[ccp4bb] AW: Re:[ccp4bb] AW: [ccp4bb] water at the same exactly position
Dear Lu, To be quite honest, before running refinement, I still use a simple program to add and delete waters based on electron density and distance criteria, which I wrote a long time ago in the dark ages of Vax computers and Evans Sutherland graphics when automatic water update did not exist. This program will guaranteed solve your problem. It is written in fortran with no dependencies, so it should be easy to get it running and I will be happy to send it with instructions so that even a GUI addict can use it. However, I am very surprised that a modern programs like Phenix would just crash and do not have options to clean-up the waters before starting refinement. Best regards, Herman Von: luzuok [mailto:luzuo...@126.com] Gesendet: Freitag, 31. Oktober 2014 01:33 An: Schreuder, Herman RD/DE Cc: CCP4BB@JISCMAIL.AC.UK Betreff: Re:[ccp4bb] AW: [ccp4bb] water at the same exactly position Dear Herman, If there are duplicate atoms in my PDB, Phenix refine GUI will post error and crash (Sorry I haven't tried other refinement program). And there is no .geo output file if program crash. Phenix refine thought them as nonbonded atoms with distance equal to 0. Because there are clash_guard parameter with nonbonded_distance_threshold = 0.5 . I think I may try to turn of the clash_guard parameter. Best Regards! Lu Zuokun -- 卢作焜 南开大学新生物站A202 At 2014-10-30 22:26:00, herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com wrote: Dear Lu, I don’t see your problem. Why don’t you use the automatic water update option, present in most modern refinement programs, as Pavel suggested? This should take care of your problem. Of course, having premium, 1st class hand-picked waters is still the gold standard, but I don’t believe the automatic waters are significantly worse. If you go down the list of waters, you start with fully occupied, well-defined waters, via partially occupied, less well-defined waters to overlapping, partially occupied waters, disordered carbohydrate chains, buffer components, cryo protectants and whatever else might have been present in your crystallization solution. I do not check my waters, but I carefully check any residual difference density. In the end it will be a subjective decision, either by yourself, or by an automatic procedure based on a number of cut-off criteria, whether or not to fit a water in some blob of density. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von luzuok Gesendet: Donnerstag, 30. Oktober 2014 04:54 An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] water at the same exactly position Dear Nicolas, It is really time-consuming! Philip told me to run the structure on PDB validation server. It will post error if there is duplicate molecules. Then I can directly find them on a text editor. I think it is better for COOT to solve this issue. Best reagards! Lu zuokun -- 卢作焜 南开大学新生物站A202 在 2014-10-29 22:29:35，FOOS Nicolas nicolas.f...@synchrotron-soleil.frmailto:nicolas.f...@synchrotron-soleil.fr 写道： Dear Lu, one simple solution is to remove the water molecules with text editor for example. It depend of how-many times you have multiply water molecules and if your model have several or more water molecules. In coot you can remove it graphically, but according to my knowledge not automatically, and it maybe time consuming. Hope to help Nicolas De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] de la part de luzuok [luzuo...@126.commailto:luzuo...@126.com] Envoyé : mercredi 29 octobre 2014 13:08 À : CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] water at the same exactly position Dear all, I found that there are some water molecules in my pdb that share the same position. This maybe cause by merging molecules in coot. It seems that I have mereged water molecules into my protein for more than one time. Does anyone tell me how to fix this problem? Best regards! Lu Zuokun -- 卢作焜 南开大学新生物站A202
[ccp4bb] fastening crystal formation
Dear all, I am trying to crystallize a 30 kD protein. Protein crystals are formed after 3 months. The crystals are formed in the following condition: 0.01M Zinc sulphate 0.1M MES monohydrate pH 6.5 25% v/v PEG monomethyl ether 550 Please suggest me how to grow these crystals faster. Thanking you -- Vijaykumar Pillalamarri, UGC-JRF, C/O: Dr. Anthony Addlagatta, Senior Scientist, CSIR-IICT, Tarnaka, Hyderabad, India-57 Mobile: +918886922975
