer Award if invited to speak. A limited amount of funds will be available for speakers from outside the U.S. Please contact Richard Gillilan for details: [EMAIL PROTECTED] The deadline for online abstract submission is December 15, 2007: www.amercrystalassn.org/AbsSubmit/ Microcrystallography Session ACA 2008 (Session 13.14, Thursday June 5) Organizers: Richard Gillilan, Ruslan Sanishvili Frontiers of structural biology are continuously expanding. Membrane proteins, hetero-molecular assemblies, multi-domain proteins, and many other important biological systems only produce microcrystals. Increasingly, synchrotron beamlines are evolving to meet the challenges of obtaining good diffraction data from samples of ever decreasing size. While x-ray optics, background scattering reduction, beam stability, and mechanical design innovations continue to improve, this session will focus on supporting technologies, data collection strategies, and important biophysical questions surrounding the occurrence of microcrystals. What causes crystals to be small? Within larger crystals, how much does diffraction quality vary from spot to spot and how can x-ray microbeam be effectively used to collect the best data? How can we avoid or mitigate radiation damage as a consequence of higher flux densities used with smaller diffracting volumes? The session will also touch upon recent advances in microcrystal recognition, manipulation, and harvesting, especially within the context of high-throughput screening.
[ccp4bb] What not to publish?
I'm sure many of you have been in this situation before, so I would be interested in your opinion. I'm about to submit a paper containing the structure of a liganded protein. The ligand itself is rather uninteresting, but it induces an important conformational change. I solved a second structure containing another different ligand which induces the same conformational change. Sadly, stereochemical inhomogeneity in the ligand results in poorly defined and ambiguous ligand density, nonetheless, the conformational change is very distinct and well- defined in the omit map (even better than the first ligand). My inclination is to at least mention the results, if not include them in, say, the supplementary materials to the paper. The structure is not technically necessary to the case, but would strengthen the argument. I don't feel that the ligand density is well resolved enough to warrant deposition. How much would you, as a reader, want to see? Crystallization conditions, unit cell, space group? Omit maps with very partially- built ligand? Nothing at all? Richard Gillilan MacCHESS Cornell
