[ccp4bb] OT: VectorNTI alternatives
Hello, After several years of offering the molecular biology software VectorNTI free to the academic community (their open access program) and building up a huge user base, Invitrogen have suddenly announced that they will no longer renew these free licences and the existing ones will be left to expire within the year. There are heavy renewal fees for anyone wishing to continue use of this software. Can anyone recommend decent alternative PC compatible alternatives? Main uses are construct and primer design, plus simple quick alignments, sequence data analysis etc. The database structure for storing sequences was pretty useful also. Ideally free, otherwise reasonably priced. I've seen CLCbio and Geneious have products, both free and paid. Any experience? Thanks, Darren EMBL Grenoble ps anyone using VNTI might consider a backup of their work by exporting files to .gb format. I don't know if a locked up (expired) version permits this and you will have no notice that it is about to expire.
Re: [ccp4bb] OT: VectorNTI alternatives
Hi Darren, much much easier than VectorNTI is ApE (http://www.biology.utah.edu/jorgensen/wayned/ape/ ), which is multi-platform and very easy to use for simple tasks. Please, could you post a summary of the answers? Thanks, ciao Sebastiano On Jan 28, 2009, at 9:47 AM, Darren Hart wrote: Hello, After several years of offering the molecular biology software VectorNTI free to the academic community (their open access program) and building up a huge user base, Invitrogen have suddenly announced that they will no longer renew these free licences and the existing ones will be left to expire within the year. There are heavy renewal fees for anyone wishing to continue use of this software. Can anyone recommend decent alternative PC compatible alternatives? Main uses are construct and primer design, plus simple quick alignments, sequence data analysis etc. The database structure for storing sequences was pretty useful also. Ideally free, otherwise reasonably priced. I've seen CLCbio and Geneious have products, both free and paid. Any experience? Thanks, Darren EMBL Grenoble ps anyone using VNTI might consider a backup of their work by exporting files to .gb format. I don't know if a locked up (expired) version permits this and you will have no notice that it is about to expire. -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094
[ccp4bb] PhD position in Computational Structural Biology at Cambridge, UK
A PhD student position is available to develop new protein modelling methods which can be applied to drug design. The successful candidate will work in collaboration with the Blundell and Hyvonen groups in the Cambridge University Biochemistry Department and industrial CASE award sponsors Eli Lilly. The optimization of a lead molecule in the early drug discovery process is greatly accelerated by knowledge of the protein-ligand binding mode. However it is not always possible to reliably predict this binding mode starting from crystal structures of related proteins and ligands, due to the conformational flexibility of the binding site. Inferring binding mode becomes particularly hard for systems like GPCRs where there are substantial difficulties in obtaining experimental protein-ligand structures. The project will aim to develop a methodology for predicting binding modes by combining approximate structural knowledge of the target with available ligand activity data. The chosen student will work to extend the Rapper program to use ligand-based pharmacophores as restraints in generating feasible protein conformations, and to validate results on experimental data. Applications should include a covering letter describing relevant research experience to date, a CV, and the names and addresses of two referees. These should be sent to Dr Mako Hyvonen (marko -at- cryst.bioc.cam.ac.uk) by email by 10th February 2009. Paper copies cannot be accepted. This studentship is open to EU nationals only. Limit of tenure: up to 3 years in first instance. See also : http://www.jobs.ac.uk/jobs/ZL434/Computational_Structural_Biology_--_CASE_PhD_Studentship/ Sent on behalf of Prof Tom Blundell and Dr Marko Hyvonen
Re: [ccp4bb] OT: VectorNTI alternatives
I've used serial cloner (http://serialbasics.free.fr/ Serial_Cloner.html) but not ApE, which I hope to try now that I know of it. One thing that serial cloner has going for it is a nice assembly tool that makes construct design much easier. Otherwise it could be a little less clunky in its design. editorial These days there are too many free alternatives for invitrogen to try to pilfer scientists. But I guess many companies are beginning to learn what a broken business model really means. If invitrogen were smart, they'd give their redundant NTI software away to whoever will take it and push to make it a convenient front- end ordering system for their other services which actually do have value beyond the configuration of bits on a hard-drive. (Hint: be like google.) /editorial James On Jan 28, 2009, at 1:14 AM, Sebastiano Pasqualato wrote: Hi Darren, much much easier than VectorNTI is ApE (http://www.biology.utah.edu/jorgensen/wayned/ape/ ), which is multi-platform and very easy to use for simple tasks. Please, could you post a summary of the answers? Thanks, ciao Sebastiano On Jan 28, 2009, at 9:47 AM, Darren Hart wrote: Hello, After several years of offering the molecular biology software VectorNTI free to the academic community (their open access program) and building up a huge user base, Invitrogen have suddenly announced that they will no longer renew these free licences and the existing ones will be left to expire within the year. There are heavy renewal fees for anyone wishing to continue use of this software. Can anyone recommend decent alternative PC compatible alternatives? Main uses are construct and primer design, plus simple quick alignments, sequence data analysis etc. The database structure for storing sequences was pretty useful also. Ideally free, otherwise reasonably priced. I've seen CLCbio and Geneious have products, both free and paid. Any experience? Thanks, Darren EMBL Grenoble ps anyone using VNTI might consider a backup of their work by exporting files to .gb format. I don't know if a locked up (expired) version permits this and you will have no notice that it is about to expire. -- Sebastiano Pasqualato, PhD IFOM-IEO Campus Dipartimento di Oncologia Sperimentale Istituto Europeo di Oncologia via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5094
Re: [ccp4bb] OT: VectorNTI alternatives
Hi Darren, My favourite program for editing sequences (apart from Vector NTI, which I suppose I'm going to have to delete soon), is BioEdit: http://www.mbio.ncsu.edu/BioEdit/bioedit.html It has an old fashioned cluttered interface, but does do sequence editing, translation into proteins, ClustalW alignments and contig assemblies (a bit like ContigExpress in Vector NTI). It opens ABI files for sequencing data, to view the chromatograms. It uses the external programs such as clustalw alignments or cap3 to do the contig assemblies, and its licence doesn't expire! BioEdit is quite impressive, and sometimes I use it instead of Vector NTI, even (honestly!). For storing everything, I put my primers, plasmid sequences, insert sequences in a MySQL database, with an HTML front end I wrote: http://plasmidb.sourceforge.net/ Plasmi::db also has a homespun feel to it, and only works with Firefox, for example (not other browsers). There is a primer designer page, for traditional cloning by restriction digestion etc.. I can't pretend it's in the same league as Vector NTI, though. The data is stored in a non-proprietary format; database tables which can be viewed with either the HTML pages, or MS Excel, for example. I never really believed that Vector NTI was going to stay free (even to universities etc.) for a long time, and I do think that they deserve some money for their (excellent) product. I hope that they can decide on a reasonable pricing scheme though, instead of vacillating between huge sums and nothing. They seem to be heading towards a moderate price nowadays, at least. Mark 2009/1/28 Darren Hart h...@embl.fr: Hello, After several years of offering the molecular biology software VectorNTI free to the academic community (their open access program) and building up a huge user base, Invitrogen have suddenly announced that they will no longer renew these free licences and the existing ones will be left to expire within the year. There are heavy renewal fees for anyone wishing to continue use of this software. Can anyone recommend decent alternative PC compatible alternatives? Main uses are construct and primer design, plus simple quick alignments, sequence data analysis etc. The database structure for storing sequences was pretty useful also. Ideally free, otherwise reasonably priced. I've seen CLCbio and Geneious have products, both free and paid. Any experience? Thanks, Darren EMBL Grenoble ps anyone using VNTI might consider a backup of their work by exporting files to .gb format. I don't know if a locked up (expired) version permits this and you will have no notice that it is about to expire. -- Mark Brooks, IBBMC, UMR8619 - Bâtiment 430, Université de Paris-Sud, 91405 Orsay CEDEX. Tel: 0169157968 Fax: 0169853715 Skype: markabrooks
[ccp4bb] Off-topic: chemical modification on thiol groups
I am looking for a reagent (and vendor) that will irreversible put a raher bulky substituent on a free SH group and that does not react with free amines (or other potential reactive groups present on a protein surface). The connection with crystallography is that it is required for an experiment asked by a referee necessary to confirms or reject a hypothesis that results from a crystal structure. For this experiment it is essential that the reaction is not reversible (so no S-S bond formation). Remy Loris Vrije Universiteit Brussel
Re: [ccp4bb] Off-topic: chemical modification on thiol groups
I am looking for a reagent (and vendor) that will irreversible put a raher bulky substituent on a free SH group and that does not react with free amines (or other potential reactive groups present on a protein surface). The connection with crystallography is that it is required for an experiment asked by a referee necessary to confirms or reject a hypothesis that results from a crystal structure. For this experiment it is essential that the reaction is not reversible (so no S-S bond formation). Any maleimide-based fluorescent dye will be bulky, irreversible and when done right will only go for thiols. Also easy to quantify extent of modification. Dima Remy Loris Vrije Universiteit Brussel
Re: [ccp4bb] Off-topic: chemical modification on thiol groups
