[ccp4bb] Reference for JLigand
Dear CCP4 Developers, What is the correct reference for JLigand to include in a publication ? Thanks best regards, Pedro Matias Industry and Medicine Applied Crystallography Macromolecular Crystallography Unit ___ Phones : (351-21) 446-9100 Ext. 1669 (351-21) 446-9669 (direct) Fax : (351-21) 441-1277 or 443-3644 email : mat...@itqb.unl.pt http://www.itqb.unl.pt/research/biological-chemistry/industry-and-medicine-applied-crystallography http://www.itqb.unl.pt/labs/macromolecular-crystallography-unit Mailing address : Instituto de Tecnologia Quimica e Biologica Apartado 127 2781-901 OEIRAS Portugal
[ccp4bb] Merging data collected at two different wavelength
Hi all, I have two datasets, both CO SAD data, one collected at CO anomalous wavelength at synchroton and the other at home source. I wish to combine these two data-sets and use for SAD phasing. Can anyone suggest how this can be done? Regards, ARKO -- *ARKA CHAKRABORTY* *CAS in Crystallography and Biophysics* *University of Madras* *Chennai,India*
Re: [ccp4bb] Merging data collected at two different wavelength
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Arko, could you not try MAD with the two different data sets? Otherwise you can check the strength of the anomalous signal for both sets separately (I am sure pointless prints the anomalous CC over resolution shell) and after merging them. If the signal increases, use the merged data set for SAD, it it does not, use them separately (but then you call it MAD...). Tim On 01/18/2012 12:03 PM, arka chakraborty wrote: Hi all, I have two datasets, both CO SAD data, one collected at CO anomalous wavelength at synchroton and the other at home source. I wish to combine these two data-sets and use for SAD phasing. Can anyone suggest how this can be done? Regards, ARKO - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPFqgcUxlJ7aRr7hoRAvTcAKCYRK3tWncS3s3WkCbRCylpWciZigCg8UG5 h7oLvHACutcAvbnBm4jknOs= =az8S -END PGP SIGNATURE-
[ccp4bb] His Purification
Hello Every one, I am trying to purify a human protein in a bacterial expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa). This TEV protease has N- terminal His tag. So I first elute my fusion protein with higher imidazole concentration and then do TEV cleavage by adding TEV protease,, but sadly It co-elutes other contamination proteins such as 72 kDa and 67 kDa, as mentioned above.. Now, I wanted to know,,, can I do On beads cleavage by directly adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? But I am worreid TEV protease which has N- terminal His tag also try to bind on Ni-NTA beads.. Please help me.. Thanks!! -- B4U
Re: [ccp4bb] Off-topic: ELNs
Hi - Here at the NKI, we had formed a committee to look at ELN solution two years ago. We had interviewed three vendors, and run two tests with twenty users. A brief description of the outcome: 1. None of the twenty test-users was satisfied with any of the two solutions - and each was annoyed for a different reason. 2. If that would work at all, better be prepared to dedicate one person for technical support (unless security considerations allow you to have cloud-based usage or remote hosting) and another person for scientific support (making templates for experiments - else in a year you would have 37 templates for how to run a gel filtration column). I would add, that for the whole thing to work at all you need an IT department that are really good and collaborative, and especially for cloud-based solutions you need a fast network. My personal conclusion - and not that of the NKI which continues to be dedicated to implementing an ELN solution may I clarify - was that I have wasted enough time, and I quit from the committee before Christmas. And, with no offense as well to my friends in the PiMS project, besides spending time and resources on that between 2004-2008, I also do not think that this is a solution for small lab like mine - admittedly it works well for bigger and better organized labs. Sorry for the negative vibes. A. On Jan 16, 2012, at 22:28, Seiji Sugiman-Marangos wrote: Hi, off-topic question regarding electronic laboratory notebooks. Our lab is planning on moving from paper to digital record keeping and I was wondering which of the available ELN platforms are being used by the ccp4 community. We are primarily a crystallography lab but we would also need some versatility in the platform as some of our lab members are more focused on biochemistry. Any suggestions or comments would be greatly appreciated! Thanks, Seiji P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
Re: [ccp4bb] His Purification
