Re: [ccp4bb] Rpim and how its related to anomalous signal
I also think Rpim, as commonly defined, is not directly useful for measuring the anomalous signal. Commenting on its use, though, I find it quite useful to judge whether one is adding useful data to already existing ones. In a multiple crystals context I always look at both Rmeas and Rpim. Suppose a given dataset shows some Rmeas and Rpim. Now add another dataset to the first one, in the hope to increase completeness and redundancy. This, of course, will depend on how isomorphic were the crystals from which the two datasets were collected. If the Rmeas stays stationary, or increases just a little, and Rpim decreases, this means thatthe crystals had a good degree of isomorphism and that scaled data will be more precise. This is what one wished for, when collecting from multiple crystals. On the other hand, an increase in Rpim definitely points at something wrong with the addition of the second dataset, quite certainly some form of non-isomorphism. J Dr James Foadi PhD Membrane Protein Laboratory Diamond Light Source Ltd. Diamond House Harwell Science and Innovation Campus Didcot Oxfordshire OX11 0DE United Kingdom office email: james.fo...@diamond.ac.uk alternative email: j.fo...@imperial.ac.uk personal web page: http://www.jfoadi.me.uk From: Jim Pflugrath jim.pflugr...@rigaku.com To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, 2 November 2012, 19:24 Subject: Re: [ccp4bb] Rpim and how its related to anomalous signal In my opinion Rpim is not related directly to anomalous signal, so perhaps that is why there is some confusion. Also I think some folks confused Rpim with Rrim. The latter is also called Rmeas. But once again, these are not related directly to anomalous signal. I do not find Rpim very useful for anything since when the data has high multiplicity Rpim gets very low, but as Michael Blum told me, Precision does not trump accuracy. I would personally rather have accurate data than highly redundant but inaccurate data. I like to look at the deltaF / sigma(deltaF) value reported by SHELXC and other programs, where deltaF is ||F+| - |F-||. This might also be called d/sig. There are other things to look at as well. Jim From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Vijayakumar.B [vijaybioscie...@gmail.com] Sent: Tuesday, September 18, 2012 4:36 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Rpim and how its related to anomalous signal Dear CCP4BB users, I am very much interested to work in Anomalous scattering technique for macromolecular structure determination. I have already gone through some literature in which they have explained about a parameter called “Rpim”. I am little bit confused about Rpim values. Can anyone tell me about the use of this parameter in structure determination by anomalous techniques and how Rpim is related to anomalous signal? Thanks in advance With kind regards B. Vijayakumar
[ccp4bb] protein cleavage
Dear CCP4 I am having a problem with cleaving my fusion protein and I would be grateful if you advice me regarding this situation, I have an MBP-DHBx fusion protein and I am trying to cleave it using TEV protease, I have tried different ratios and different temperatures with different incubation time but still it will not cleave, all I observe on the gel is the bands of the fusion protein which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa and no sign of the DHBx which is 17kDa,I have also checked the sequence if there was any problem but I could not find anything unusual the sequence was fine , so if you have any suggestions regarding this situation I will be thankful Best Regards Rana
Re: [ccp4bb] protein cleavage
You do not mention what buffer you are trying to do your cleavage in. You need a reducing agent for TEV to work (e.g. reduced gluthionine, DTT, mercaptoethanol). EDTA (0.5-1mM) is recommended as TEV is a cysteine protease and the presence of divalent metal ions will/eventually inhibit TEV. TEV becomes less active as salt concentration increases (50% active at 0.5M NaCl) If your protein contains disulphide bridges and you want to keep them intact you can use a ratio of 3 mM glutathione/0.3 mM oxidized glutathione which provides enough reducing power for TEV to work but should not disrupt disulphides. If none of these reasons are why your protein fails to cleave, there is a strong possibility that the TEV site is inaccessible. You could try 1M urea in this case. TEV again will be less active, but you could at least test this theory.