Dear Remy, Iodoacetamide might be a good candidate as it alkylates thiol groups and is also used as cysteine protease inhibitor. good luck, hannes - Hannes Uchtenhagen, Ph.D student Karolinska Institutet Center for Infectious Medicine (CIM) Karolinska University Hospital Huddinge, F59 SE-141 86 Stockholm, Sweden CIM: +46-(0)8-585 896 88 MTC: +46-(0)8-524 86216 Mobile: +46-(0)7-36901461 - Original Message - From: Remy Loris relo...@vub.ac.be Date: Wednesday, January 28, 2009 2:47 pm Subject: [ccp4bb] Off-topic: chemical modification on thiol groups To: CCP4BB@JISCMAIL.AC.UK I am looking for a reagent (and vendor) that will irreversible put a raher bulky substituent on a free SH group and that does not react with free amines (or other potential reactive groups present on a protein surface). The connection with crystallography is that it is required for an experiment asked by a referee necessary to confirms or reject a hypothesis that results from a crystal structure. For this experiment it is essential that the reaction is not reversible (so no S-S bond formation). Remy Loris Vrije Universiteit Brussel
Re: [ccp4bb] OT: VectorNTI alternatives
I like BioEdit too. It is PC based, downloadable, and very easy to use. It allows copy-paste of a word or text file, and does alignment, translation, back translation, etc, and more. Fabulous program. I also use Lasergene which has the long standing DNA Star, Megalign, but you have to buy a license. It also requires changing format of files to text and saving with a specific suffix such as .seq which is inconvenient. You cannot copy and paste, and when you see a good alignment, you cannot copy and paste out either. Yong-Fu Li On Wed, Jan 28, 2009 at 7:43 AM, Mark Brooks mark.x.bro...@gmail.comwrote: Hi Darren, My favourite program for editing sequences (apart from Vector NTI, which I suppose I'm going to have to delete soon), is BioEdit: http://www.mbio.ncsu.edu/BioEdit/bioedit.html It has an old fashioned cluttered interface, but does do sequence editing, translation into proteins, ClustalW alignments and contig assemblies (a bit like ContigExpress in Vector NTI). It opens ABI files for sequencing data, to view the chromatograms. It uses the external programs such as clustalw alignments or cap3 to do the contig assemblies, and its licence doesn't expire! BioEdit is quite impressive, and sometimes I use it instead of Vector NTI, even (honestly!). For storing everything, I put my primers, plasmid sequences, insert sequences in a MySQL database, with an HTML front end I wrote: http://plasmidb.sourceforge.net/ Plasmi::db also has a homespun feel to it, and only works with Firefox, for example (not other browsers). There is a primer designer page, for traditional cloning by restriction digestion etc.. I can't pretend it's in the same league as Vector NTI, though. The data is stored in a non-proprietary format; database tables which can be viewed with either the HTML pages, or MS Excel, for example. I never really believed that Vector NTI was going to stay free (even to universities etc.) for a long time, and I do think that they deserve some money for their (excellent) product. I hope that they can decide on a reasonable pricing scheme though, instead of vacillating between huge sums and nothing. They seem to be heading towards a moderate price nowadays, at least. Mark 2009/1/28 Darren Hart h...@embl.fr: Hello, After several years of offering the molecular biology software VectorNTI free to the academic community (their open access program) and building up a huge user base, Invitrogen have suddenly announced that they will no longer renew these free licences and the existing ones will be left to expire within the year. There are heavy renewal fees for anyone wishing to continue use of this software. Can anyone recommend decent alternative PC compatible alternatives? Main uses are construct and primer design, plus simple quick alignments, sequence data analysis etc. The database structure for storing sequences was pretty useful also. Ideally free, otherwise reasonably priced. I've seen CLCbio and Geneious have products, both free and paid. Any experience? Thanks, Darren EMBL Grenoble ps anyone using VNTI might consider a backup of their work by exporting files to .gb format. I don't know if a locked up (expired) version permits this and you will have no notice that it is about to expire. -- Mark Brooks, IBBMC, UMR8619 - Bâtiment 430, Université de Paris-Sud, 91405 Orsay CEDEX. Tel: 0169157968 Fax: 0169853715 Skype: markabrooks
Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac?
Hi Anybody have suggestions for Mac OsX alternatives? Thanks in advance, Mark
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Hi everyone, Thanks for answer my question. Just to add some more notes regarding to my expression system. The insert-vector (pET28) has been sequenced and the his-tag is N-terminal. The anti his-tag WB is positive and the binding buffer's pH is 8.2 (double-checked). I had already experienced the same problem before, which I solved just increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no binding at all. I'm currently running a SDS-PAGE with samples eluted from Talon and let you know the results. All the Best, Fred --- Fred /ccp4bb.l...@gmail.com/ schrieb am *Di, 27.1.2009: * *Von: Fred ccp4bb.l...@gmail.com Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind An: CCP4BB@JISCMAIL.AC.UK Datum: Dienstag, 27. Januar 2009, 22:00 * *Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred *
Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac?
On Wed, 28 Jan 2009, Jovine Luca wrote: On 28 Jan 2009, at 16:02, Mark Collins wrote: Hi Anybody have suggestions for Mac OsX alternatives? Thanks in advance, Mark Hi Mark, The latest version of DNA Strider (1.4) runs just fine on both Tiger and Leopard. For more info, you can contact the author directly at christian.ma...@cea.fr You could also try Christian Biertuempfel's suggestion of pDRAW32: if it works under Wine on Linux, it should work under Wine (or the commercial equivalent Codeweavers Crossover) on OS X as well. Regards, Peter. -- Peter Keller Tel.: +44 (0)1223 353033 Global Phasing Ltd., Fax.: +44 (0)1223 366889 Sheraton House, Castle Park, Cambridge CB3 0AX United Kingdom
Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac?
Hi all, I've recently come across the program PlasmaDNA which seems pretty good for the basics - http://research.med.helsinki.fi/plasmadna/ . It works under Mac OS X and Windows... and probably Wine on Linux too. Cheers, Roger Original Message Subject: Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac? From: Mark Collins mcoll...@convex.hhmi.columbia.edu To: CCP4BB@JISCMAIL.AC.UK Date: Wed Jan 28 15:02:17 2009 Hi Anybody have suggestions for Mac OsX alternatives? Thanks in advance, Mark -- Roger Dodd PhD The Institute of Cancer Research Chester Beatty Laboratories 237 Fulham Road London SW3 6JB UK The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] OT: VectorNTI alternatives
Hello, Thanks for all the emails (only some of which reached the bb). It is clear that I am not alone in feeling aggrieved by Invitrogen suddenly introducing licence fees, having first persuaded us to put our files and time into their free product over several years. Once the thread goes quiet, I'll summarise the suggestions for everyone's benefit. I've tried several packages today but it would be good if suggestions were accompanied by brief strengths and weaknesses. From a first view, there are a number of clunky programs that do the job. And some that are really just viewers that are difficult to use for sequence manipulation. As a lab with hundreds of constructs and primers in our database, we also appreciate the file arrangement/storage as well as the sequence analysis function. Thanks Darren 2009/1/28 Darren Hart h...@embl.fr Hello, After several years of offering the molecular biology software VectorNTI free to the academic community (their open access program) and building up a huge user base, Invitrogen have suddenly announced that they will no longer renew these free licences and the existing ones will be left to expire within the year. There are heavy renewal fees for anyone wishing to continue use of this software. Can anyone recommend decent alternative PC compatible alternatives? Main uses are construct and primer design, plus simple quick alignments, sequence data analysis etc. The database structure for storing sequences was pretty useful also. Ideally free, otherwise reasonably priced. I've seen CLCbio and Geneious have products, both free and paid. Any experience? Thanks, Darren EMBL Grenoble ps anyone using VNTI might consider a backup of their work by exporting files to .gb format. I don't know if a locked up (expired) version permits this and you will have no notice that it is about to expire. -- ** Dr. Darren Hart, Team Leader High Throughput Protein Lab Grenoble Outstation European Molecular Biology Laboratory (EMBL) ** EMBL webpages: http://tinyurl.com/6xdltl http://tinyurl.com/667jrp Email: h...@embl.fr Tel: +33 4 76 20 77 68 Fax: +33 4 76 20 71 99 Skype: hartdarren Postal address: EMBL, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex 9, France **
Re: [ccp4bb] small lines in diffraction pattern
Margriet This looks like stacking or shift disorder which can occur when perfect 3 dimensional order breaks down. For example one can have a situation where the lattice is preserved in 2 dimensions but the planes can slide with respect to one another destroying the order in the 3rd dimension, thereby giving rods in reciprocal space rather than spots. Preservation of order in 1 dimension but with the 1 D lattices shifted with respect to each other gives diffuse planes in reciprocal space. If displacements are non random, than the diffuse scatter might only occur for certain reflections. As sharp spots are also present, it is likely that the 3D order is preserved over much of the sample. To analyse properly one would have to record diffraction over small angular increments (similar to 3D reciprocal space mapping) and perhaps identify the hkl value of reflections which exhibit strong, weak or absent effects of this type. The reason for the behaviour can become clear when the structure is known and weak lattice interactions identified. However, if these effects are preventing you determining the structure then this statement is not particularly useful. If really interested you can of course google for such types of disorders and find examples with similar types of streaking (e.g. in J. Appl. Cryst. (1971). 4, 329 http://journals.iucr.org/j/issues/1971/04/00/a08549/a08549.pdf http://journals.iucr.org/j/issues/1971/04/00/a08549/a08549.pdf ). Alternatively read ancient text books such as Hosemann and Bagchi. Others will be far better at suggesting means of preventing the effects, which I suspect is what you would really like to know! Colin From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Margriet Ovaere Sent: 28 January 2009 13:52 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] small lines in diffraction pattern Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. Has anybody seen this phenomena before and has got an explanation for it please..? Many thanks Margriet Ovaere DIVFONT size=1 color=grayThis e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom /FONT/DIV -- Scanned by iCritical.
