[ccp4bb] SUMMARY [ccp4bb] List of conserved waters
This is the summary for my question from Fri, 29 Aug 2008, when I wanted to learn about programs that produce a list of overlapping (conserved) waters after superpositioning of two structures. - Somebody who did not want to answer in public offered to send a small unpublished program written particularly for this purpose. - Jens T. Kaiser menstioned that Rasmol, CNS, MAIN and similar programs have such featured and listed the lines for Rasmol select (water and *:W) and within(0.7,(water and *:X)) write pdb conserved.pdb to select waters that are closer than .7A. I found this code very interesting for I like rasmol - G. Kleywegt mentioned the WAters command in lsqman http://xray.bmc.uu.se/usf/lsqman_man.html#S71 which was handy since I was using lsqman to superpose the two structures. - G. Sheldrick mentioned (during coffee break) that the envi command in xp (that's NOT a product by Microsoft) would also do the job. Thank you to everyone who considered my question. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Fri, 29 Aug 2008, Tim Gruene wrote: Hello, I am (lazily ;-)) looking for a program that list all conserved waters between to structures (after superposition), i.e. all waters between both structures within a certain distance cut-off. Could anyone please point me to a program that does this? Thanks a lot, Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] regarding cloning
Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning. The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful in subcloning (T/A vector) and getting my insert at 1.2kb after double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. Connect with friends all over the world. Get Yahoo! India Messenger at http://in.messenger.yahoo.com/?wm=n/
Re: [ccp4bb] SUMMARY [ccp4bb] List of conserved waters
I forgot to mention K. Sekar who suggested 3dss at http://cluster.physics.iisc.ernet.in/3dss/ I am sorry about this. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Mon, 1 Sep 2008, Tim Gruene wrote: This is the summary for my question from Fri, 29 Aug 2008, when I wanted to learn about programs that produce a list of overlapping (conserved) waters after superpositioning of two structures. - Somebody who did not want to answer in public offered to send a small unpublished program written particularly for this purpose. - Jens T. Kaiser menstioned that Rasmol, CNS, MAIN and similar programs have such featured and listed the lines for Rasmol select (water and *:W) and within(0.7,(water and *:X)) write pdb conserved.pdb to select waters that are closer than .7A. I found this code very interesting for I like rasmol - G. Kleywegt mentioned the WAters command in lsqman http://xray.bmc.uu.se/usf/lsqman_man.html#S71 which was handy since I was using lsqman to superpose the two structures. - G. Sheldrick mentioned (during coffee break) that the envi command in xp (that's NOT a product by Microsoft) would also do the job. Thank you to everyone who considered my question. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Fri, 29 Aug 2008, Tim Gruene wrote: Hello, I am (lazily ;-)) looking for a program that list all conserved waters between to structures (after superposition), i.e. all waters between both structures within a certain distance cut-off. Could anyone please point me to a program that does this? Thanks a lot, Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] List of conserved waters
You can use the ancient but useful watertidy script - that assigns water names coded according to the residue they are linked to. So ones with the same link code are related.. But I also use distang. Make a file with the two or more sets of waters - then do a distance search with the appropriate water radius. Eleanor Thus Tim Gruene wrote: Hello, I am (lazily ;-)) looking for a program that list all conserved waters between to structures (after superposition), i.e. all waters between both structures within a certain distance cut-off. Could anyone please point me to a program that does this? Thanks a lot, Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] Postdoctoral positions - Birkbeck
Postdoctoral Research Assistants – Ref: 10142 School of Crystallography, Faculty of Science Full time, fixed term appointment for up to five years The School of Crystallography/Institute of Structural and Molecular Biology is seeking Post-doctoral Research Assistants to carry out structural analysis (x-ray crystallography or cryo-electron microscopy) of complexes formed during pilus biogenesis and type IV secretion. The group is led by Professor Gabriel Waksman and has over the years produced numerous high profile publications in the highest impact journals. The research programme is funded by 5-year grants from the MRC and Wellcome Trust to Prof Gabriel Waksman. Further details on the research group can be found at http://people.cryst.bbk.ac.uk/~ubcg54a/ . Applicants should have a PhD in Structural Molecular Biology, Biophysics or a related area, postdoctoral research experience and relevant research publications. Salary range will be from £31,907 to £40,788 per annum inclusive of London Allowance on Grade 7 or 8, initial salary will be dependent on the skills and experience of the successful applicant. Closing date for completed applications: 01 October 2008 To apply for this position please go to www.bbk.ac.uk/jobs and search using reference number 10142. If you have difficulties accessing this site please email, [EMAIL PROTECTED] , quoting the appropriate reference in the subject header. Birkbeck is an equal opportunities employer and encourages applications from all candidates irrespective of gender, ethnicity, age, disability, religious belief and sexual preference.
