Re: [ccp4bb] Protein expression
Hi Reza, In addition to the many useful suggestions already made, I would suggest lowering the final concentrations of IPTG. In many cases, 1mM IPTG interferes with expression levels and/or solubility. This suggestion does not address your concern for why things become ugly in going from 3mL to 500mL (for which there can be many many explanations), but its worth a try before you head off to make many more clones. Cheers and best wishes, Raji On Thu, Mar 19, 2015 at 3:33 PM, Reza Khayat rkha...@ccny.cuny.edu wrote: Hi, We can express quite a bit of soluble protein when growing 3ml cultures. However, the protein becomes insoluble (inclusion bodies) when we scale up to 500ml cultures. Has anyone experienced such a problem, and found a solution to it? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor Department of Chemistry City College of New York New York, NY 10031 http://www.khayatlab.org/ 212-650-6070
Re: [ccp4bb] Searching for DNA structures in the PDB
Hi Sophie, The crystal structure of winged helix protein/transcription factor HNF-3gamma bound to DNA comes to mind. Also, other transcription factors of that ilk would be worth looking at. Also, if you are looking for proteins that bend DNA in a sequence-independent manner, one of the best examples to my knowledge is the crystal structure of the nucleosome core particle (PDB id, 1aoi). With histones having the job of compressing all of our genomic DNA into discs-like nucleosome core particles (NCP), you may find it very informative to see the many specifics of how histones bind the 146-bp DNA in a sequence-independent fashion and bend the 146-bp DNA every or so base pairs throughout the length of 146 base pairs. Look up Luger et al., 1997 (Nature) and related publications where the sequence-independent bending of DNA is discussed in great detail. Hope this helps. Raji On Tue, Jun 3, 2014 at 9:25 AM, Sophie Bliss mbp1...@sheffield.ac.uk wrote: Hello, I was wondering if anyone knows of a method that can be used to search the PDB for DNA/RNA structures (essentially a Dali search for DNA/RNA)? I have recently obtained a 2.3 A protein structure bound in complex with DNA. X3DNA has shown the DNA to be B-form generally but the 3DNA output and the structure both show a significant bend in the DNA chain. Other than apparently non-specific nuclease activity, we do not know the function of the protein in vivo. It would be very useful to be able to look at other proteins that bind DNA/RNA that target/produce similar bends in DNA/RNA structure, independent of base sequence. However, with thousands of structures in the PDB containing DNA it would be impossible to do this by eye! Thanks for reading, I look forward to hearing your suggestions. Sophie, Sheffield University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Difficult MR with MBP fusion protein
Hi Niu, Several things come to mind. First, it may not be trivial when the first component to be placed is ~20kDa and the second component (SU) is ~43kDa. The signal after placing the first component may be weak. Also, if the model for the smaller SU has low sequence identity with the target and you have 4 Angstrom data, it would not be surprising to encounters problems after searching with the smaller SU. But the question of why MR with MBP failed is worth investigating. Here are couple of quick questions: (1) When you say you got some better results with the smaller component, what are the TFZ and LLG values? (2) Assuming you have very good 4A data, the correct space group, and a successful rotation and translation search with the first component so that maps are not entirely crappy, do you actually see a big mass of unaccounted density that could potentially fit MBP when you look at the maps after placing the first component? If yes, how about physically placing MBP in there yourself and starting from there. (3) What is the sequence identity of model with the target 20kDa component? (4) Do you know for sure that you have MBP in the crystal? Unlikely concern but I ask nonetheless. Also, I'm curious whether you ran Phaser jobs with the default settings or whether you tried tweaking some of the parameters? I'm happy to speak further about this offline. Good luck! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University On Fri, May 16, 2014 at 11:03 AM, Niu Tou niutou2...@gmail.com wrote: Dear All, Recently we collected some data of a MBP fusion protein, at around 4A resolution. The protein itself is about half of the MBP size. However when we tried to solve it with MR, it failed. We tried to use MBP alone, homology model of target protein alone, and MBP+model. It is very strange that MBP alone can not yield any reasonable solution at all, so does searching with MBP and model together. While searching with model alone could get some better results, but when fix it to search MBP, it failed. There are 1 molecule per ASU with solvent content 55%. The spacegroup should be right and we tried to search all possible alternatives in each run, we also tried to lower it down, but did not work either. When running Phenix.phaser, there is a warning at the beginning saying eLLG suggests placing of ensembles will be very difficult. I wonder if anybody has encountered similar situation before. Any suggestions will be greatly appreciated! Regards, Niu