Re: [ccp4bb] OT: VectorNTI alternatives
Hi Darren, I can recommend another free tool: pDRAW32 ( http://www.acaclone.com/ ). It runs natively under Windows but I am using it with the emulator wine on Linux. Cheers, christian Darren Hart wrote: Hello, After several years of offering the molecular biology software VectorNTI free to the academic community (their open access program) and building up a huge user base, Invitrogen have suddenly announced that they will no longer renew these free licences and the existing ones will be left to expire within the year. There are heavy renewal fees for anyone wishing to continue use of this software. Can anyone recommend decent alternative PC compatible alternatives? Main uses are construct and primer design, plus simple quick alignments, sequence data analysis etc. The database structure for storing sequences was pretty useful also. Ideally free, otherwise reasonably priced. I've seen CLCbio and Geneious have products, both free and paid. Any experience? Thanks, Darren EMBL Grenoble ps anyone using VNTI might consider a backup of their work by exporting files to .gb format. I don't know if a locked up (expired) version permits this and you will have no notice that it is about to expire. ___ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA ___
Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac?
There is GENtle which has a whole slew of tools associated with it. There are versions for several platforms. http://gentle.magnusmanske.de/ If you're used to Vector NTI, it is pretty similar (and open source for the ambitious). _ Cynthia Kinsland, Ph. D. Director, Protein Production and Characterization Cornell University Core Lab Center Ithaca, NY 14853 On Jan 28, 2009, at 10:02 AM, Mark Collins wrote: Hi Anybody have suggestions for Mac OsX alternatives? Thanks in advance, Mark
Re: [ccp4bb] sticky crystals
Hi Savvas, If the very good suggestions you have already got from the ccp4bb do not help, try crystallization with agarose as an additive. Crystals form inside the very soft gel and they are hold in place by this meshwork. So, they are mechanically protected and do not fall down onto the bottom of the sitting-drop well. A final concentration of 0.1-0.2 % (w/v) agarose is sufficient. When you harvest a crystal cut generously around it with a microtool, pick it up (e.g. using a nylon loop) and do not mind if some agarose comes with it. for example: Biertümpfel, C.; Basquin, J.; Suck, D. Sauter, C. Crystallization of biological macromolecules using agarose gel. Acta Crystallogr D Biol Crystallogr, 2002, 58, 1657-9 PMID: 12351881 Good luck! christian Savvas Savvides wrote: Dear colleagues, we have been growing crystals of a protein complex in sitting-drop geometry that stick to the bottom of the drop remarkably well. It’s as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, loops, all kinds of micro-tools, and pipetting techniques to no avail. I can say at the outset that we have been unsuccessful in growing these crystals in hanging-drops or at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are only able to get crystals from homogeneously glycosylated protein produced in HEK293S/I- cells. In the meantime we are playing with the idea of siliconizing the sitting-drop depressions to alter the crystal/plate interface. But then again, nucleation events on the plastic may be the reason we are getting crystals in the first place. We have also thought of trying microseeding to have more control on nucleation issues. Our protein production is quite limiting and forces us to be very selective with our experimentation. Nonetheless, while we are waiting for fresh material to explore some of these ideas we would like to make the most out of the crystals we have grown thus far. We would therefore very much appreciate any input/ideas on manipulating these crystals for data collection. Best wishes Savvas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of *Katarina Moravcevic *Sent:* Tuesday, January 27, 2009 10:52 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] pseudo translation Hi all, here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K * E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11630 http://www.pctools.com/spyware-doctor-antivirus/ http://www.pctools.com/en/spyware-doctor-antivirus/ * ___ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA ___
Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac?
Hi all, Geneouis also runs on OS X. From my experience (past two years), the program works just fine. You can download the trial at http://www.apple.com/downloads/macosx/math_science/index1.html (second page). Cheers, Yann ir. Yann Sterckx Pre-doctoral student Structural Biology Brussels, Vrije Universiteit Brussel (http://www.structuralbiology.be ) Molecular and Cellular Interactions, Vlaams Instituut Biotechnologie (http://www.vib.be ) VUB - SBB Building E, 4th 5th floor Pleinlaan 2 B-1050 Brussels Belgium e-mail: yann.ster...@vub.ac.be skype: yannsterckx On 28 Jan 2009, at 16:18, Roger Dodd wrote: Hi all, I've recently come across the program PlasmaDNA which seems pretty good for the basics - http://research.med.helsinki.fi/plasmadna/ . It works under Mac OS X and Windows... and probably Wine on Linux too. Cheers, Roger Original Message Subject: Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac? From: Mark Collins mcoll...@convex.hhmi.columbia.edu To: CCP4BB@JISCMAIL.AC.UK Date: Wed Jan 28 15:02:17 2009 Hi Anybody have suggestions for Mac OsX alternatives? Thanks in advance, Mark -- Roger Dodd PhD The Institute of Cancer Research Chester Beatty Laboratories 237 Fulham Road London SW3 6JB UK The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] small lines in diffraction pattern
Hi Margriet It's almost certainly due to diffuse scattering as a result of correlated atomic displacements. See this: http://www.nature.com/nsmb/journal/v1/n2/pdf/nsb0294-124.pdf . Are they lines or sheets, in other words do they appear only on one image, or are they also on adjacent images, i.e. are you looking at a slice through more extensive diffuse scattering? According to the above paper there are at least 3 types of DS: haloes around each Bragg spot (thermal diffuse or acoustic scattering) due to long range displacements correlated over different unit cells (this is very common); DS located along reciprocal lattice planes which results from anisotropic intermolecular displacements correlated over a few unit cells (which is I think what you are seeing), and low-intensity very diffuse background patches (optic scattering), but I don't see much of that. -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Margriet Ovaere Sent: 28 January 2009 13:52 To: ccp...@dl.ac.uk Subject: small lines in diffraction pattern Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. Has anybody seen thisphenomenabefore and has got an explanation for it please..? Many thanks Margriet Ovaere Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] sticky crystals
I second Chris's suggestions. These have worked well for me in the past. You only need a very thin layer of the grease (i.e. keep wiping until its almost completely gone) and it usually has no affect on the crystallization. Jeff On Jan 27, 2009, at 3:51 PM, Christopher Colbert wrote: If you have good and bad crystals in the same drop, I've had success pushing a crummy crystal into a good crystal and having it release that way. Additionally, once I realized this was going to be a long term problem, I started coating the sitting drop depressions with a thin layer of vacuum grease. The crystals just slid right off the grease and I never saw any changes in the diffraction data to suggest the grease was giving me issues. Chris On Wed, 28 Jan 2009, Savvas Savvides wrote: Dear colleagues, we have been growing crystals of a protein complex in sitting- drop geometry that stick to the bottom of the drop remarkably well. It's as if they are glued onto the plastic. This makes crystal handling next to impossible without destroying the crystals. We have tried whiskers, loops, all kinds of micro-tools, and pipetting techniques to no avail. I can say at the outset that we have been unsuccessful in growing these crystals in hanging-drops or at 4 degrees. Deglycosylating the complex also leads to nowhere. In fact, we are only able to get crystals from homogeneously glycosylated protein produced in HEK293S/I- cells. In the meantime we are playing with the idea of siliconizing the sitting-drop depressions to alter the crystal/plate interface. But then again, nucleation events on the plastic may be the reason we are getting crystals in the first place. We have also thought of trying microseeding to have more control on nucleation issues. Our protein production is quite limiting and forces us to be very selective with our experimentation. Nonetheless, while we are waiting for fresh material to explore some of these ideas we would like to make the most out of the crystals we have grown thus far. We would therefore very much appreciate any input/ ideas on manipulating these crystals for data collection. Best wishes Savvas Savvas Savvides L-ProBE, Unit for Structural Biology Ghent University K.L. Ledeganckstraat 35 9000 Ghent, BELGIUM office: +32-(0)9-264.51.24 ; mobile: +32-(0)472-92.85.19 Email: savvas.savvi...@ugent.be http://www.lprobe.ugent.be/xray.html From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Katarina Moravcevic Sent: Tuesday, January 27, 2009 10:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] pseudo translation Hi all, here is a question from a beginner. I have a home source data set that indexed and scaled in a P2 space group (a=46.704,b=59.362, c=48.783, alpha=90.0, beta=104.469, gamma=90.0) with predicted 2mol/au. After failing to get a MR solution with Phaser I ran the phenix.xtriage which showed that I have a large (89.18) Patterson peak at 0.5 0 0.5 which indicates pseudo translational symmetry. I was wondering if there is anything I could do with this data to get around this problem. Given that I don't have a lot of experience any suggestion/explanation would be fantastic. Thanks in advance K E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11630 http://www.pctools.com/en/spyware-doctor-antivirus/ Christopher L. Colbert, Ph.D. InstructorPhone: (214) 645 5944 University of Texas Southwestern Medical Center FAX: (214) 645 5945 6001 Forest Park Lane Dallas, TX 75390