Re: [ccp4bb] protein cleavage
Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. It may be that your protein is not stable/soluble without the MBP fusion. Different buffer conditions may help if your protein is being lost post-cleavage. The efficiency of cleavage may also be aided by different buffer conditions. Since you didn't report the buffer you're using, it's impossible to judge if it is appropriate for maximum TEV activity. Cynthia Sent from my iPhone On Nov 4, 2012, at 10:24 AM, rana ibd rna19792...@yahoo.commailto:rna19792...@yahoo.com wrote: Dear CCP4 I am having a problem with cleaving my fusion protein and I would be grateful if you advice me regarding this situation, I have an MBP-DHBx fusion protein and I am trying to cleave it using TEV protease, I have tried different ratios and different temperatures with different incubation time but still it will not cleave, all I observe on the gel is the bands of the fusion protein which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa and no sign of the DHBx which is 17kDa,I have also checked the sequence if there was any problem but I could not find anything unusual the sequence was fine , so if you have any suggestions regarding this situation I will be thankful Best Regards Rana
Re: [ccp4bb] low-resolution data and SG
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear SDY, if you can see extra density after MR into which you can even build or correct the model it is a good sign your chose the correct space group. Check the geometry of your model. I suppose it is very distorted - a matrix weight of 0.03 sounds high for 3.4A data - You can go down by a factor of 10 at least. You may need to run refmac for many cycles - I have used 200-300 cycles with weight matrix 0.001 and the LL would still not converge. Regards, Tim On 11/04/2012 04:03 PM, SD Y wrote: Dear All, I have few basic questions for which I need help. I have a 3.4 A data and I have processed it to P4. 1. I used pointless to find SG, it suggests P41 21 2. But I see two strong intensities in systematic absences Intensities of systematic absences h k l Intensity Sigma I/Sigma 0 0 2 -0.7 0.3 -2.0 0 0 3 1.0 0.4 2.3 0 0 5 0.3 0.7 0.4 0 0 6 -0.7 0.9 -0.8 0 0 7 -0.4 0.9 -0.4 0 0 9 -0.2 0.9 -0.2 0 0 10 1.3 1.2 1.1 0 0 11 -0.8 2.1 -0.4 0 0 13 1.2 2.1 0.6 0 0 14 2.3 1.8 1.3 0 0 15 -1.0 1.9 -0.5 0 0 17 2.4 2.0 1.2 0 0 18 21.1 4.5 4.7 0 0 19 90.2 6.0 15.0 3 0 0 -0.1 0.2 -0.8 5 0 0 0.2 0.2 0.9 7 0 0 -0.3 0.2 -1.3 9 0 0 0.0 0.5 0.0 11 0 0 -0.2 0.6 -0.4 13 0 0 0.8 0.7 1.1 15 0 0 -1.2 0.6 -1.9 17 0 0 -0.3 0.8 -0.4 19 0 0 -1.4 0.6 -2.6 21 0 0 -2.2 1.2 -1.9 23 0 0 -0.8 1.3 -0.6 25 0 0 -1.2 1.1 -1.1 27 0 0 -0.9 1.6 -0.5 29 0 0 -0.4 1.7 -0.2 31 0 0 -7.1 1.3 -5.3 33 0 0 -2.4 2.1 -1.1 2. When I used phaser for MR, it gave weak solution in p43, so I scaled data in p43 21 2 (this also two intesities high like above in systamatic absences) and used for Phaser to get the following solution SINGLE solution SOLU SET RFZ=4.5 TFZ=9.4 PAK=0 LLG=105 TFZ==10.1 RF++ TFZ=17.7 PAK=0 LLG=282 TFZ==15.6 LLG=285 TFZ==12.4 SOLU SPAC P 43 21 2 SOLU 6DIM ENSE ensemble1 EULER 153.1 50.3 73.2 FRAC -0.11 0.03 -0.94 BFAC -2.65 SOLU 6DIM ENSE ensemble1 EULER 148.4 129.9 252.8 FRAC -0.32 -0.35 1.07 BFAC 4.01 Ensemble ensemble1 RMS variance(s): 1.13 3. I used this solution to further refine the model in refmac, using local ncs, with/without jelly, optimized weight/weight of 0.03, map sharpening with B=20 in several rounds. I noticed that R factor R factor stayed around 33% while R free keeps floating around 42%. I could see some density for missing loop in the model and I could build but the R work and R free moving apart. By reading, I understand that this is very common for low resolution data unless I use appropriate restraints. I am wondering if my space group is correct? I had understood that if it’s right SG, high intensity reflections will not be found in systematic absences but I started doubting if I have understood correctly. This is my first low resolution data, I want use this opportunity to learn refmac well. So could you please let me know if my doubt is right regarding SG and how do I troubleshoot. Thanks SDY - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFQlpsuUxlJ7aRr7hoRAgUMAKCJNhlDW4q2Lgmer4lZJoi+GpxDmACg9sRW a5HeDN5HHK/Wdy1sEY+9vbE= =aefJ -END PGP SIGNATURE-
Re: [ccp4bb] protein cleavage