Have you tried reverse purification over the Ni-NTA column? That is the typical next step in purification after His-tag cleavage. Or did you mean to say that the impurities elute off with your cleaved protein during the reverse purification? If this is the case, you could try adding a reducing agent to help prevent unwanted interactions. You could also try a subsequent purification using ion exchange. As for on-column digestion... it is possible but comparatively inefficient. Hope this helps. --- Greg Costakes PhD Candidate Department of Structural Biology Purdue University Hockmeyer Hall, Room 320 240 S. Martin Jischke Drive, West Lafayette, IN 47907 ** Hard work often pays of in time, but Procrastination always pays off now ** - Original Message - From: PULSARSTRIAN bhanu.hydpri...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, January 18, 2012 7:56:39 AM Subject: [ccp4bb] His Purification Hello Every one, I am trying to purify a human protein in a bacterial expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa). This TEV protease has N- terminal His tag. So I first elute my fusion protein with higher imidazole concentration and then do TEV cleavage by adding TEV protease,, but sadly It co-elutes other contamination proteins such as 72 kDa and 67 kDa, as mentioned above.. Now, I wanted to know,,, can I do On beads cleavage by directly adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? But I am worreid TEV protease which has N- terminal His tag also try to bind on Ni-NTA beads.. Please help me.. Thanks!! -- B4U
Re: [ccp4bb] His Purification
On Wed, 2012-01-18 at 18:26 +0530, PULSARSTRIAN wrote: The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). This is only a problem if you plan to have imac purification as your only step. If the goal is crystallization, such products can definitely be used in initial trials (in fact, impurities may provide a benefit of nucleation), but you would have to introduce a second step eventually, which is often the ion-exchange. The co-purified proteins will be definitely removed at that step, so perhaps your time is better spent optimizing some high-res ion-exchange gradient. Two ways I can think of if you'd rather stick with IMAC is to try cobalt instead of nickel (might have different non-specific binding profile) or overload the resin with your protein (the presumption here is that it has higher affinity than impurities and you will get rid of them by simple competition). Or maybe the imidazole gradient could help. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] His Purification
May be this one helps The current study presents the design, engineering, and characterization of two E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. http://www.ncbi.nlm.nih.gov/pubmed/21602383 Regards,Raj Date: Wed, 18 Jan 2012 18:26:39 +0530 From: bhanu.hydpri...@gmail.com Subject: [ccp4bb] His Purification To: CCP4BB@JISCMAIL.AC.UK Hello Every one, I am trying to purify a human protein in a bacterial expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa). This TEV protease has N- terminal His tag. So I first elute my fusion protein with higher imidazole concentration and then do TEV cleavage by adding TEV protease,, but sadly It co-elutes other contamination proteins such as 72 kDa and 67 kDa, as mentioned above.. Now, I wanted to know,,, can I do On beads cleavage by directly adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? But I am worreid TEV protease which has N- terminal His tag also try to bind on Ni-NTA beads.. Please help me.. Thanks!! -- B4U
Re: [ccp4bb] His Purification
Another option is to try NEB NiCo21(DE3) cells. http://www.neb.com/nebecomm/products/productC2529.asp I have no relation to NEB beyond being a customer. They've mutated GlmS to eliminate binding to IMAC resins and have added chitin affinity tags to SlyD, ArnA and Can to allow simple post- IMAC removal of those common contaminants. I've just started testing them and would be interested in feedback from others. Best, Cynthia On Jan 18, 2012, at 10:21 AM, Ed Pozharski wrote: On Wed, 2012-01-18 at 18:26 +0530, PULSARSTRIAN wrote: The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). This is only a problem if you plan to have imac purification as your only step. If the goal is crystallization, such products can definitely be used in initial trials (in fact, impurities may provide a benefit of nucleation), but you would have to introduce a second step eventually, which is often the ion-exchange. The co-purified proteins will be definitely removed at that step, so perhaps your time is better spent optimizing some high-res ion-exchange gradient. Two ways I can think of if you'd rather stick with IMAC is to try cobalt instead of nickel (might have different non-specific binding profile) or overload the resin with your protein (the presumption here is that it has higher affinity than impurities and you will get rid of them by simple competition). Or maybe the imidazole gradient could help. Cheers, Ed. -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Merging data collected at two different wavelength