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Two things to be mentioned. * IDA columns bear an overall negative charge. I expect this behavior holds true with NTA gels. Hence salt, (= 0.5 M NaCl) must be present in your adsoprtion buffer to quench possible repulsive electrostatic interactions. * You are dealing with protein adsoption by coordination bond formation to a metal-chelate. Coordination bond lentghs decrease (and binding improves) as ionic strength increases, so a 1-2 M salt concentration in you buffer may turn out to be appropriate. HTH, Nadir Mrabet -- Pr. Nadir T. Mrabet Cellular Molecular Biochemistry INSERM U-724 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 Fred wrote: Hi everyone, Thanks for answer my question. Just to add some more notes regarding to my expression system. The insert-vector (pET28) has been sequenced and the his-tag is N-terminal. The anti his-tag WB is positive and the binding buffer's pH is 8.2 (double-checked). I had already experienced the same problem before, which I solved just increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no binding at all. I'm currently running a SDS-PAGE with samples eluted from Talon and let you know the results. All the Best, Fred --- Fred /ccp4bb.l...@gmail.com/ schrieb am *Di, 27.1.2009: * *Von: Fred ccp4bb.l...@gmail.com Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind An: CCP4BB@JISCMAIL.AC.UK Datum: Dienstag, 27. Januar 2009, 22:00 * *Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred *
Re: [ccp4bb] small lines in diffraction pattern
Couldn't the lines be explained most simply by extreme mosaicity, perhaps severely anisotropic? If not, why not? What were the values obtained in integration? Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Margriet Ovaere To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 7:51 AM Subject: [ccp4bb] small lines in diffraction pattern Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. �Has anybody seen this�phenomena�before and has got an explanation for it please..? Many thanks Margriet Ovaere -- -- Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 -- Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. Has anybody seen this phenomena before and has got an explanation for it please..? Many thanks Margriet Ovaere  Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm
[ccp4bb] Problem running BALBES
Hello All, I'm trying to use BALBES locally to perform molecular replacement on data of a two protein complex. Protein 1 is a truncated version of a two domain protein of known structure. Protein 2 has a homologous protein of known structure and ~50% identity. I'm having two related problems - 1 If I include the truncated PDB of Protein 1 as an input model, along with a .seq file containing the two sequences, I get the following error: #---# # Search for Structures with Similar Cell and Space Group # #---# No structure of similar cell and space group was found #---# # Model Database Analysis # #---# number of amino acids in the input sequence file is 615 1 structures found to be above 20% identity with the given sequence, of which the best identity is 100.0 Error message : no structure was found ** | All of search model PDB files are in:| |Subdirectory: template_models | ** BALBES exits after searching the internal database 2 If I repeat this, but use a .seq file that has the two sequences merged as one, I get this error: #---# # Search for Structures with Similar Cell and Space Group # #---# No structure of similar cell and space group was found #---# # Model Database Analysis # #---# number of amino acids in the input sequence file is 615 2 structures found to be above 20% identity with the given sequence, of which the best identity is 100.0 can not create a reader object for XML file for /Users/B/Test/xml/get_structure_DB_Protein1.xml Traceback (most recent call last): File /apps/ccp4-6.1.0/bin/balbes, line 154, in module sfAnalysisIni.Sfcheck.info_strucFactor.get_b_overall()) File /apps/BALBES_0.0.1/bin_py/exeCodeClasses.py, line 532, in execute self.SearchDB.controller() File /apps/BALBES_0.0.1/bin_py/exeCodeClasses.py, line 1378, in controller self.structDB_info_analysis() File /apps/BALBES_0.0.1/bin_py/exeCodeClasses.py, line 881, in structDB_info_analysis rootElement = StripXml(struct_xml_document.documentElement) UnboundLocalError: local variable 'struct_xml_document' referenced before assignment Ideas? Any help would be greatly appreciated -Jesse
Re: [ccp4bb] small lines in diffraction pattern
I recommend you have a look at a book from OUP called Diffuse X-Ray Scattering and Models of Disorder by T. R. Welberry. The first chapter explains quite well (I think) where all these streaky things come from. It will also make you feel better about having it when you see all the small molecule structures that have horrible diffuse scattering! (such as urea). This looks to me like a fairly classic case of correlated static disorder. Best way to think about it is this: Imagine you have two different kinds of unit cells: A an B. Doesn't really matter what the difference between A and B is, could be a two-headed side chain in conformer A vs conformer B, or it could be as complicated as a domain motion. But, for simplicity, lets assume it is two rotamers of a side chain and also assume that each unit cell in your crystal can only be one or the other (no in betweens). Now, if the arrangement of these unit cells is perfectly correlated and an A always occurs right next door to a B along the c-axis (say), then what you really have is a bigger unit cell than you think. That is, you can draw a unit cell around each A-B pair and call it a supercell with the contents of B as a simple NCS mate of A (with one side chain in a different rotamer). Some people might call this a pseudotranslation. The effect on the diffraction pattern in this case would be the appearance of a very weak spot in between each old spot along your c axis. That is, your supercell is twice as big along c so the reciprocal-space lattice has twice as many spots in it. The new spots are weak because they only correspond to the differences between A and B, which in this case is only a few atoms. Now lets say A and B are not perfectly correlated, but only slightly. That is, in some parts of the crystal A and B are side-by-side, but in other parts you get AAB, ABBA, BABBA, etc. In each of these cases the supercell you must draw is 3, 4 and 5x your original unit cell. Each of these will produce new weak spots with progressively tighter spacings. As the supercell becomes very long, these rows of tight spots will become a streak. The streaks are particularly prominent if the A-B disorder is along only one axis. In that case, you must have a whole a-b layer of A and other whole a-b layers of B, and the ordering of these layers along c is fairly random. Colin just described this as a stacking disorder which is probably a good name for it. The final case is when A and B are completely uncorrelated and occur absolutely at random locations in your crystal. In this case the supercell can be anything and the streaks are in every direction (including every diagonal) so they simply show up as increased background. Every crystal does this. In fact, this is the origin of the B-factor as no two unit cells are exactly alike. Ever wonder where those photons go that scatter off protein atoms but don't go into spots? They go into the background. Now, since these streaks represent correlations of neighboring unit cells this means that the diffuse scattering can tell you something about how your molecule moves. There is something about your structure that forces its neighbors to be the same in at least one direction. There are a class of people who study this for a living. I am not one of them. BTW. This is definitely NOT a mosaic spread. Mosaicity occurs on length scales thousands of times larger than this. By definition, a mosaic spread is the width of the distribution of relative rotation angles of mosaic domains and these domains all scatter independently of each other. An infinite mosaic spread (or at least 180 degrees) corresponds to a powder diffraction pattern, and the fact that powder lines are sharp demonstrates how mosaicity cannot smear spots in anything but the tangential direction. That is, no rotation can change the d-spacing of a spot. Changes in unit cell size can do this, but that is a very different phenomenon than mosaic spread as mosaic domains are much much bigger than unit cells. The good news is, it is highly unlikely that this will prevent you from solving the structure. Indeed I think there are many structures in the PDB that had streaks in their diffraction pattern like this. The reason it won't hurt you is that the intensity of the Bragg peaks is the same in the perfectly-correlated, partially-correlated and completely uncorrelated cases. The electron density will simply have a two-headed side chain in it. So, I would suggest doing what most crystallographers do and completely ignore any potentially informative weirdness along the way and sally forth. But save these pictures (and the above book) for when your reviewer tells you your R-merge is too high. -James Holton MAD Scientist Margriet Ovaere wrote: Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've
Re: [ccp4bb] small lines in diffraction pattern
Jacob Traditional mosaic spread (ordered mosaic blocks imperfectly aligned with respect to one another) gives spherical caps in reciprocal space. These would appear as arcs on a single crystal rotation photograph. If anisotropic, the arcs would be more extensive in some directions. The patterns do not appear to fit this case. Different integration software could have different models for mosaicity but they often simply ensure that most of the intensity is captured in the integration volume by choosing some appropriate reflection range for the reflection. This could capture broadening of the reflections due to many types of disorder, including some of the ones discussed by Ian and myself. However, for the case discussed here, I think most integration programs would struggle. Regards Colin From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: 28 January 2009 17:05 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] small lines in diffraction pattern Couldn't the lines be explained most simply by extreme mosaicity, perhaps severely anisotropic? If not, why not? What were the values obtained in integration? Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: Margriet Ovaere mailto:margriet.ova...@chem.kuleuven.be To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 7:51 AM Subject: [ccp4bb] small lines in diffraction pattern Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. �Has anybody seen this�phenomena�before and has got an explanation for it please..? Many thanks Margriet Ovaere Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. Has anybody seen this phenomena before and has got an explanation for it please..? Many thanks Margriet Ovaere  Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm DIVFONT size=1 color=grayThis e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom /FONT/DIV