Re: [ccp4bb] regarding cloning
Hi Vijay, I have heard of TOPO-TA cloning. Not sure what T/A cloning is. I have a couple to check based on your description: 1) Do you CIP-treat your vector? If not, that might be a step to add. Also, you could include a 'vector only' transformation control to determine how many colonies are obtained in the 'vector+insert' plate above background levels. 2) For NheI/BamHI, a sequential digestion (rather than a double digestion) is recommended. 3) Make sure the primers have sufficient extensions (6-8nt) outside of the RE site. 4) Make sure both enzymes work by doing single digestion controls with the vector. It's obviously hard to tell with the PCRT product 4) Titrate vector:insert ratios -- lower and higher than you mention 5)... Once ALL possibilities are exhausted and when nothing else works, I have seen people reorder the exact same primers and then things have worked like a charm! Hope that helps. Raji -Included Message-- Date: 1-sep-2008 03:06:29 -0400 From: vijay srivastava [EMAIL PROTECTED] Reply-To: [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning.#194; The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful#194; in subcloning (T/A vector) and getting my insert at 1.2kb after#194; double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how#194; when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. Connect with friends all over the world. Get Yahoo! India Messenger at http://in.messenger.yahoo.com/?wm=n/ -End of Included Message--
Re: [ccp4bb] regarding cloning
Hi, First of all - I am curious why did you decide to put in an extra step (the T/A cloning into an intermediate vector)? You can happily digest your PCR product with NheI/BamHI, clean up and ligate into the appropriately digested pET-23a(+). If you have issues, you should definitely try this. Now, since you do have an intermediate step - did you verify that everything was OK after havig subcloned your insert into whatever vector you're using? Did you sequence the insert and most importantly did the sequencing confirm the nature of the linker regions? The enzyme pair that you chose has a slight issue with digestion buffer - most people would choose NEB buffer 2 (since buffer 3 is bad for Nhe) where Bam still has '100% activity' - however, in buffer 2 you can have star activity of the Bam due to the somewhat lower salt concentration (50 mM instead of the optimum 100 mM). It's not impossible to imagine that you have issues with digestion. This can be easily avoided by sequential digestion although of course it's slightly more work (but if you cut out the T/A cloning step that's actually still faster). So, in conclusion the most likely issue is digesiton (probably of the pET vector, to be more specific). Next likely issue could be ligation - make sure that you base your ligation ratio on the gel intensity of the bands as well as on the OD260 of your DNA. Faulty primers are not likely to be an issue since you seem to be able to restrict your insert out of the intermediate vector. Please note that you can often use SpeI or XbaI instead of Nhe since they have compatible sticky ends. Clearly this depends on the vector you're working with and I am too lazy to look up pET23 polylinker. Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of vijay srivastava Sent: Monday, September 01, 2008 3:06 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning. The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful in subcloning (T/A vector) and getting my insert at 1.2kb after double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. _ Be the first one to try the new Messenger 9 Beta! Click http://in.rd.yahoo.com/tagline_messenger_7/*http:/in.messenger.yahoo.com/wi n/ here.
