[ccp4bb] Refmac specific questions
Hi, Sorry to sound stupid, could someone explain what does refmac do if one chooses mixed (bref MIXED) or overall (bref OVER) B factor refinement in the context of TLS refinement? Is it something like: Isotropic for the main chain and/or waters and anisotropic for the side chain? Regards, Partha -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: pcha...@nimr.mrc.ac.uk Phone: + 44 208 816 2515
Re: [ccp4bb] Refmac specific questions
Hi Partha, It should be depended on the input model : MIXEd Some atoms with isotropic, some with anisotropic B-values. In this case input file (PDB) defines which atom should be refined isotropicly and which anisotropicly. The atoms with ANISOU card are refined anisotropicly. ta leo Partha Chakrabarti wrote: Hi, Sorry to sound stupid, could someone explain what does refmac do if one chooses mixed (bref MIXED) or overall (bref OVER) B factor refinement in the context of TLS refinement? Is it something like: Isotropic for the main chain and/or waters and anisotropic for the side chain? Regards, Partha -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: pcha...@nimr.mrc.ac.uk mailto:pcha...@nimr.mrc.ac.uk Phone: + 44 208 816 2515
Re: [ccp4bb] images
I started a poll to find out whether crystallographers need and are interested in an X-ray diffraction data bank. Will crystallographers find this resource helpful and be willing to submit their structures? I hope you will take a moment to share your opinion via the poll and/or by posting any questions or comments you may have. I will follow up with results in about a month. http://www.p212121.com/2009/06/04/do-we-need-an-x-ray-diffraction-image-data-bank/ Thanks. Sean Seaver
[ccp4bb] TEV nucleotude sequence with restriction site
Dear Crystallographers, Does anybody have a TEV-protease-site-coding nucleotide sequence with a commonly-used restriction site in it, preferably right at the end? Alternatively, does some somebody know of a program to determine all equivalent codon permutations for a small coding region, filtered for resulting restriction site possibilities? It seems like it would be an easy enough script to write... (I have already done some googling around for such a program, with not much luck.) 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] TEV nucleotude sequence with restriction site
You seem to be describing the MCS found in many TEV-site-containing expression plasmids (am I missing something?) E.g., look at the sequences in Sheffield et al., Protein Expression and Purification 15, 34 –39 (1999) (let me know if you want a PDF, I don't want to send it to the whole bb) On 5 Jun 2009, at 5:41 PM, Jacob Keller wrote: Dear Crystallographers, Does anybody have a TEV-protease-site-coding nucleotide sequence with a commonly-used restriction site in it, preferably right at the end? Alternatively, does some somebody know of a program to determine all equivalent codon permutations for a small coding region, filtered for resulting restriction site possibilities? It seems like it would be an easy enough script to write... (I have already done some googling around for such a program, with not much luck.) 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 *** --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] TEV nucleotude sequence with restriction site
I'm not quite sure what you want, but I have a series of vectors encoding various N-terminal tags and fusions, all followed by a TEV site. They have an MCS standard to many pET vectors. Therefore, they are designed to clone your gene in using the NdeI site at the 5' end (which will, after proteolysis, leave you with GH at the N-terminus of your protein). Other restriction enzymes in the MCS can be used, but more amino acids will be left at your N-terminus. I've used WatCut (from the U. Waterloo) for the silent mutagenesis question: http://watcut.uwaterloo.ca/watcut/watcut/template.php Best, Cynthia On Jun 5, 2009, at 5:41 PM, Jacob Keller wrote: Dear Crystallographers, Does anybody have a TEV-protease-site-coding nucleotide sequence with a commonly-used restriction site in it, preferably right at the end? Alternatively, does some somebody know of a program to determine all equivalent codon permutations for a small coding region, filtered for resulting restriction site possibilities? It seems like it would be an easy enough script to write... (I have already done some googling around for such a program, with not much luck.) 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] TEV nucleotude sequence with restriction site