Re: [ccp4bb] small lines in diffraction pattern
Hi, it's clearly not a detector problem (at least not an obvious one!), since the 'lines' clearly follow the *curved* path of the reciprocal lattice lines as they are projected from the Ewald sphere onto the detector. A saturated pixel would presumably affect other pixels only in the same row and would appear in *straight* lines unrelated to the reciprocal lattice. I'm sticking with diffuse scattering! Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Stephan Ginell Sent: 28 January 2009 17:43 To: Margriet Ovaere; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] small lines in diffraction pattern Hi Such line can occur on a CCD detector if reflection are saturating a pixel and is generally in the direction of the detector readout. 1) what detector are you using, 2) are reflections in the black center saturated i.e. Greater than the dynamic range of the detector. 3) what is your exposure time, 4) do you see such streaks on short / attenuated exposures? 5 do you see such streaks on dark images of (no x-rays) but same time. Steve Stephan L. Ginell, Ph.D. Coordinator, SBC User Program Biosciences Argonne National Laboratory 9700 S. Cass Ave Argonne, IL 60439 (630)252-3972 office (630)218-8122 pager (630)252-6126 Fax gin...@anl.gov Email On 1/28/09 7:51 AM, Margriet Ovaere margriet.ova...@chem.kuleuven.be wrote: Dear all, In the diffraction pattern of crystals of an RNA decamer, small lines appeared (see pictures attached). We've tried different crystals but they all showed the same small lines. Has anybody seen this phenomena before and has got an explanation for it please..? Many thanks Margriet Ovaere Margriet Ovaere Chemistry Department K.U.Leuven Biomolecular Architecture Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] small lines in diffraction pattern
Re: [ccp4bb] small lines in diffraction patternI had thought that in a previous thread, we had all come to a consensus that actually the largest source of what is normally explained as mosaicity is really differences in unit cell size, due perhaps to uneven shrinkage in crystals upon freezing or otherwise. I believe that there was actually an acta cryst paper which investigated all of the various ingredients of mosaicity which supports this (this is why I said it.) Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] small lines in diffraction pattern
Re: [ccp4bb] small lines in diffraction patternActa Cryst. (1998). D54, 848-853 [ doi:10.1107/S0907444998001875 ] A Description of Imperfections in Protein Crystals C. Nave Abstract: An analysis is given of the contribution of various crystal imperfections to the rocking widths of reflections and the divergence of the diffracted beams. The crystal imperfections are the angular spread of the mosaic blocks in the crystal, the size of the mosaic blocks and the variation in cell dimensions between blocks. The analysis has implications for improving crystal perfection, defining data-collection requirements and for data-processing procedures. Measurements on crystals of tetragonal lysozyme at room temperature and 100 K were made in order to illustrate how parameters describing the crystal imperfections can be obtained. At 100 K, the dominant imperfection appeared to be a variation in unit-cell dimensions in the crystal. *** 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: Jacob Keller To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 11:57 AM Subject: Re: [ccp4bb] small lines in diffraction pattern I had thought that in a previous thread, we had all come to a consensus that actually the largest source of what is normally explained as mosaicity is really differences in unit cell size, due perhaps to uneven shrinkage in crystals upon freezing or otherwise. I believe that there was actually an acta cryst paper which investigated all of the various ingredients of mosaicity which supports this (this is why I said it.) Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] OT: VectorNTI alternatives
From: Darren Hart After several years of offering the molecular biology software VectorNTI free to the academic community (their open access program) and building up a huge user base, Invitrogen have suddenly announced that they will no longer renew these free licences and the existing ones will be left to expire within the year. There are heavy renewal fees for anyone wishing to continue use of this software. This is reminiscent of what happened with both MDL's Chime and Accelrys' WebLab/DSViewer, and it serves as yet another compelling anecdote for why scientists should: 1. Be wary of relying upon free tools not based on open-source code. 2. Be extremely wary of free tools which come with a license manager. 3. Instead favor free software tools which strictly meet the established definitions of: Open Source: http://www.opensource.org/docs/osd, Free Software: http://www.gnu.org/philosophy/free-sw.html, or Public Domain: http://en.wikipedia.org/wiki/Public_domain since it is *only* those tools that can be safely taken for granted over the long haul. Of course, if taking software tools for granted isn't your top priority, then please help to improve scientific software by purchasing high-quality commercial tools, by sponsoring and/or participating in open-source projects, or by becoming a developer yourself. Cheers, Warren
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Just to let you know. No way, Talon also don't work. I am gonna try the GE His-trap column. Fred wrote: Hi everyone, Thanks for answer my question. Just to add some more notes regarding to my expression system. The insert-vector (pET28) has been sequenced and the his-tag is N-terminal. The anti his-tag WB is positive and the binding buffer's pH is 8.2 (double-checked). I had already experienced the same problem before, which I solved just increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no binding at all. I'm currently running a SDS-PAGE with samples eluted from Talon and let you know the results. All the Best, Fred --- Fred /ccp4bb.l...@gmail.com/ schrieb am *Di, 27.1.2009: * *Von: Fred ccp4bb.l...@gmail.com Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind An: CCP4BB@JISCMAIL.AC.UK Datum: Dienstag, 27. Januar 2009, 22:00 * *Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred *
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Hi Fred, I am not sure if this has been brought up already. Another possibility might be to generously pipet your Ni- or Co-matrix of choice directly to buffered solubilized inclusion bodies, incubate for some time (even overnight if you feel its necessary), and then use the slurry to pack a column for further chromatographic manipulations. To quickly check if this approach might be promising, you can do this on a small scale in epps. IN fact working on a small scale like this might allow you to test a number of buffer choices and incubation protocols. You can spin down your beads followed by several washing steps with your column buffer. You can then go two ways: (1) either pellet with a hard spin and just add your SDS-PAGE sample buffer to the pellet to visualize binding on gel (make sure you boil your sample) OR (2) 'elute' your protein with your imidazole-containing buffer followed by analysis of the supernatant via SDS-PAGE. We have found that this approach works quite well for difficult metal-affinity chromatography cases. Best wishes Savvas -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Fred Sent: Wednesday, January 28, 2009 9:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind Just to let you know. No way, Talon also don't work. I am gonna try the GE His-trap column. Fred wrote: Hi everyone, Thanks for answer my question. Just to add some more notes regarding to my expression system. The insert-vector (pET28) has been sequenced and the his-tag is N-terminal. The anti his-tag WB is positive and the binding buffer's pH is 8.2 (double-checked). I had already experienced the same problem before, which I solved just increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no binding at all. I'm currently running a SDS-PAGE with samples eluted from Talon and let you know the results. All the Best, Fred --- Fred /ccp4bb.l...@gmail.com/ schrieb am *Di, 27.1.2009: * *Von: Fred ccp4bb.l...@gmail.com Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind An: CCP4BB@JISCMAIL.AC.UK Datum: Dienstag, 27. Januar 2009, 22:00 * *Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred * E-mail message checked by Spyware Doctor (6.0.0.386) Database version: 5.11640 http://www.pctools.com/en/spyware-doctor-antivirus/
Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind
Hi Fred, I doubt His-trap column be any better. I never found much a difference between these resins or columns. If you want to try, use His-Trap HP, not His-Trap FF. Doing batch binding may help you figure out the problem. In some cases, protein binds better in batch mode. You can take beads out at time intervals up to overnight for binding analysis. I guess not working means the protein in the flow-through. Sometimes for native purification, proteins don't come out of a column. But never experienced anything like this under denatured conditions. Good luck, Chun -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Fred Sent: Wednesday, January 28, 2009 12:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind Just to let you know. No way, Talon also don't work. I am gonna try the GE His-trap column. Fred wrote: Hi everyone, Thanks for answer my question. Just to add some more notes regarding to my expression system. The insert-vector (pET28) has been sequenced and the his-tag is N-terminal. The anti his-tag WB is positive and the binding buffer's pH is 8.2 (double-checked). I had already experienced the same problem before, which I solved just increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no binding at all. I'm currently running a SDS-PAGE with samples eluted from Talon and let you know the results. All the Best, Fred --- Fred /ccp4bb.l...@gmail.com/ schrieb am *Di, 27.1.2009: * *Von: Fred ccp4bb.l...@gmail.com Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind An: CCP4BB@JISCMAIL.AC.UK Datum: Dienstag, 27. Januar 2009, 22:00 * *Hi ccp4 list, I am trying to purify a his-tag protein by metal affinity chromatography. The protein was expressed in inclusion bodies and its his-tag doesn't bind the Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with NaCl and detergents didn't help much. Any help is appreciated. Fred *