How to merge two or more runs depends on the software you used to process the images. If you used MOSFLM/CCP4, then you would use the programs REBATCH (perhaps REINDEX) and SORTMTZ to combine the unmerged mtz files (the ones that come out of MOSFLM) before feeding them to SCALA. My program Scaler Elves will do this automatically if you just put the unmerged mtz file names on the command line: http://bl831.als.lbl.gov/~jamesh/elves/manual/scaler.html Once they are scaled, by default Elves will merge different wavelengths separately, but you can run the merge.com script they produce with all on the command line to just average everything together into one merged mtz file. You can also feed the unmerged mtz files to POINTLESS and it can be told to do pretty much the same thing. POINTLESS will also take XDS_ASCII.HKL files as input, but I don't think it should surprise anyone that XSCALE was designed to combine data from as many XDS runs as you like, as well as do zero-dose extrapolation. If you are using the HKL Research Inc. suite, then you can put the *.sca files back into scalepack as individual frames (which is what you want to do to check for non-isomorphism), or you can input all the *.x files into one scalepack run and obtain a single merged *.sca file that way. There are plenty of other processing packages out there as well, but I will make no attempt to make this post a comprehensive list. Suffice it to say, the exact procedure for combining two runs depend on the software you are using (and the manuals are remarkably helpful). An important thing that is not done automatically, however, is to check if your space group has more than one indexing solution. Basically, if merohedral twinning is possible, then it is also possible that your two datasets were indexed differently. If so, they will not merge well! Until you re-index one of them. A nice table to use is here: http://www.ccp4.ac.uk/html/reindexing.html Again, POINTLESS can be used to try and re-index one dataset to match a reference, but if non-isomorphism is high, then it can be hard to tell. In general, it is better to merge/average data together when the sets are isomorphous and it is not a good idea to merge/average if they are not! How much non-isomorphism is too much depends on how small of a difference signal you are trying to measure. For example if you are trying to measure a 3% dispersive difference, then 15% non-isomorphism is way too much. It is an under-appreciated fact that radiation damage is a serious source of non-isomorphism. Banumathi et al. (2006) found about 1% increase in non-isomorphism per MGy of dose. If you don't know what a MGy is, then I recommend you have a look at the open-access article: http://dx.doi.org/10.1107/S0909049509004361 There are plenty of other sources of error as well, and although there is no a-priori reason to think that data taken from one instrument on one day would be any different from data taken from the same crystal on a completely different instrument on a different day, it is never surprising when they don't merge very well. By the way, I wouldn't use MAD to describe the mergeing of non-isomorphous datasets. Strictly speaking, MAD is at least an attempt to measure both anomalous (f) and dispersive (f') differences, and I don't think it is appropriate to use the term MAD when you know the dispersive signal is washed out by non-isomorphism. I call such attempts MSAD (mult-SAD), which I think helps differentiate them from actual MAD data collections where you at least try not to fry the crystal between measurements that you need to subtract to get your phasing signal. Unless, of course, you are doing RIP! Just my humble opinion, -James Holton MAD Scientist On 1/18/2012 3:08 AM, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Arko, could you not try MAD with the two different data sets? Otherwise you can check the strength of the anomalous signal for both sets separately (I am sure pointless prints the anomalous CC over resolution shell) and after merging them. If the signal increases, use the merged data set for SAD, it it does not, use them separately (but then you call it MAD...). Tim On 01/18/2012 12:03 PM, arka chakraborty wrote: Hi all, I have two datasets, both CO SAD data, one collected at CO anomalous wavelength at synchroton and the other at home source. I wish to combine these two data-sets and use for SAD phasing. Can anyone suggest how this can be done? Regards, ARKO - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPFqgcUxlJ7aRr7hoRAvTcAKCYRK3tWncS3s3WkCbRCylpWciZigCg8UG5 h7oLvHACutcAvbnBm4jknOs= =az8S -END PGP SIGNATURE-