Re: [ccp4bb] fastening crystal formation
I recommend to try seeding! On 31.10.2014 14:05, Vijaykumar Pillalamarri wrote: Dear all, I am trying to crystallize a 30 kD protein. Protein crystals are formed after 3 months. The crystals are formed in the following condition: 0.01M Zinc sulphate 0.1M MES monohydrate pH 6.5 25% v/v PEG monomethyl ether 550 Please suggest me how to grow these crystals faster. Thanking you -- Vijaykumar Pillalamarri, UGC-JRF, C/O: Dr. Anthony Addlagatta, Senior Scientist, CSIR-IICT, Tarnaka, Hyderabad, India-57 Mobile: +918886922975 -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] fastening crystal formation
Dear, the conditions you list should equilibrate within a couple of days. A growth time of three months could be a sign of some proteolytic reaction happening. You might want to sequence the crystal and subclone the fragment that actually crystallises. Best, Tim On 10/31/2014 11:05 AM, Vijaykumar Pillalamarri wrote: Dear all, I am trying to crystallize a 30 kD protein. Protein crystals are formed after 3 months. The crystals are formed in the following condition: 0.01M Zinc sulphate 0.1M MES monohydrate pH 6.5 25% v/v PEG monomethyl ether 550 Please suggest me how to grow these crystals faster. Thanking you -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] fastening crystal formation
Although three months is a long time, it is no completely unheard of, and does not require the invocation of proteolysis. The longest time I have heard of is ~1 yr, so count yourself lucky. However to get good advice, as well as to use it, you need to ask yourself several questions: 1. What kind of crystals are they? Protein, salt, etc? If they are salt, don't pursue this condition. 2. How many crystals did you get? One or 2 in a drop or a microcrystal shower. And of what kind? Single, well shaped, rosettes, needle clusters, or something that looks crystalline. Screen more broadly around your initial hit. 3. How many times have your tried to repeat this? Once, twice, or more? Did you try setups in duplicate? If so, did you get reproducible results? Have you actually screened around these conditions, varying each component systematically (PEG, salt, pH, buffer, etc.)? 4. What method did you use? And in what kind of container? For one thing, we don't completely trust the integrity of our setups for longer than 2 months. All containers leak water slowly, so when crystals take longer than 2 months to grow (a) the real conditions are at much higher values than you naively think (i.e., the drop has dried out more than you expected) or (b) other components are crystallizing, for example a zinc salt. It depends what else is in your protein buffer, as well. To quicken protein crystallization (which is not always a good thing), increase your protein concentration (by 1.5-2x) and/or PEG concentration (such as screening up to 40% PEGmme 550). Sadly, crystallization is a combination of thermodynamic and kinetic factors: you can get crystals (sometimes a single crystal only) when just outside the truly optimal conditions, but this may be only a sporadic event. You got to keep screening. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri vijaypkuma...@gmail.com wrote: Dear all, I am trying to crystallize a 30 kD protein. Protein crystals are formed after 3 months. The crystals are formed in the following condition: 0.01M Zinc sulphate 0.1M MES monohydrate pH 6.5 25% v/v PEG monomethyl ether 550 Please suggest me how to grow these crystals faster. Thanking you -- Vijaykumar Pillalamarri, UGC-JRF, C/O: Dr. Anthony Addlagatta, Senior Scientist, CSIR-IICT, Tarnaka, Hyderabad, India-57 Mobile: +918886922975
Re: [ccp4bb] fastening crystal formation