[ccp4bb] SUMMARY: Sticky crystals
Dear colleagues, I would like to thank all of you who responded so generously (either privately or publically on the ccp4bb) to my question on how to deal with 'sticky crystals'. I provide below a consensus of all the many answers grouped according to the proposed solution. Best wishes Savvas OPERATE ON THE PLASTIC RATHER THAN ON THE CRYSTAL! --- -I dig the microtool into the plastic as close as I can get to the crystal without touching the crystal. Usually a small deformation of the plastic causes the crystal to pop off intact. -I have had similar problems with crystals sticking to the plastic of the crystallisation plates (96-well Grenier plates). I use an acupuncture needle and dig into the plastic right next to the crystal, directing the needle below the crystal. By digging and twisting, you can generate little movements across the plastic which can be enough to free the crystal. If you slip, you end up playing golf with your crystals, but with a steady hand, it has worked well for me, though I guess it might be quite dependant on the strength of the plastic of the plate. Worth trying though if you have crystals. -Take a sturdy needle (like one of the microneedles from a Hampton kit or a very thin syringe needle) and (while observing the whole thing under a scope) stick the needle into the plastic a bit away from the crystal. Push hard. If you're using polarizers, you may be abe to visualize the stress forces in the plastic by the shifting of the colors. The basic idea here is to stress the plastic under the crystal w/o touching the crystal in any way. In my case the bloody things just popped off. Sometimes you have to push quite hard, and to wiggle the needle a bit - and beware, they can slip and ruin your drop. But this really worked quite well! COAT THE CRYSTALLIZATION SURFACE WITH A THIN LAYER OF GREASE - -Additionally, once I realized this was going to be a long term problem, I started coating the sitting drop depressions with a thin layer of vacuum grease. The crystals just slid right off the grease and I never saw any changes in the diffraction data to suggest the grease was giving me issues. -You only need a very thin layer of the grease (i.e. keep wiping until its almost completely gone) and it usually has no affect on the crystallization. -Another approach is to grease the wells of the plate. If you then gently warm and melt the grease (use Vaseline or other petroleum jelly rather than silicone grease) it will set almost clear. This is sometimes used with microbatch. COAT THE CRYSTALLIZATION SURFACE WITH SILICON You can try various siliconizing fluids. Hampton used to sell one called Aquasil which you mix up in water. This will not melt the plastic as eg. Repelcote will. CHANGE CRYSTALLIZATION PLATE - You can also try plates made from COC (cyclic olefins), such as the UVP plates made by SwissCi (sold by lots of companies including us). They are less sticky than polystyrene plates. COC is halfway between polystyrene and polypropylene. Polypropylene is even less sticky than COC but is not rigid, therefore harder to dispense to automatically. You can get plates made of clarified polypropylene from Emerald, and you can also get polypropylene bridges that you place in Linbro wells. I think Hampton still sells them. CRYSTAL BOWLING --- If you have good and bad crystals in the same drop, I've had success pushing a crummy crystal into a good crystal and having it release that way. CRYSTALLIZATION WITH AGAROSE AS AN ADDITIVE -- Crystals form inside the very soft gel and they are hold in place by this meshwork. So, they are mechanically protected and do not fall down onto the bottom of the sitting-drop well. A final concentration of 0.1-0.2 % (w/v) agarose is sufficient. When you harvest a crystal cut generously around it with a microtool, pick it up (e.g. using a nylon loop) and do not mind if some agarose comes with it. Reference: Biertmpfel, C.; Basquin, J.; Suck, D. Sauter, C. Crystallization of biological macromolecules using agarose gel. Acta Crystallogr D Biol Crystallogr, 2002, 58, 1657-9 PMID: 12351881 FLOATING DROP CRYSTALLIZATION METHOD -- References 1. Application of a two-liquid system to sitting-drop vapour-diffusion protein crystallization. Adachi, H. et al, Acta Cryst. (2003) D59, 194-196 2. Promotion of large protein crystal growth with stirring solution. Adachi, H. et al. Jpn. J. Appl. Phys. Vol. 41 (2002) pp.1025-1027 3. Two-liquid hanging-drop vapour-diffusion technique of protein crystallization. Hiroaki Adachi et
Re: [ccp4bb] OT: VectorNTI alternatives - 4 Mac?
For basic analysis, editing, etc, i really like ApE, A Plasmid Editor. Versions are available for OS X, linux and Windows. http://www.biology.utah.edu/jorgensen/wayned/ape/ i find it does most everything MacVector does, and a few things MacVector does not do. What VectorNTI functionality are you looking for? Mike Michael Giffin The Scripps Research Institute Department of Molecular and Experimental Medicine 10550 North Torrey Pines Road, MEM-131 La Jolla, CA 92037 email: mjgif...@scripps.edu lab: 858-784-7758 On Wed, Jan 28, 2009 at 7:02 AM, Mark Collins mcoll...@convex.hhmi.columbia.edu wrote: Hi Anybody have suggestions for Mac OsX alternatives? Thanks in advance, Mark
Re: [ccp4bb] small lines in diffraction pattern
There is something in the unit cell, aligned with the long axis of the cell, with a periodicity corresponding to ~1/5 of the long axis. This can be seen as greater intensities along the long axis every fifth spot. Without knowing the unit cell parameters, I would guess it is either the interplanar spacings of the nucleotides (probably this is too small) or the periodic twist of the helix itself. Interesting that the RNA is a decamer ( = 2 x 5). I would be curious to know what the unit cell parameters are, or more generally, what is causing that noticeable periodicity... Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 12:20 PM Subject: Re: [ccp4bb] small lines in diffraction pattern Hmm. I don't remember that thread. However, I personally think it is a good idea to keep the mosaic crystal as Ewald and Darwin defined it. Just because current integration software lumps things together into a mosaicity does not mean that every mechanism contributing to the rocking width of a spot should be given the same name. Especially when it is difficult to describe the mosaic crystal using any other words. Perhaps Colin could come up with a cool word for unit cell non-uniformity? Or is he waiting for us to name it after him? Nonuniform Anisotropic Variance of Elasticity? or Cells Of Loose INdex? Comments and suggestions are welcome. -James Holton MAD Scientist Jacob Keller wrote: I had thought that in a previous thread, we had all come to a consensus that actually the largest source of what is normally explained as mosaicity is really differences in unit cell size, due perhaps to uneven shrinkage in crystals upon freezing or otherwise. I believe that there was actually an acta cryst paper which investigated all of the various ingredients of mosaicity which supports this (this is why I said it.) Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu mailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] small lines in diffraction pattern
I'm coming in late here, having only now found time to look at the images. It's facinating, isn't it? Since the lines are not arcs centered on the origin, this isn't mosaic spread. For those who haven't seen the image and the zoom, the diffraction pattern clearly shows one very long axis and a couple of much shorter ones. The rotation image is taken rotating around the long one. The small lines are perpendicular to the long axis, and run fairly uniformly, narrowly and evenly spaced at the intervals of the reflections along this axis, throughout the diffraction pattern. Also it appears that this is a rotation of about a degree; Margriet doesn't give us clues for any of this. I'm guessing that whoever said it's a diffuse scatter effect is close to the mark. I think diffuse scatter comes from the contents of each unit cell being essentially identical, but that within the molecule things are waving around a bit (where are Don Caspar and George Phillips when we need them?). I'll go a touch farther and suggest that it's really disorder -- each unit cell is well aligned to the others, but each one is different in some way. I'll guess that the RNA decamer is aligned along this long unit cell axis, but in some way either there's an opportunity for the register along the RNA axis to slip from one unit cell to the other or each is rotated slightly. On the other hand, the fact that there's a wide distribution of intensities in the Bragg spots, which are quite sharp, is confusing -- there must be a lot of contrast in the averaged structure for this to be true. Ok, it's interesting, but I have no idea. Bob On Wed, 28 Jan 2009, James Holton wrote: Hmm. I don't remember that thread. However, I personally think it is a good idea to keep the mosaic crystal as Ewald and Darwin defined it. Just because current integration software lumps things together into a mosaicity does not mean that every mechanism contributing to the rocking width of a spot should be given the same name. Especially when it is difficult to describe the mosaic crystal using any other words. Perhaps Colin could come up with a cool word for unit cell non-uniformity? Or is he waiting for us to name it after him? Nonuniform Anisotropic Variance of Elasticity? or Cells Of Loose INdex? Comments and suggestions are welcome. -James Holton MAD Scientist Jacob Keller wrote: I had thought that in a previous thread, we had all come to a consensus that actually the largest source of what is normally explained as mosaicity is really differences in unit cell size, due perhaps to uneven shrinkage in crystals upon freezing or otherwise. I believe that there was actually an acta cryst paper which investigated all of the various ingredients of mosaicity which supports this (this is why I said it.) Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu mailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] small lines in diffraction pattern