Re: [ccp4bb] Off-topic: ELNs
On Jan 18, 2012, at 6:17 AM, Anastassis Perrakis wrote: Here at the NKI, we had formed a committee to look at ELN solution two years ago. We had interviewed three vendors, and run two tests with twenty users. A brief description of the outcome: 1. None of the twenty test-users was satisfied with any of the two solutions - and each was annoyed for a different reason. Would you mind elaborating on any of the reasons that resonated most with you? Are these ELNs simply over-engineered? You mention the requirement to make experiment templates. This sounds cumbersome and a potential for duplicated effort. Do ELNs generally require templates or other overhead in the form of end-user effort? James
Re: [ccp4bb] Merging data collected at two different wavelength
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Comments on the comments ;-): On 01/18/2012 05:54 PM, James Holton wrote: [...] An important thing that is not done automatically, however, is to check if your space group has more than one indexing solution. Basically, if merohedral twinning is possible, then it is also possible that your two datasets were indexed differently. If so, they will not merge well! Until you re-index one of them. A nice table to use is here: http://www.ccp4.ac.uk/html/reindexing.html both pointless (as you point out) and XDS do this automatically (whether or not they do it correctly is a different matter). [...] By the way, I wouldn't use MAD to describe the mergeing of non-isomorphous datasets. I agree, neither would I. Just to be on the save side and avoid confusion by less experienced readers of the list: I used the term MAD because there are two data sets collected at two different wavelenghts, both of which should give rise to a measurable anomalous signal from the Co in the sample. Cheers, Tim -James Holton MAD Scientist On 1/18/2012 3:08 AM, Tim Gruene wrote: Dear Arko, could you not try MAD with the two different data sets? Otherwise you can check the strength of the anomalous signal for both sets separately (I am sure pointless prints the anomalous CC over resolution shell) and after merging them. If the signal increases, use the merged data set for SAD, it it does not, use them separately (but then you call it MAD...). Tim On 01/18/2012 12:03 PM, arka chakraborty wrote: Hi all, I have two datasets, both CO SAD data, one collected at CO anomalous wavelength at synchroton and the other at home source. I wish to combine these two data-sets and use for SAD phasing. Can anyone suggest how this can be done? Regards, ARKO -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPFv9tUxlJ7aRr7hoRAqC/AJ48y5F8PP10HYVbUznPKcXKXG++zQCg70Ok xOQB6YcE1/6eP1he0wXfxyQ= =xY+/ -END PGP SIGNATURE-
Re: [ccp4bb] Merging data collected at two different wavelength
By the way, I wouldn't use MAD to describe the mergeing of non-isomorphous datasets. Strictly speaking, MAD is at least an attempt to measure both anomalous (f) and dispersive (f') differences, and I don't think it is appropriate to use the term MAD when you know the dispersive signal is washed out by non-isomorphism. I call such attempts MSAD (mult-SAD), which I think helps differentiate them from actual MAD data collections where you at least try not to fry the crystal between measurements that you need to subtract to get your phasing signal. Unless, of course, you are doing RIP! Isn't it true that we cannot even agree on what MAD stands for? Is the following right? M = Multiple-wavelength. I think everyone agrees to this, although I believe I've seen the occasional (and sometime non-sensical) variant A = Anomalous (I think everyone agrees, although this term should really be changed to resonant, as there is no anomaly to it anymore...) D = Diffraction, Dispersion, Destruction, Dissolution...? JPK
Re: [ccp4bb] Merging data collected at two different wavelength
Isn't it true that we cannot even agree on what MAD stands for? Is the following right? M = Multiple-wavelength. I think everyone agrees to this, although I believe I've seen the occasional (and sometime non-sensical) variant A = Anomalous (I think everyone agrees, although this term should really be changed to resonant, as there is no anomaly to it anymore...) D = Diffraction, Dispersion, Destruction, Dissolution...? JPK D =Discussion?