I assume you were using vapour diffusion. In that case, you could also try microbatch to cover the possibility of the plate slowly drying out and concentrations increasing beyond those of the original screen. Dmitry On Fri, Oct 31, 2014 at 12:07 PM, R. M. Garavito rmgarav...@gmail.com wrote: Although three months is a long time, it is no completely unheard of, and does not require the invocation of proteolysis. The longest time I have heard of is ~1 yr, so count yourself lucky. However to get good advice, as well as to use it, you need to ask yourself several questions: 1. What kind of crystals are they? Protein, salt, etc? If they are salt, don't pursue this condition. 2. How many crystals did you get? One or 2 in a drop or a microcrystal shower. And of what kind? Single, well shaped, rosettes, needle clusters, or something that looks crystalline. Screen more broadly around your initial hit. 3. How many times have your tried to repeat this? Once, twice, or more? Did you try setups in duplicate? If so, did you get reproducible results? Have you actually screened around these conditions, varying each component systematically (PEG, salt, pH, buffer, etc.)? 4. What method did you use? And in what kind of container? For one thing, we don't completely trust the integrity of our setups for longer than 2 months. All containers leak water slowly, so when crystals take longer than 2 months to grow (a) the real conditions are at much higher values than you naively think (i.e., the drop has dried out more than you expected) or (b) other components are crystallizing, for example a zinc salt. It depends what else is in your protein buffer, as well. To quicken protein crystallization (which is not always a good thing), increase your protein concentration (by 1.5-2x) and/or PEG concentration (such as screening up to 40% PEGmme 550). Sadly, crystallization is a combination of thermodynamic and kinetic factors: you can get crystals (sometimes a single crystal only) when just outside the truly optimal conditions, but this may be only a sporadic event. You got to keep screening. Good luck, Michael ** *R. Michael Garavito, Ph.D.* *Professor of Biochemistry Molecular Biology* *603 Wilson Rd., Rm. 513* *Michigan State University * *East Lansing, MI 48824-1319* *Office:* *(517) 355-9724 %28517%29%20355-9724 Lab: (517) 353-9125 %28517%29%20353-9125* *FAX: (517) 353-9334 %28517%29%20353-9334 Email: rmgarav...@gmail.com garav...@gmail.com* ** On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri vijaypkuma...@gmail.com wrote: Dear all, I am trying to crystallize a 30 kD protein. Protein crystals are formed after 3 months. The crystals are formed in the following condition: 0.01M Zinc sulphate 0.1M MES monohydrate pH 6.5 25% v/v PEG monomethyl ether 550 Please suggest me how to grow these crystals faster. Thanking you -- Vijaykumar Pillalamarri, UGC-JRF, C/O: Dr. Anthony Addlagatta, Senior Scientist, CSIR-IICT, Tarnaka, Hyderabad, India-57 Mobile: +918886922975
Re: [ccp4bb] fastening crystal formation
Michael (and some others) You haven't mentioned - and I guess don't use regularly - the random MMS approach, where crushed seed-crystals are added to random screens. This really is a terrific method, and it will on average roughly double productivity. It's the first thing I would think of in a case like Vijaykumar's (as I told him this morning!). Galina Obmolova and her colleagues published a great paper earlier this year about MMS. They were working with a set of 16 Fab fragments that comprised all combinations of 4 different heavy chains and 4 different light chains. Three structures were generated without MMS, but by various very creative uses of microseeding they were able to get all 16/16 structures. Ref below. Best wishes, Patrick __ Obmolova, G., Malia, T. J., Teplyakov, A., Sweet, R. W., Gilliland, G. L. (2014). Protein crystallization with microseed matrix screening: application to human germline antibody Fabs. *Structural Biology and Crystallization Communications*, *70*(8). On 31 October 2014 16:07, R. M. Garavito rmgarav...@gmail.com wrote: Although three months is a long time, it is no completely unheard of, and does not require the invocation of proteolysis. The longest time I have heard of is ~1 yr, so count yourself lucky. However to get good advice, as well as to use it, you need to ask yourself several questions: 1. What kind of crystals are they? Protein, salt, etc? If they are salt, don't pursue this condition. 2. How many crystals did you get? One or 2 in a drop or a microcrystal shower. And of what kind? Single, well shaped, rosettes, needle clusters, or something that looks crystalline. Screen more broadly around your initial hit. 3. How many times have your tried to repeat this? Once, twice, or more? Did you try setups in duplicate? If so, did you get reproducible results? Have you actually screened around these conditions, varying each component systematically (PEG, salt, pH, buffer, etc.)