Hi James, what your descriptions aims at is I think shown in this publication Borgstahl, G. E. O. Incommensurate Crystallography by Sander van Smaalen Crystallography reviews 14 , 259-260 (2008). Or am I misunderstanding something here ? Jürgen On 28 Jan 2009, at 12:39, James Holton wrote: I recommend you have a look at a book from OUP called Diffuse X-Ray Scattering and Models of Disorder by T. R. Welberry. The first chapter explains quite well (I think) where all these streaky things come from. It will also make you feel better about having it when you see all the small molecule structures that have horrible diffuse scattering! (such as urea). This looks to me like a fairly classic case of correlated static disorder. Best way to think about it is this: Imagine you have two different kinds of unit cells: A an B. Doesn't really matter what the difference between A and B is, could be a two-headed side chain in conformer A vs conformer B, or it could be as complicated as a domain motion. But, for simplicity, lets assume it is two rotamers of a side chain and also assume that each unit cell in your crystal can only be one or the other (no in betweens). Now, if the arrangement of these unit cells is perfectly correlated and an A always occurs right next door to a B along the c-axis (say), then what you really have is a bigger unit cell than you think. That is, you can draw a unit cell around each A-B pair and call it a supercell with the contents of B as a simple NCS mate of A (with one side chain in a different rotamer). Some people might call this a pseudotranslation. The effect on the diffraction pattern in this case would be the appearance of a very weak spot in between each old spot along your c axis. That is, your supercell is twice as big along c so the reciprocal-space lattice has twice as many spots in it. The new spots are weak because they only correspond to the differences between A and B, which in this case is only a few atoms. Now lets say A and B are not perfectly correlated, but only slightly. That is, in some parts of the crystal A and B are side-by-side, but in other parts you get AAB, ABBA, BABBA, etc. In each of these cases the supercell you must draw is 3, 4 and 5x your original unit cell. Each of these will produce new weak spots with progressively tighter spacings. As the supercell becomes very long, these rows of tight spots will become a streak. The streaks are particularly prominent if the A-B disorder is along only one axis. In that case, you must have a whole a-b layer of A and other whole a-b layers of B, and the ordering of these layers along c is fairly random. Colin just described this as a stacking disorder which is probably a good name for it. The final case is when A and B are completely uncorrelated and occur absolutely at random locations in your crystal. In this case the supercell can be anything and the streaks are in every direction (including every diagonal) so they simply show up as increased background. Every crystal does this. In fact, this is the origin of the B-factor as no two unit cells are exactly alike. Ever wonder where those photons go that scatter off protein atoms but don't go into spots? They go into the background. Now, since these streaks represent correlations of neighboring unit cells this means that the diffuse scattering can tell you something about how your molecule moves. There is something about your structure that forces its neighbors to be the same in at least one direction. There are a class of people who study this for a living. I am not one of them. BTW. This is definitely NOT a mosaic spread. Mosaicity occurs on length scales thousands of times larger than this. By definition, a mosaic spread is the width of the distribution of relative rotation angles of mosaic domains and these domains all scatter independently of each other. An infinite mosaic spread (or at least 180 degrees) corresponds to a powder diffraction pattern, and the fact that powder lines are sharp demonstrates how mosaicity cannot smear spots in anything but the tangential direction. That is, no rotation can change the d-spacing of a spot. Changes in unit cell size can do this, but that is a very different phenomenon than mosaic spread as mosaic domains are much much bigger than unit cells. The good news is, it is highly unlikely that this will prevent you from solving the structure. Indeed I think there are many structures in the PDB that had streaks in their diffraction pattern like this. The reason it won't hurt you is that the intensity of the Bragg peaks is the same in the perfectly-correlated, partially-correlated and completely uncorrelated cases. The electron density will simply have a two- headed side chain in it. So, I would suggest doing what most crystallographers do and completely ignore any potentially informative weirdness along the way and sally forth.
Re: [ccp4bb] small lines in diffraction pattern
I'm coming in late here, having only now found time to look at the images. It's facinating, isn't it? Since the lines are not arcs centered on the origin, this isn't mosaic spread. For those who haven't seen the image and the zoom, the diffraction pattern clearly shows one very long axis and a couple of much shorter ones. The rotation image is taken rotating around the long one. The small lines are perpendicular to the long axis, and run fairly continuously, narrowly and evenly spaced at the intervals of the reflections along this axis, throughout the diffraction pattern. They're all faint and about the same intensity, and modulated along their length only slightly. Also it appears that this is a rotation of about one degree; Margriet doesn't give us clues for any of this. I'm guessing that whoever said it's a diffuse scatter effect is close to the mark. I think diffuse scatter comes from the contents of each unit cell being essentially identical, but that within the molecule things are waving around a bit (where are Don Caspar and George Phillips when we need them?), that is, different in each unit cell. I'll go a touch farther and suggest that it's really disorder -- each unit cell is well aligned to the others, but each one is different in a more significant way. I'll guess that the RNA decamer is aligned along this long unit cell axis, but in some way either there's an opportunity for the register along the RNA axis to slip from one unit cell to the other or each is rotated slightly. On the other hand, the fact that there's a wide distribution of intensities in the Bragg spots, which are quite sharp, is confusing -- there must be a lot of contrast in the averaged structure for this to be true. Ok, it's interesting, but I have no idea. Bob On Wed, 28 Jan 2009, Jacob Keller wrote: There is something in the unit cell, aligned with the long axis of the cell, with a periodicity corresponding to ~1/5 of the long axis. This can be seen as greater intensities along the long axis every fifth spot. Without knowing the unit cell parameters, I would guess it is either the interplanar spacings of the nucleotides (probably this is too small) or the periodic twist of the helix itself. Interesting that the RNA is a decamer ( = 2 x 5). I would be curious to know what the unit cell parameters are, or more generally, what is causing that noticeable periodicity... Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 12:20 PM Subject: Re: [ccp4bb] small lines in diffraction pattern Hmm. I don't remember that thread. However, I personally think it is a good idea to keep the mosaic crystal as Ewald and Darwin defined it. Just because current integration software lumps things together into a mosaicity does not mean that every mechanism contributing to the rocking width of a spot should be given the same name. Especially when it is difficult to describe the mosaic crystal using any other words. Perhaps Colin could come up with a cool word for unit cell non-uniformity? Or is he waiting for us to name it after him? Nonuniform Anisotropic Variance of Elasticity? or Cells Of Loose INdex? Comments and suggestions are welcome. -James Holton MAD Scientist Jacob Keller wrote: I had thought that in a previous thread, we had all come to a consensus that actually the largest source of what is normally explained as mosaicity is really differences in unit cell size, due perhaps to uneven shrinkage in crystals upon freezing or otherwise. I believe that there was actually an acta cryst paper which investigated all of the various ingredients of mosaicity which supports this (this is why I said it.) Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu mailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] small lines in diffraction pattern (fwd)
I'm coming in late here, having only now found time to look at the images. It's facinating, isn't it? Since the lines are not arcs centered on the origin, this isn't mosaic spread. For those who haven't seen the image and the zoom, the diffraction pattern clearly shows one very long axis and a couple of much shorter ones. The rotation image is taken rotating around the long one. The small lines are perpendicular to the long axis, and run fairly continuously, narrowly and evenly spaced at the intervals of the reflections along this axis, throughout the diffraction pattern. They're all faint and about the same intensity, and modulated along their length only slightly. Also it appears that this is a rotation of about one degree; Margriet doesn't give us clues for any of this. I'm guessing that whoever said it's a diffuse scatter effect is close to the mark. I think diffuse scatter comes from the contents of each unit cell being essentially identical, but that within the molecule things are waving around a bit (where are Don Caspar and George Phillips when we need them?), that is, different in each unit cell. I'll go a touch farther and suggest that it's really disorder -- each unit cell is well aligned to the others, but each one is different in a more significant way. I'll guess that the RNA decamer is aligned along this long unit cell axis, but in some way either there's an opportunity for the register along the RNA axis to slip from one unit cell to the other or each is rotated slightly. On the other hand, the fact that there's a wide distribution of intensities in the Bragg spots, which are quite sharp, is confusing -- there must be a lot of contrast in the averaged structure for this to be true. Ok, it's interesting, but I have no idea. Bob On Wed, 28 Jan 2009, Jacob Keller wrote: There is something in the unit cell, aligned with the long axis of the cell, with a periodicity corresponding to ~1/5 of the long axis. This can be seen as greater intensities along the long axis every fifth spot. Without knowing the unit cell parameters, I would guess it is either the interplanar spacings of the nucleotides (probably this is too small) or the periodic twist of the helix itself. Interesting that the RNA is a decamer ( = 2 x 5). I would be curious to know what the unit cell parameters are, or more generally, what is causing that noticeable periodicity... Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 12:20 PM Subject: Re: [ccp4bb] small lines in diffraction pattern Hmm. I don't remember that thread. However, I personally think it is a good idea to keep the mosaic crystal as Ewald and Darwin defined it. Just because current integration software lumps things together into a mosaicity does not mean that every mechanism contributing to the rocking width of a spot should be given the same name. Especially when it is difficult to describe the mosaic crystal using any other words. Perhaps Colin could come up with a cool word for unit cell non-uniformity? Or is he waiting for us to name it after him? Nonuniform Anisotropic Variance of Elasticity? or Cells Of Loose INdex? Comments and suggestions are welcome. -James Holton MAD Scientist Jacob Keller wrote: I had thought that in a previous thread, we had all come to a consensus that actually the largest source of what is normally explained as mosaicity is really differences in unit cell size, due perhaps to uneven shrinkage in crystals upon freezing or otherwise. I believe that there was actually an acta cryst paper which investigated all of the various ingredients of mosaicity which supports this (this is why I said it.) Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu mailto:j-kell...@northwestern.edu ***