Re: [ccp4bb] Merging data collected at two different wavelength
On Jan 18, 2012, at 10:20 AM, Tim Gruene wrote: Comments on the comments ;-): Ditto [...] By the way, I wouldn't use MAD to describe the mergeing of non-isomorphous datasets. I agree, neither would I. Just to be on the save side and avoid confusion by less experienced readers of the list: I used the term MAD because there are two data sets collected at two different wavelenghts, both of which should give rise to a measurable anomalous signal from the Co in the sample. Using the terms 'MAD' and 'SAD' have always been confusing to me when considering more complex phasing cases. What happens if you have intrinsic Zn's, collect a 3wvl experiment and then derivatize it with SeMet or a heavy atom? Or the MAD+native scenario (SHARP) ? Instead of using MAD/SAD nomenclature I favor explicitly stating whether dispersive/anomalous/isomorphous differences (and what heavy atoms for each ) were used in phasing. Aren't analyzing the differences (independent of source) the important bit anyway? F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Merging data collected at two different wavelength
That is excellent! You refer obviously to the multiple anomalous discussions on the bb? (Maybe d = disagreement?) JPK On Wed, Jan 18, 2012 at 11:42 AM, D Bonsor dbon...@ihv.umaryland.edu wrote: Isn't it true that we cannot even agree on what MAD stands for? Is the following right? M = Multiple-wavelength. I think everyone agrees to this, although I believe I've seen the occasional (and sometime non-sensical) variant A = Anomalous (I think everyone agrees, although this term should really be changed to resonant, as there is no anomaly to it anymore...) D = Diffraction, Dispersion, Destruction, Dissolution...? JPK D =Discussion? -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Merging data collected at two different wavelength
Hi, Regardless of what the consensus on naming for the technique, I'd suggest you combine these datasets during phasing (I'm aware of MLPHARE, SHARP, PHASIT supporting multiple anomalous datasets during phasing; others probably do as well). Combining at the merging step (pointless/scalepack/etc) might result in averaging amplitudes collected at different wavelengths (and therefore with different anomalous signal): and since your phases will come from amplitude differences this may result in less reliable phases. Pete arka chakraborty wrote: Hi all, I have two datasets, both CO SAD data, one collected at CO anomalous wavelength at synchroton and the other at home source. I wish to combine these two data-sets and use for SAD phasing. Can anyone suggest how this can be done? Regards, ARKO -- ARKA CHAKRABORTY CAS in Crystallography and Biophysics University of Madras Chennai,India
Re: [ccp4bb] His Purification
Make sure the imidazole is removed before you apply the TEV digested sample back to the Ni column for the reverse Ni step. Sounds like the expression level of your protein is low. You many consider to improve the expression. Those contamination proteins disappear from Ni elution when the target protein is abundant. As mentioned by Greg Costakes, on beads/column cleavage is less efficient comparing to solution digestion. However on column digestion is useful for some difficult-to-purify proteins. We have used our product TurboTEV (http://www.accelagen.com/TurboTEV.htm) for on column digestions. We found that keeping the TurboTEV to target protein ratio at 1:20 to 1:100 (same ratio for digestion in solution) gives good digestion, although never complete. That means you need to use 0.2 mg/ml TurboTEV for a resin with binding capacity of 20 mg/ml. We made TurboTEV storage buffer compatible with Ni resins. TurboTEV has dual GST- and His-tags. The geometry could be different from His-tagged TEV products. TurboTEV also has very high specific activity due to a novel stabilizing mechanism that is independent of S219 mutations. These