? 4. What method did you use? And in what kind of container? For one thing, we don't completely trust the integrity of our setups for longer than 2 months. All containers leak water slowly, so when crystals take longer than 2 months to grow (a) the real conditions are at much higher values than you naively think (i.e., the drop has dried out more than you expected) or (b) other components are crystallizing, for example a zinc salt. It depends what else is in your protein buffer, as well. To quicken protein crystallization (which is not always a good thing), increase your protein concentration (by 1.5-2x) and/or PEG concentration (such as screening up to 40% PEGmme 550). Sadly, crystallization is a combination of thermodynamic and kinetic factors: you can get crystals (sometimes a single crystal only) when just outside the truly optimal conditions, but this may be only a sporadic event. You got to keep screening. Good luck, Michael ** *R. Michael Garavito, Ph.D.* *Professor of Biochemistry Molecular Biology* *603 Wilson Rd., Rm. 513* *Michigan State University * *East Lansing, MI 48824-1319* *Office:* *(517) 355-9724 Lab: (517) 353-9125* *FAX: (517) 353-9334Email: rmgarav...@gmail.com garav...@gmail.com* ** On Oct 31, 2014, at 6:05 AM, Vijaykumar Pillalamarri vijaypkuma...@gmail.com wrote: Dear all, I am trying to crystallize a 30 kD protein. Protein crystals are formed after 3 months. The crystals are formed in the following condition: 0.01M Zinc sulphate 0.1M MES monohydrate pH 6.5 25% v/v PEG monomethyl ether 550 Please suggest me how to grow these crystals faster. Thanking you -- Vijaykumar Pillalamarri, UGC-JRF, C/O: Dr. Anthony Addlagatta, Senior Scientist, CSIR-IICT, Tarnaka, Hyderabad, India-57 Mobile: +918886922975 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] Nova F- (topic off in left field)
Apparently these are not for sale anymore by EMD Millipore yet it is what is recommended for plasmid preps with pETcoco-2. Does anyone know of a source of these cells or a substitute cell line we could use? Thanks, Gloria
[ccp4bb] off-topic: binding site comparison
Dear all, I'm sorry, this is a bit off-topic. I'm looking for a tool to compare pairs of binding sites. It doesn't need to be high-throughput, since I'll only be comparing ~100 pairs, but I'd like it to be robust and, more importantly, alignment-independent. Indeed, the binding sites I want to compare are not necessarily related in terms of amino-acid composition, but I'd like to still be able to detect similarities between them. Does anyone have suggestions? Thank you. Sarah -- Sarah Barelier, Ph.D. Postdoctoral Researcher Shoichet Lab, UCSF sarah.barel...@blur.compbio.ucsf.edu Department of Pharmaceutical Chemistry 1700 4th Street, Byers Hall, Room N501 San Francisco, CA 94158-2550
Re: [ccp4bb] off-topic: binding site comparison
Dear Sarah, we don't have a server for you to quickly use but our lab has methods for alignment-free binding sites comparison by comparing pocket surface shape, named Pocket-Surfer and Patch-Surfer. These are literature: http://www.ncbi.nlm.nih.gov/pubmed/20455259 http://www.ncbi.nlm.nih.gov/pubmed/22275074 http://bioinformatics.oxfordjournals.org/content/early/2014/10/29/bioinformatics.btu724.abstract If you are interested, I am happy to discuss what we can do for your problem. Sincerely, Daisuke Daisuke Kihara, Ph.D. Professo Department of Biological Sciences/Computer Science Purdue University West Lafayette, IN 47907 http://kiharalab.org Tel: 1-765-496-2284 On Fri, Oct 31, 2014 at 3:48 PM, Sarah Barelier sarah.barel...@blur.compbio.ucsf.edu wrote: Dear all, I'm sorry, this is a bit off-topic. I'm looking for a tool to compare pairs of binding sites. It doesn't need to be high-throughput, since I'll only be comparing ~100 pairs, but I'd like it to be robust and, more importantly, alignment-independent. Indeed, the binding sites I want to compare are not necessarily related in terms of amino-acid composition, but I'd like to still be able to detect similarities between them. Does anyone have suggestions? Thank you. Sarah -- Sarah Barelier, Ph.D. Postdoctoral Researcher Shoichet Lab, UCSF sarah.barel...@blur.compbio.ucsf.edu Department of Pharmaceutical Chemistry 1700 4th Street, Byers Hall, Room N501 San Francisco, CA 94158-2550 -- Daisuke