I checked out the Sheffield et al paper, and the restriction sites there are all just after the TEV site, thereby including, as Cynthia mentioned, at least an extra H beyond the obligatory G from the TEV site. I was hoping to be able to have only the G. (Since I am cloning in the TEV site with my PCR primer, I have free choice about what codons to choose, and therefore think it would be nice to have the restriction site in the TEV site itself, if possible. Also, this will keep my primer a little shorter.) 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: Cynthia Kinsland cl...@cornell.edu To: Jacob Keller j-kell...@md.northwestern.edu Cc: CCP4BB@JISCMAIL.AC.UK Sent: Friday, June 05, 2009 5:19 PM Subject: Re: [ccp4bb] TEV nucleotude sequence with restriction site I'm not quite sure what you want, but I have a series of vectors encoding various N-terminal tags and fusions, all followed by a TEV site. They have an MCS standard to many pET vectors. Therefore, they are designed to clone your gene in using the NdeI site at the 5' end (which will, after proteolysis, leave you with GH at the N-terminus of your protein). Other restriction enzymes in the MCS can be used, but more amino acids will be left at your N-terminus. I've used WatCut (from the U. Waterloo) for the silent mutagenesis question: http://watcut.uwaterloo.ca/watcut/watcut/template.php Best, Cynthia On Jun 5, 2009, at 5:41 PM, Jacob Keller wrote: Dear Crystallographers, Does anybody have a TEV-protease-site-coding nucleotide sequence with a commonly-used restriction site in it, preferably right at the end? Alternatively, does some somebody know of a program to determine all equivalent codon permutations for a small coding region, filtered for resulting restriction site possibilities? It seems like it would be an easy enough script to write... (I have already done some googling around for such a program, with not much luck.) 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] images
Hi Sean Ash Buckle has already developed one! Tools for general deposition will be released shortly. http://tardis.edu.au/ Cheers J Sean Seaver s...@p212121.com wrote: I started a poll to find out whether crystallographers need and are interested in an X-ray diffraction data bank. Will crystallographers find this resource helpful and be willing to submit their structures? I hope you will take a moment to share your opinion via the poll and/or by posting any questions or comments you may have. I will follow up with results in about a month. http://www.p212121.com/2009/06/04/do-we-need-an-x-ray-diffraction-image-data-bank/ Thanks. Sean Seaver -- Professor James Whisstock ARC Federation Fellow Honorary NHMRC Principal Research Fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] TEV nucleotude sequence with restriction site
Hi Jacob, If you're including the TEV site in your primer, then you won't need a restriction site after it (unless you're planning to recycle the vector afterwards for other inserts). Having the protease site too close to the protein can often make it difficult to cut. Cheers, Charlie Quotfintg Jacob Keller j-kell...@md.northwestern.edu: I checked out the Sheffield et al paper, and the restriction sites there are all just after the TEV site, thereby including, as Cynthia mentioned, at least an extra H beyond the obligatory G from the TEV site. I was hoping to be able to have only the G. (Since I am cloning in the TEV site with my PCR primer, I have free choice about what codons to choose, and therefore think it would be nice to have the restriction site in the TEV site itself, if possible. Also, this will keep my primer a little shorter.) 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: Cynthia Kinsland cl...@cornell.edu To: Jacob Keller j-kell...@md.northwestern.edu Cc: CCP4BB@JISCMAIL.AC.UK Sent: Friday, June 05, 2009 5:19 PM Subject: Re: [ccp4bb] TEV nucleotude sequence with restriction site I'm not quite sure what you want, but I have a series of vectors encoding various N-terminal tags and fusions, all followed by a TEV site. They have an MCS standard to many pET vectors. Therefore, they are designed to clone your gene in using the NdeI site at the 5' end (which will, after proteolysis, leave you with GH at the N-terminus of your protein). Other restriction enzymes in the MCS can be used, but more amino acids will be left at your N-terminus. I've used WatCut (from the U. Waterloo) for the silent mutagenesis question: http://watcut.uwaterloo.ca/watcut/watcut/template.php Best, Cynthia On Jun 5, 2009, at 5:41 PM, Jacob Keller wrote: Dear Crystallographers, Does anybody have a TEV-protease-site-coding nucleotide sequence with a commonly-used restriction site in it, preferably right at the end? Alternatively, does some somebody know of a program to determine all equivalent codon permutations for a small coding region, filtered for resulting restriction site possibilities? It seems like it would be an easy enough script to write... (I have already done some googling around for such a program, with not much luck.) 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 *** -- Charlie Bond Professorial Fellow University of Western Australia School of Biomedical, Biomolecular and Chemical Sciences M310, 35 Stirling Highway Crawley WA 6009, Australia