properties probably make TurboTEV more efficient for on column digestion than other TEV products. You need to experiment a little bit with His-tagged TEV products. Chun Accelagen From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of PULSARSTRIAN Sent: Wednesday, January 18, 2012 4:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] His Purification Hello Every one, I am trying to purify a human protein in a bacterial expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein is 87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able to get rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene (67 kDa). I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa). This TEV protease has N- terminal His tag. So I first elute my fusion protein with higher imidazole concentration and then do TEV cleavage by adding TEV protease,, but sadly It co-elutes other contamination proteins such as 72 kDa and 67 kDa, as mentioned above.. Now, I wanted to know,,, can I do On beads cleavage by directly adding TEV enzyme when the fusion protein is still bound to Ni-NTA beads?? But I am worreid TEV protease which has N- terminal His tag also try to bind on Ni-NTA beads.. Please help me.. Thanks!! -- B4U
Re: [ccp4bb] Merging data collected at two different wavelength
Can I be dogmatic about this ? Multiwavelength anomalous diffraction from Hendrickson (1991) Science Vol. 254 no. 5028 pp. 51-58 Multiwavelength anomalous diffraction (MAD) from the CCP4 proceedings http://www.ccp4.ac.uk/courses/proceedings/1997/j_smith/main.html Multi-wavelength anomalous-diffraction (MAD) from Terwilliger Acta Cryst. (1994). D50, 11-16 etc. I don't see where the problem lies: a SAD experiment is a single wavelength experiment where you are using the anomalous/dispersive signals for phasing a MAD experiment is a multiple wavelength version of SAD. Hopefully one picks an appropriate range of wavelengths for whatever complex case one has. One can have SAD and MAD datasets that exploit anomalous/dispersive signals from multiple difference sources. This after all is one of the things that SHARP is particularly good at accommodating. If you're not using the anomalous/dispersive signals for phasing, you're collecting native data. After all C,N,O,S etc all have a small anomalous signal at all wavelengths, and metalloproteins usually have even larger signals so the mere presence of a theoretical d difference does not make it a SAD dataset. ALL datasets contain some anomalous/dispersive signals, most of the time way down in the noise. Phil Jeffrey Princeton On 1/18/12 12:48 PM, Francis E Reyes wrote: Using the terms 'MAD' and 'SAD' have always been confusing to me when considering more complex phasing cases. What happens if you have intrinsic Zn's, collect a 3wvl experiment and then derivatize it with SeMet or a heavy atom? Or the MAD+native scenario (SHARP) ? Instead of using MAD/SAD nomenclature I favor explicitly stating whether dispersive/anomalous/isomorphous differences (and what heavy atoms for each ) were used in phasing. Aren't analyzing the differences (independent of source) the important bit anyway? F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Merging data collected at two different wavelength
Can I be dogmatic about this ? I wish you could, but I don't think so, because even though those sources call it that, others don't. I agree with your thinking, but usage is usage. a SAD experiment is a single wavelength experiment where you are using the anomalous/dispersive signals for phasing I think dispersive usually refers to differences caused by changes in f'/f between wavelengths, no? JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Merging data collected at two different wavelength
Can I be dogmatic about this ? I wish you could, but I don't think so, because even though those sources call it that, others don't. I agree with your thinking, but usage is usage. And 10,000 lemmings can't be wrong?