Re: [ccp4bb] small lines in diffraction pattern
I'm sure Gloria would be delighted if that were the case, but I don't think this is an incommensurate lattice. These actually don't so much give you diffuse scattering as little satellite spots near the main spots at spacings that don't make any sense given the lattice repeat. My understanding is that these arise from something that is slowly varying from unit cell to unit cell (could be as simple as a side chain waving back and forth) in a repetitive pattern that just doesn't line up in any way with the repeat of the unit cells. Still, I'll ask her. However, I think that the difference is that modulated lattices are gradual changes of structure across many unit cells and what I was talking about is a more simplistic case of two different kinds of unit cells with varying degrees of randomness in their arrangement. That is, using the formalism I described below, a modulated lattice would have unit cells that go: ABCDEFGHGFEDCBABCDEFG... etc. where A is not that different from B, B similar to C, etc., but A and H are very different. -James Holton MAD Scientist Jürgen Bosch wrote: Hi James, what your descriptions aims at is I think shown in this publication Borgstahl, G. E. O. Incommensurate Crystallography by Sander van Smaalen Crystallography reviews 14 , 259-260 (2008). Or am I misunderstanding something here ? Jürgen On 28 Jan 2009, at 12:39, James Holton wrote: I recommend you have a look at a book from OUP called Diffuse X-Ray Scattering and Models of Disorder by T. R. Welberry. The first chapter explains quite well (I think) where all these streaky things come from. It will also make you feel better about having it when you see all the small molecule structures that have horrible diffuse scattering! (such as urea). This looks to me like a fairly classic case of correlated static disorder. Best way to think about it is this: Imagine you have two different kinds of unit cells: A an B. Doesn't really matter what the difference between A and B is, could be a two-headed side chain in conformer A vs conformer B, or it could be as complicated as a domain motion. But, for simplicity, lets assume it is two rotamers of a side chain and also assume that each unit cell in your crystal can only be one or the other (no in betweens). Now, if the arrangement of these unit cells is perfectly correlated and an A always occurs right next door to a B along the c-axis (say), then what you really have is a bigger unit cell than you think. That is, you can draw a unit cell around each A-B pair and call it a supercell with the contents of B as a simple NCS mate of A (with one side chain in a different rotamer). Some people might call this a pseudotranslation. The effect on the diffraction pattern in this case would be the appearance of a very weak spot in between each old spot along your c axis. That is, your supercell is twice as big along c so the reciprocal-space lattice has twice as many spots in it. The new spots are weak because they only correspond to the differences between A and B, which in this case is only a few atoms. Now lets say A and B are not perfectly correlated, but only slightly. That is, in some parts of the crystal A and B are side-by-side, but in other parts you get AAB, ABBA, BABBA, etc. In each of these cases the supercell you must draw is 3, 4 and 5x your original unit cell. Each of these will produce new weak spots with progressively tighter spacings. As the supercell becomes very long, these rows of tight spots will become a streak. The streaks are particularly prominent if the A-B disorder is along only one axis. In that case, you must have a whole a-b layer of A and other whole a-b layers of B, and the ordering of these layers along c is fairly random. Colin just described this as a stacking disorder which is probably a good name for it. The final case is when A and B are completely uncorrelated and occur absolutely at random locations in your crystal. In this case the supercell can be anything and the streaks are in every direction (including every diagonal) so they simply show up as increased background. Every crystal does this. In fact, this is the origin of the B-factor as no two unit cells are exactly alike. Ever wonder where those photons go that scatter off protein atoms but don't go into spots? They go into the background. Now, since these streaks represent correlations of neighboring unit cells this means that the diffuse scattering can tell you something about how your molecule moves. There is something about your structure that forces its neighbors to be the same in at least one direction. There are a class of people who study this for a living. I am not one of them. BTW. This is definitely NOT a mosaic spread. Mosaicity occurs on length scales thousands of times larger than this. By definition, a mosaic spread is the width of the distribution of relative rotation angles of mosaic domains and these domains all scatter
Re: [ccp4bb] small lines in diffraction pattern
Hi Bob et al: I think that this is the convolution of the helical transform with the Bragg diffraction, which you often see in nucleic acid diffraction patters. I think I first saw it, in fact, at your beam-line in 1994, with ribozyme crystals. (Aaron Klug had to explain it to me.) The part that puzzled me was that the dark spots seem to be modulo 10, whereas for A-form RNA it should be 11 (due to 11 bases per helical turn). But then I read the initial email, and it says RNA decamer. So I think what is happening is that the RNA is slightly distorted to 10 bases per turn to accommodate the crystal lattice repeat. The horizontal lines aren't diffuse scatter, but rather the layer line scatter of a helical transform described by increasing order cylindrical Bessel functions (mod 10). All the best, Bill On Jan 28, 2009, at 6:57 PM, Robert Sweet wrote: I'm coming in late here, having only now found time to look at the images. It's facinating, isn't it? Since the lines are not arcs centered on the origin, this isn't mosaic spread. For those who haven't seen the image and the zoom, the diffraction pattern clearly shows one very long axis and a couple of much shorter ones. The rotation image is taken rotating around the long one. The small lines are perpendicular to the long axis, and run fairly continuously, narrowly and evenly spaced at the intervals of the reflections along this axis, throughout the diffraction pattern. They're all faint and about the same intensity, and modulated along their length only slightly. Also it appears that this is a rotation of about one degree; Margriet doesn't give us clues for any of this. I'm guessing that whoever said it's a diffuse scatter effect is close to the mark. I think diffuse scatter comes from the contents of each unit cell being essentially identical, but that within the molecule things are waving around a bit (where are Don Caspar and George Phillips when we need them?), that is, different in each unit cell. I'll go a touch farther and suggest that it's really disorder -- each unit cell is well aligned to the others, but each one is different in a more significant way. I'll guess that the RNA decamer is aligned along this long unit cell axis, but in some way either there's an opportunity for the register along the RNA axis to slip from one unit cell to the other or each is rotated slightly. On the other hand, the fact that there's a wide distribution of intensities in the Bragg spots, which are quite sharp, is confusing -- there must be a lot of contrast in the averaged structure for this to be true. Ok, it's interesting, but I have no idea. Bob On Wed, 28 Jan 2009, Jacob Keller wrote: There is something in the unit cell, aligned with the long axis of the cell, with a periodicity corresponding to ~1/5 of the long axis. This can be seen as greater intensities along the long axis every fifth spot. Without knowing the unit cell parameters, I would guess it is either the interplanar spacings of the nucleotides (probably this is too small) or the periodic twist of the helix itself. Interesting that the RNA is a decamer ( = 2 x 5). I would be curious to know what the unit cell parameters are, or more generally, what is causing that noticeable periodicity... Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** - Original Message - From: James Holton jmhol...@lbl.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 28, 2009 12:20 PM Subject: Re: [ccp4bb] small lines in diffraction pattern Hmm. I don't remember that thread. However, I personally think it is a good idea to keep the mosaic crystal as Ewald and Darwin defined it. Just because current integration software lumps things together into a mosaicity does not mean that every mechanism contributing to the rocking width of a spot should be given the same name. Especially when it is difficult to describe the mosaic crystal using any other words. Perhaps Colin could come up with a cool word for unit cell non-uniformity? Or is he waiting for us to name it after him? Nonuniform Anisotropic Variance of Elasticity? or Cells Of Loose INdex? Comments and suggestions are welcome. -James Holton MAD Scientist Jacob Keller wrote: I had thought that in a previous thread, we had all come to a consensus that actually the largest source of what is normally explained as mosaicity is really differences in unit cell size, due perhaps to uneven shrinkage in crystals upon freezing or otherwise. I believe that there was actually an acta cryst paper which investigated all of the various ingredients of mosaicity which