[ccp4bb] Como Crystallography School registration deadline extended for the last time!
There are still a limited number of places available at the Como Crystallography School in late September. Visit the web site for the detailed program and registration details! The registration deadline has been extended for the last time, to 16th September. http://www.crystallographyschool.org/ The 9th International School on the Crystallography of Biological Macromolecules Società del Casino, Como, Italy September 29th–October 3rd, 2008 ORGANISERS Prof. Keith Wilson (York University, UK) Dr. Marjolein Thunnissen (Lund University, Sweden) Dr. Derek Logan (Lund University, Sweden) AIMS The school focuses on the application of X-ray crystallographic methods to the study of biological macromolecules, with emphasis on the latest developments. The programme includes lectures on protein expression and purification, crystallisation, data collection and processing, synchrotron sources, phasing, phase improvement, model building, refinement and analysis of structural data. These methods sections will be complemented with seminars about new structures. Participants who are encouraged to present a poster; nine of the oral contributions will be selected from the poster abstracts.
Re: [ccp4bb] regarding cloning
T/A cloning utilizes the overhangs left by certain polymerases as cloning handles. To lower background, the vectors for TA cloning are often designed to contain a rare cutter which is used during ligation to constantly re-cut the self-ligated vector, so this way only the insert ligation product is supposed to survive. A. -Original Message- I have heard of TOPO-TA cloning. Not sure what T/A cloning is.
Re: [ccp4bb] regarding cloning
I haven't done TA cloning in a very long time. Typically, TA cloning is done with blunt-ended DNA fragments that have been modified by terminal transferase. Normally, a blunt-ended vector digest is treated with terminal transferase and TTP. (If the vector is cut by non-blunt-ended restriction endonucleases, it must be filled in with Klenow or equivalent before adding the T. PCR fragments must be treated with terminal transferase and ATP, or if using Taq (not recommended these days with high-fidelity polymerases available) a significant fraction of PCR products will already have the A overhang. Ligation will result in the insert being inserted (supposedly) randomly in forward and reverse orientations. We don't do this method anymore because it is too problematic and too time-consuming. A double digest with two different overhangs (both vector and PCR product) is much preferred, and results in a high percentage of recombinants with the correct orientation. We usually overdigest PCR products and vectors for at least 3 hr to ensure double-cutting. Phosphatase treatment of the vector after double digest prevents re-ligation of empty plasmid that has only been cleaved by one enzyme. RE digestion of PCR products requires at least 3 additional nucleotides in front of the recognition site. We usually add TGC in front of the recognition site for all PCR primers. Unfortunately in your case, NheI and BamHI have the same 3' C overhang, so double digestion with these two enzymes will result in a random orientation of your PCR product in the vector. I would recommend using a different RE for one of the sites to ensure correct orientation of the PCR product when ligated. We routinely use NdeI and PstI for most of our expression plasmid constructs (assuming these recognition sites are not present in our vector outside the cloning region--this is typically a good pair for pET vectors, and is a good double digest), as NdeI has a start codon within it. (NcoI is another possibility, if your first codon after the start begins with G). For details, see our lab wiki with time-tested methods: http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Protein+Engineering+Protocols Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED] Raji Edayathumangalam wrote: Hi Vijay, I have heard of TOPO-TA cloning. Not sure what T/A cloning is. I have a couple to check based on your description: 1) Do you CIP-treat your vector? If not, that might be a step to add. Also, you could include a 'vector only' transformation control to determine how many colonies are obtained in the 'vector+insert' plate above background levels. 2) For NheI/BamHI, a sequential digestion (rather than a double digestion) is recommended. 