Rana, I understood that these proteases are very efficient in cleavage. One time, I had a construct MBP-3C-protein, I never able to cleave this particular construct while I could do well with other truncations.The MBP you are seeing might be co-purified with DHBx and unless gel band intensity suggest that its coming from fusion protein. I feel cleavage site may not be accessible for the protease. Might be adding few more residues might help. Good luckSDY Date: Sun, 4 Nov 2012 07:24:42 -0800 From: rna19792...@yahoo.com Subject: [ccp4bb] protein cleavage To: CCP4BB@JISCMAIL.AC.UK Dear CCP4 I am having a problem with cleaving my fusion protein and I would be grateful if you advice me regarding this situation, I have an MBP-DHBx fusion protein and I am trying to cleave it using TEV protease, I have tried different ratios and different temperatures with different incubation time but still it will not cleave, all I observe on the gel is the bands of the fusion protein which is 59kDa and the MBP which is 42kDa and the TEV protease which is 27kDa and no sign of the DHBx which is 17kDa,I have also checked the sequence if there was any problem but I could not find anything unusual the sequence was fine , so if you have any suggestions regarding this situation I will be thankful Best Regards Rana
[ccp4bb] protein cleavage
Dear All Thank you for all your replies, the buffer for the TEV protease that I have used contains 50mM Tris-HCl, 150mM NaCl, 1mM EDTA, and 1mM DTT at PH= 8.0. I have tried using this buffer without NaCl but the TEV protease precipitates when dialyzing over night, as for using glutathione (reduced alone, also with the oxidized) still had the same result Rana
Re: [ccp4bb] low-resolution data and SG
This looks like an output from SCALEPACK. Unfortunately, one has no way to know from the output if 21 and 90 are strong intensities or not. One cannot go by the I/sigmaI alone. For example, suppose there is thermal diffuse scatter at these positions or perhaps there is a cosmic ray or radioactive decay (zinger) or a spot from a split crystal or the tail of a nearby streaky spot or other error during integrating of these Bragg reflections. I recommend you look at (a) neighboring reflections to get a sense of what a strong reflection value is. Maybe it is 20,000 or more so that 90 would be a weak reflection, and (b) the raw image itself at these reflection positions to see what the actual appearance of the pixel values in these positions are. Jim ... 0 0 17 2.4 2.0 1.2 0 0 18 21.1 4.5 4.7 0 0 19 90.2 6.0 15.0 ...
Re: [ccp4bb] protein cleavage
@Cynthia, On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote: Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. I think that is a wrong assumption. He did not specify if he sees the MBP band also just before TEV addition - it might also be truncation products which we happen to see all the time. The ratio varies depending on the construct but it can be as bad as a 1:1 ratio. You can really only tell if TEV cleaves if you do a time course experiment at RT with a defined amount of your protein and see if the fusion construct decreases. An alternative for the lack of your 17kDa desired band is simply your fusion construct is cleaved but your cleaved product might a) not be soluble at that pH or b) aggregates and precipitates. You might be able to perform the cleavage on the Amylose column keeping a constant flow cycling. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] protein cleavage
I assumed, perhaps wrongly, that the fusion was homogeneous prior to cleavage as the existence of the MBP band post-cleavage would not have been noteworthy if it had also been there pre-cleavage. A time course or at least a time zero sample is always a good idea. Truncation products prior to proteolysis can certainly complicate interpretation of results. In my experience, precipitation of the target protein upon proteolysis from an MBP fusion is a fairly common problem and not necessarily fixable. Cynthia Sent from my iPhone On Nov 4, 2012, at 1:19 PM, Bosch, Juergen jubo...@jhsph.edumailto:jubo...@jhsph.edu wrote: @Cynthia, On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote: Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. I think that is a wrong assumption. He did not specify if he sees the MBP band also just before TEV addition - it might also be truncation products which we happen to see all the time. The ratio varies depending on the construct but it can be as bad as a 1:1 ratio. You can really only tell if TEV cleaves if you do a time course experiment at RT with a defined amount of your protein and see if the fusion construct decreases. An alternative for the lack of your 17kDa desired band is simply your fusion construct is cleaved but your cleaved product might a) not be soluble at that pH or b) aggregates and precipitates. You might be able to perform the cleavage on the Amylose column keeping a constant flow cycling. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] protein cleavage