Re: [ccp4bb] TEV nucleotude sequence with restriction site
Try type II restriction enzymes. They cleave outside of their recognition sites. So you can use one vector for different inserts. There may be a type I enzyme site for TEV site, I haven't found a good one yet. It's likely some inserts will have the same site internally, making the vector less useful. BTW, Accelagen has TurboTEV with dual GST- and His-tags. TurboTEV is stabilized through a mechanism different from the common S219 mutations. So it does not infringe the recently issued Yale patent. Cheers, Chun Chun Luo, Ph.D. Accelagen, Inc. 6044 Cornerstone Court West, Suite C San Diego, CA 92121 P: 858-678-8618 ext 111 F: 858-678-8628 c...@accelagen.com www.accelagen.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Charlie Bond Sent: Friday, June 05, 2009 4:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] TEV nucleotude sequence with restriction site Hi Jacob, If you're including the TEV site in your primer, then you won't need a restriction site after it (unless you're planning to recycle the vector afterwards for other inserts). Having the protease site too close to the protein can often make it difficult to cut. Cheers, Charlie Quotfintg Jacob Keller j-kell...@md.northwestern.edu: I checked out the Sheffield et al paper, and the restriction sites there are all just after the TEV site, thereby including, as Cynthia mentioned, at least an extra H beyond the obligatory G from the TEV site. I was hoping to be able to have only the G. (Since I am cloning in the TEV site with my PCR primer, I have free choice about what codons to choose, and therefore think it would be nice to have the restriction site in the TEV site itself, if possible. Also, this will keep my primer a little shorter.) 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: Cynthia Kinsland cl...@cornell.edu To: Jacob Keller j-kell...@md.northwestern.edu Cc: CCP4BB@JISCMAIL.AC.UK Sent: Friday, June 05, 2009 5:19 PM Subject: Re: [ccp4bb] TEV nucleotude sequence with restriction site I'm not quite sure what you want, but I have a series of vectors encoding various N-terminal tags and fusions, all followed by a TEV site. They have an MCS standard to many pET vectors. Therefore, they are designed to clone your gene in using the NdeI site at the 5' end (which will, after proteolysis, leave you with GH at the N-terminus of your protein). Other restriction enzymes in the MCS can be used, but more amino acids will be left at your N-terminus. I've used WatCut (from the U. Waterloo) for the silent mutagenesis question: http://watcut.uwaterloo.ca/watcut/watcut/template.php Best, Cynthia On Jun 5, 2009, at 5:41 PM, Jacob Keller wrote: Dear Crystallographers, Does anybody have a TEV-protease-site-coding nucleotide sequence with a commonly-used restriction site in it, preferably right at the end? Alternatively, does some somebody know of a program to determine all equivalent codon permutations for a small coding region, filtered for resulting restriction site possibilities? It seems like it would be an easy enough script to write... (I have already done some googling around for such a program, with not much luck.) 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 *** -- Charlie Bond Professorial Fellow University of Western Australia School of Biomedical, Biomolecular and Chemical Sciences M310, 35 Stirling Highway Crawley WA 6009, Australia
Re: [ccp4bb] TEV nucleotude sequence with restriction site
Does anybody have a TEV-protease-site-coding nucleotide sequence with a commonly-used restriction site in it, preferably right at the end? Alternatively, does some somebody know of a program to determine all equivalent codon permutations for a small coding region, filtered for resulting restriction site possibilities? It seems like it would be an easy enough script to write... Our main vectors (His- and His-MBP cleavable fusions) use blunt site right after TEV site. This way, you are not dependent on whether the same site is present in your gene and the cloning can be done without introducing any artefacts (besides Gly leftover). Here is the sequence: AGTGCCTGTACAAGGCCT AGGCCT is a StuI blunt cutter site (cheap and good enzyme) and GGC is a Gly So your insert is typically a PCR product that supplies the last letter in the Gly codon and you have a complete freedom of what follows. Blunt/sticky directional ligation, if done right, is extremely efficient and you only need to cut PCR product with one enzyme (no need to phosphorylate primers, the single non-ligatable joint gets repaired in E.coli). But then, we have by now almost completely switched to QuickChange cloning - clone anything anywhere anytime completely artefact-free (at least as long as the final plasmid is not larger than ~ 9 kbp). Dima
[ccp4bb] Jeff Christensen is out of the office.
I will be out of the office starting 06/05/2009 and will not return until 06/08/2009. I will respond to your message when I return. Thanks!