3) Make sure the primers have sufficient extensions (6-8nt) outside of the RE site. 4) Make sure both enzymes work by doing single digestion controls with the vector. It's obviously hard to tell with the PCRT product 4) Titrate vector:insert ratios -- lower and higher than you mention 5)... Once ALL possibilities are exhausted and when nothing else works, I have seen people reorder the exact same primers and then things have worked like a charm! Hope that helps. Raji -Included Message-- Date: 1-sep-2008 03:06:29 -0400 From: vijay srivastava [EMAIL PROTECTED] Reply-To: [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning.#194; The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful#194; in subcloning (T/A vector) and getting my insert at 1.2kb after#194; double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how#194; when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. Connect with friends all over the world. Get Yahoo! India Messenger at http://in.messenger.yahoo.com/?wm=n/ -End of Included Message--
Re: [ccp4bb] regarding cloning
Alternatively, you could skip troubleshooting digestion/ligation/etc. and use a kit based on site-specific recombination, like the In-Fusion kit that Clontech sells. (I don't have any financial interest here--I'm just a graduate student, but I've had good results using it.) The kit is not without its own downsides--it's a bit pricier than traditional cloning, you'd have to design another set of specific primers, and you have to be very careful about the vector:insert ratios you use. Good luck, Brian Artem Evdokimov wrote: Hi, First of all -- I am curious why did you decide to put in an extra step (the T/A cloning into an intermediate vector)? You can happily digest your PCR product with NheI/BamHI, clean up and ligate into the appropriately digested pET-23a(+). If you have issues, you should definitely try this. Now, since you do have an intermediate step -- did you verify that everything was OK after havig subcloned your insert into whatever vector you're using? Did you sequence the insert and most importantly did the sequencing confirm the nature of the linker regions? The enzyme pair that you chose has a slight issue with digestion buffer -- most people would choose NEB buffer 2 (since buffer 3 is bad for Nhe) where Bam still has '100% activity' -- however, in buffer 2 you can have star activity of the Bam due to the somewhat lower salt concentration (50 mM instead of the optimum 100 mM). It's not impossible to imagine that you have issues with digestion. This can be easily avoided by sequential digestion although of course it's slightly more work (but if you cut out the T/A cloning step that's actually still faster). So, in conclusion the most likely issue is digesiton (probably of the pET vector, to be more specific). Next likely issue could be ligation -- make sure that you base your ligation ratio on the gel intensity of the bands as well as on the OD260 of your DNA. Faulty primers are not likely to be an issue since you seem to be able to restrict your insert out of the intermediate vector. Please note that you can often use SpeI or XbaI instead of Nhe since they have compatible sticky ends. Clearly this depends on the vector you're working with and I am too lazy to look up pET23 polylinker. Artem *From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf Of *vijay srivastava *Sent:* Monday, September 01, 2008 3:06 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning. The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful in subcloning (T/A vector) and getting my insert at 1.2kb after double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. Be the first one to try the new Messenger 9 Beta! Click here. http://in.rd.yahoo.com/tagline_messenger_7/*http:/in.messenger.yahoo.com/win/
[ccp4bb] snippet of code for coordinate fit
Dear ccp4ers, Does someone by any chance have a snippet of code (in whatever language but PERL, C, or FORTRAN for preference) that would perform coordinate fit for two sets of atoms? I don't need anything fancy, just some simple code to fit two peptides of equal length and composition. Yes, I can do it in one of the many programs such as lsqcab, pymol, lsqman, etc. but I need to iterate this process many thousands of times so I'd rather not execute thousands of time-wasting system calls. I can write the code myself but this is a quick and dirty attempt to answer a question that's been bothering me for some time and I'd rather not spend an evening trying to figure out why my stuff does not work :-) Thank you :-) Artem
[ccp4bb] thank you, code found