We once had a more-or-less MBP-sized fragment before cleavage, but this turned to be a spontaneous mutation. This expression experiment had been started from a glycerol stock with an unknown number of growth cycles prior to expression. Starting from a fresh transformation with the purified and sequenced plasmid solved the problem. Since then, I insist everybody does a fresh transformation before every expression experiment and not generate extra growth/dilution cycles beyond the normal transformation, growth on plate, overnight culture, dilution into large-scale expression culture. Quoting Bosch, Juergen: @Cynthia, On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote: Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. I think that is a wrong assumption. He did not specify if he sees the MBP band also just before TEV addition - it might also be truncation products which we happen to see all the time. The ratio varies depending on the construct but it can be as bad as a 1:1 ratio. You can really only tell if TEV cleaves if you do a time course experiment at RT with a defined amount of your protein and see if the fusion construct decreases. An alternative for the lack of your 17kDa desired band is simply your fusion construct is cleaved but your cleaved product might a) not be soluble at that pH or b) aggregates and precipitates. You might be able to perform the cleavage on the Amylose column keeping a constant flow cycling. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Extra electron density
Could it be a fatty acid? It looks like it has a (hydrophobic?) tail with a (charged?) head. Partially occupied perhaps, as they're so close together. On Sun, Nov 4, 2012 at 7:38 PM, yogesh khandokar yogesh.khando...@gmail.com wrote: Dear All I am working on an enzyme which involved in fatty acid biosynthesis. We solved the crystal structure of it. Biological unit of this enzyme is Hexamer and showing extra electron density in the center of Hexamer. Wincoot software doesn't recognize this extra electron density as water molecules/ substrate. My question is:Is there any way to find the molecules responsible for extra electron density? Picture is attached with email. Thanks in advance for your comments. Regards -- Yogesh Khandokar PhD Student School of Biomedical Sciences Charles Sturt University Wagga Wagga, NSW, Australia http://www.csu.edu.au/faculty/science/biomed/
Re: [ccp4bb] protein cleavage
Do you see the same MBP band (or corresponding to the same MW, in case it isn't MBP) before cleavage, at the same total concentration of protein? If not, your protein could be crashing out. Even so, if you use SDS and heat up the sample, I am surprised that your protein just disappeared. TEV protease has a pretty long recognition sequence. It doesn't appear as is in your protein, does it? Is it possible to attach the tag at the other terminus? Perhaps it is more accessible? Of course, it still doesn't solve the mystery of the disappearing protein if the band is indeed MBP that appears after the cleavage reaction. On Sun, Nov 4, 2012 at 3:07 PM, VAN RAAIJ , MARK JOHAN mjvanra...@cnb.csic.es wrote: We once had a more-or-less MBP-sized fragment before cleavage, but this turned to be a spontaneous mutation. This expression experiment had been started from a glycerol stock with an unknown number of growth cycles prior to expression. Starting from a fresh transformation with the purified and sequenced plasmid solved the problem. Since then, I insist everybody does a fresh transformation before every expression experiment and not generate extra growth/dilution cycles beyond the normal transformation, growth on plate, overnight culture, dilution into large-scale expression culture. Quoting Bosch, Juergen: @Cynthia, On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote: Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. I think that is a wrong assumption. He did not specify if he sees the MBP band also just before TEV addition - it might also be truncation products which we happen to see all the time. The ratio varies depending on the construct but it can be as bad as a 1:1 ratio. You can really only tell if TEV cleaves if you do a time course experiment at RT with a defined amount of your protein and see if the fusion construct decreases. An alternative for the lack of your 17kDa desired band is simply your fusion construct is cleaved but your cleaved product might a) not be soluble at that pH or b) aggregates and precipitates. You might be able to perform the cleavage on the Amylose column keeping a constant flow cycling. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Unusually low B factors with phenix
Pavel, 1.8.1-1168 also has this problem. I found this problem existed with some dataset, but not all dataset. 1.8.1 give better map, so I built model with 1.8.1 map and go back to 1.7.3 before I deposit the structure to PDB. When I compared the B factor from 1.8.x and 1.7.3, I found B(1.7.3)=B(1.8.x)+Boverall(1.8.x). Perhaps this is the problem. 2012/11/3, Pavel Afonine pafon...@gmail.com: Joao, I am using the latest version of phenix 1.8.1-1168 1.8.1-1168 should not have that problem. If you suspect there is still a problem, you can send me the data and model files off list, explain what exactly the problem is, and I will have a look right away. FYI: there is Phenix mailing list for Phenix-specific questions, where I can also explain in great details the behavior with the B-factors you observe. All the best, Pavel