Re: [ccp4bb] Merging data collected at two different wavelength
This begs the question* whether you want the lemmings to understand you. One theory of language, gotten more or less from Strunk and White's Elements of Style, is that the most important feature of language is its transparency to the underlying thoughts. Bad language breaks the transparency, reminds you that you are reading and not simply thinking the thoughts of the author, who should also usually be invisible. Bad writing calls attention to itself and to the author, whereas good writing guides the thoughts of the reader unnoticeably. For Strunk and White, it seems that all rules of writing follow this principle, and it seems to be the right way to think about language. So, conventions, even when somewhat inaccurate, are important in that they are often more transparent, and the reader does not get stuck on them. Anyway, a case in point of lemmings is that once Wayne Hendrickson himself suggested that the term anomalous be decommissioned in favor of resonant. I don't hear any non-lemmings jumping on that bandwagon... JPK *Is this the right use of beg the question? On Wed, Jan 18, 2012 at 1:57 PM, Phoebe Rice pr...@uchicago.edu wrote: Can I be dogmatic about this ? I wish you could, but I don't think so, because even though those sources call it that, others don't. I agree with your thinking, but usage is usage. And 10,000 lemmings can't be wrong? -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Merging data collected at two different wavelength
But if we were to follow that convention we would have been stuck with Multi-wavelength Resonant Diffraction Experimental Results, or, quite simply, MuRDER. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Wednesday, January 18, 2012 3:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data collected at two different wavelength This begs the question* whether you want the lemmings to understand you. One theory of language, gotten more or less from Strunk and White's Elements of Style, is that the most important feature of language is its transparency to the underlying thoughts. Bad language breaks the transparency, reminds you that you are reading and not simply thinking the thoughts of the author, who should also usually be invisible. Bad writing calls attention to itself and to the author, whereas good writing guides the thoughts of the reader unnoticeably. For Strunk and White, it seems that all rules of writing follow this principle, and it seems to be the right way to think about language. So, conventions, even when somewhat inaccurate, are important in that they are often more transparent, and the reader does not get stuck on them. Anyway, a case in point of lemmings is that once Wayne Hendrickson himself suggested that the term anomalous be decommissioned in favor of resonant. I don't hear any non-lemmings jumping on that bandwagon... JPK *Is this the right use of beg the question? On Wed, Jan 18, 2012 at 1:57 PM, Phoebe Rice pr...@uchicago.edu wrote: Can I be dogmatic about this ? I wish you could, but I don't think so, because even though those sources call it that, others don't. I agree with your thinking, but usage is usage. And 10,000 lemmings can't be wrong? -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Merging data collected at two different wavelength
On Wednesday, 18 January 2012, Soisson, Stephen M wrote: But if we were to follow that convention we would have been stuck with Multi-wavelength Resonant Diffraction Experimental Results, or, quite simply, MuRDER. You could switch that to Multiple Energy Resonant Diffraction Experiment but I don't think that would help any. As to anomalous - the term comes from the behaviour of the derivative delta_(optical index) / delta_(wavelength) This term is positive nearly everywhere, but is anomalously negative at the absorption edge. Ethan -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Wednesday, January 18, 2012 3:13 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Merging data collected at two different wavelength This begs the question* whether you want the lemmings to understand you. One theory of language, gotten more or less from Strunk and White's Elements of Style, is that the most important feature of language is its transparency to the underlying thoughts. Bad language breaks the transparency, reminds you that you are reading and not simply thinking the thoughts of the author, who should also usually be invisible. Bad writing calls attention to itself and to the author, whereas good writing guides the thoughts of the reader unnoticeably. For Strunk and White, it seems that all rules of writing follow this principle, and it seems to be the right way to think about language. So, conventions, even when somewhat inaccurate, are important in that they are often more transparent, and the reader does not get stuck on them. Anyway, a case in point of lemmings is that once Wayne Hendrickson himself suggested that the term anomalous be decommissioned in favor of resonant. I don't hear any non-lemmings jumping on that bandwagon... JPK *Is this the right use of beg the question? On Wed, Jan 18, 2012 at 1:57 PM, Phoebe Rice pr...@uchicago.edu wrote: Can I be dogmatic about this ? I wish you could, but I don't think so, because even though those sources call it that, others don't. I agree with your thinking, but usage is usage. And 10,000 lemmings can't be wrong?