Thank you, Courtesy of Peter Zwart, William Scott, Phil Evans, and Gerard Kleywegt - I now have *four* different ways to do what I need! I can use Clipper, cctbx, Fortran+numerical recipes, or Clipper :-) This goes to show that crystallographers are: a) overall very nice and b) do not sleep or take days off at all Now I can go burn my Linux CPU. Artem
Re: [ccp4bb] regarding cloning
2008/9/1 Artem Evdokimov [EMAIL PROTECTED] Hi, First of all – I am curious why did you decide to put in an extra step (the T/A cloning into an intermediate vector)? You can happily digest your PCR product with NheI/BamHI, clean up and ligate into the appropriately digested pET-23a(+). If you have issues, you should definitely try this. Hello, Artem is right in making this point of course, but why? I think the number of moles of DNA molecule that you get is important. With PCR you can get a huge number of moles, especially for a very intense 600bp band. As you go up in size of product, the yield often becomes lower, and it becomes more difficult to clone, but still, you'll probably have more moles of insert compared to using restriction enzymes to cleave a fragment out of a vector. So when is using a TA cloning vector step justifiable? If you have a meagre PCR product which you really can't clone any other way, TA cloning is the most efficient with these low yields (for me). Can high PCR yields inhibit restriction enzyme-based ligations? Yes, if you don't cleave these products to completion. If you have PCR products where a significant proportion are not cleaved properly, the non-cleaved molecules will have an inhibitory effect on your reaction- one can imagine that they'll mop up a large proportion of your vector and make it unusable. So, if you have a huge PCR product, try cleaving 10-30% of the entire reaction either 4 hours or over night to make sure it's fully cleaved. (Again, as Artem says, choose your buffer carefully here). As a final piece of advice, make sure you have a copy of Molecular Cloning, a Laboratory Guide (Cold Sring Harbor Press) nearby. http://books.google.fr/books?id=YTxKwWUiBeUCdq=molecular+cloning+a+lab+manualpg=PP1ots=FVO87K4uEtsig=xTYcKpFP45dBqwsfRWZ0UXR0wb8hl=ensa=Xoi=book_resultresnum=1ct=result ...You will probably have a recent or not-so-recent edition (A.K.A. Maniatis for previous editions IIRC) in a University Library nearby. I recently frog-marched a technician here to read it when he was having cloning problems. After persuading him that the PEG-additive protocol might be helpful to help him with a particularly nasty cloning problem, he tried it. As he said, the results were miraculous. (I have no affiliation to CSHL press nor the authors BTW) Good luck, Mark Now, since you do have an intermediate step – did you verify that everything was OK after havig subcloned your insert into whatever vector you're using? Did you sequence the insert and most importantly did the sequencing confirm the nature of the linker regions? The enzyme pair that you chose has a slight issue with digestion buffer – most people would choose NEB buffer 2 (since buffer 3 is bad for Nhe) where Bam still has '100% activity' – however, in buffer 2 you can have star activity of the Bam due to the somewhat lower salt concentration (50 mM instead of the optimum 100 mM). It's not impossible to imagine that you have issues with digestion. This can be easily avoided by sequential digestion although of course it's slightly more work (but if you cut out the T/A cloning step that's actually still faster). So, in conclusion the most likely issue is digesiton (probably of the pET vector, to be more specific). Next likely issue could be ligation – make sure that you base your ligation ratio on the gel intensity of the bands as well as on the OD260 of your DNA. Faulty primers are not likely to be an issue since you seem to be able to restrict your insert out of the intermediate vector. Please note that you can often use SpeI or XbaI instead of Nhe since they have compatible sticky ends. Clearly this depends on the vector you're working with and I am too lazy to look up pET23 polylinker. Artem -- *From:* CCP4 bulletin board [mailto:[EMAIL PROTECTED] *On Behalf Of *vijay srivastava *Sent:* Monday, September 01, 2008 3:06 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning. The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful in subcloning (T/A vector) and getting my insert at 1.2kb after double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. -- Be the first one to try the new Messenger 9 Beta! Click here.http://in.rd.yahoo.com/tagline_messenger_7/*http:/in.messenger.yahoo.com/win/ -- Mark BROOKS Telephone: 0169157968 Fax: 0169853715 Institut de Biochmie et de Biophysique Moleculaire et Cellulaire