[ccp4bb] How to tell Refmac it is a fixed double bond?
Hi every one I have made a Cif file for the restraints of my ligand with Jligand, which is attached to my protein via a lysine-aldehyde Schiff base formation. The problem is that whenever I run the refmac with the Cif file with torsions and link description, it changes the distance of the Lysine and the Carbon of my ligand. The density is there, but it does not recognize it as a C=N bond and puts them up to 2 angstrom away from each other. I do not know, if I should change the link description to make the Refmac regonize the double bond or what else I can do. This is the description of link in my Cif file: _chem_link_bond.link_id _chem_link_bond.atom_1_comp_id _chem_link_bond.atom_id_1 _chem_link_bond.atom_2_comp_id _chem_link_bond.atom_id_2 _chem_link_bond.type _chem_link_bond.value_dist _chem_link_bond.value_dist_esd LYS-MER 1 NZ 2 C18 double 1.2600.020 I appriciate your help beforehand. Regards Sam
Re: [ccp4bb] Reference for JLigand
Hopefully by the time your paper accepted it will be out. Here is the reference: JLigand: a graphical tool for CCP4 template restraint library Lebedev AA, Young P, Isupov MN, Moroz OV, Vagin AA and Murshudov GN (2012) Acta Cryst D68, in press (submitted, in consideration or whatever) I hope it helps regards Garib On 18 Jan 2012, at 09:47, Pedro M. Matias wrote: Dear CCP4 Developers, What is the correct reference for JLigand to include in a publication ? Thanks best regards, Pedro Matias Industry and Medicine Applied Crystallography Macromolecular Crystallography Unit ___ Phones : (351-21) 446-9100 Ext. 1669 (351-21) 446-9669 (direct) Fax : (351-21) 441-1277 or 443-3644 email : mat...@itqb.unl.pt http://www.itqb.unl.pt/research/biological-chemistry/industry-and-medicine-applied-crystallography http://www.itqb.unl.pt/labs/macromolecular-crystallography-unit Mailing address : Instituto de Tecnologia Quimica e Biologica Apartado 127 2781-901 OEIRAS Portugal Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] How to tell Refmac it is a fixed double bond?
Dear Sam double bond is only used to find bon lengths and other parameters. Refmac uses bond length. As I see you would like to make a link between two residues. There is a tutorial written by Andrey Lebedev that addresses exactly this type of problems. Please have a look this site: http://www.ysbl.york.ac.uk/mxstat/JLigand/index.html there are three tutorials: 1) simple ligand, 2) links (that is what you want) and 3) simple metal containing ligand treatment. I hope it helps. If it does not please let us know. regards Garib On 18 Jan 2012, at 21:07, Sam Arnosti wrote: Hi every one I have made a Cif file for the restraints of my ligand with Jligand, which is attached to my protein via a lysine-aldehyde Schiff base formation. The problem is that whenever I run the refmac with the Cif file with torsions and link description, it changes the distance of the Lysine and the Carbon of my ligand. The density is there, but it does not recognize it as a C=N bond and puts them up to 2 angstrom away from each other. I do not know, if I should change the link description to make the Refmac regonize the double bond or what else I can do. This is the description of link in my Cif file: _chem_link_bond.link_id _chem_link_bond.atom_1_comp_id _chem_link_bond.atom_id_1 _chem_link_bond.atom_2_comp_id _chem_link_bond.atom_id_2 _chem_link_bond.type _chem_link_bond.value_dist _chem_link_bond.value_dist_esd LYS-MER 1 NZ 2 C18 double 1.2600.020 I appriciate your help beforehand. Regards Sam Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
[ccp4bb] conversion of IU/ml to mcg
We are involved in R D of recombinant filgrastim and the standard sample label mentions it as 30MiOU/ml i.e 300 mcg/ml. How can we determine the IU/ml as we know our protein is 300 mcg/ml. can anyone please guide me on the corelation. regards, megha