Re: [ccp4bb] Trouble cleaving SUMO tag off of membrane protein
Thanks to everyone who responded to my post (especially, John Lee and Brad Bennett) with helpful comments and suggestions. The good news is that I was able to get the Ulp1 protease cleavage step to 100% completion at 4C by using between 1:3-to-1:2 enzyme:substrate ratios and incubating the reactions overnight on an orbital shaker to ensure constant mixing (instead of dialysis). It turns out that I didn't have to switch from beta-mercaptoethanol to TCEP. I also kept NaCl concentration at 150mM Nacl and that was okay as well. Many thanks that I can now be off to the next hurdle! Raji On Sun, Feb 16, 2014 at 10:58 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Everyone, After several attempts to cleave the SUMO tag off my membrane protein under various conditions (different reducing agents, enzyme-to-substrate ratios, etc.) and after reading the manual and troubleshooting guide, I'm reaching out to the ccp4bb community. Experiment: I dialyze the mixture of SUMO-membrane protein and Ulp1 Express protease against buffer containing 20mM Tris pH 7, 150mM NaCl, 1mM DTT or 2mM beta-mercaptoethanol and 0.02% DDM at 4C overnight (14-18 hours). I am currently using an enzyme-to-substrate molar ratio of 1-to-15-20. Problem: With buffer containing 1mM DTT, I get about 50% tag-cleaved protein and 50% tagged protein. With buffer containing 2mM bME, I get about 30% tag-cleaved protein and 70% tagged protein. Couple of things: (1) Side-by-side cleavage of a SUMO-tagged control soluble protein by the same batch of Ulp1 works to 100% completion. (2) Detergent (0.02% DDM) did not interfere at all with cleavage of the SUMO-tagged control soluble protein. (3) I cannot set up the cleavage reaction at 30C or 37C and must stick with 4C, a protocol that I have used successfully in the past with SUMO-tagged soluble proteins. Although membrane proteins supposedly form a protein-detergent complex, I wonder if some of my protein is in micelles and if the random orientation of my SUMO-tagged protein in micelles may be the cause for incomplete digestion. I've also suspected that some of my membrane protein may be misfolded and oligomeric/aggregated, making the cleavage site inaccessible to the protease. But suppose the above explanations are not the problem in my case and that it's a technical issue and I am missing something very simple. Therefore, I am planning to set up more reactions ramping up the ratio of enzyme-to-substrate, increasing amount of bME (I prefer bME over DTT since I need to rebind the cleaved mixture to His-affinity resin) and decreasing the NaCl concentration to 100mM or lower (although 250mM NaCl did not interfere with cleavage of control protein). Have folks working with SUMO-tagged membrane protein encountered similar problems? I am purifying membrane protein from 30L bacterial culture and the yields are not all that great. So, if possible, I'd like to get the cleavage reaction to completion so that I don't have to suffer a 50% loss of protein at this step. I have a construct for my membrane protein without a SUMO tag and the expression is abysmal. Thanks very much for your time and suggestions! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Determining concentration of membrane protein
Hi Everyone, Thank you so much for your additional tips about various kits. My protein is tagged and when I cleave off the tag (a step that needs reducing agent), I will unfortunately have both detergent and reducing agent in my protein buffer. Nicolas, thanks for your word of caution. I can't therefore use Pierce RAC BCA kit but it looks like I should be able to use the 660 nm kit recommended by Ho (Thanks very much, Ho!). By extension, is it then the case that the BCA assay is not recommended when both detergent and reducing agent are present or is that just a peculiarity of the Pierce RAC BCA kit? Thanks very much to everyone who responded! Raji On Fri, Feb 14, 2014 at 9:49 AM, Patrick Loll pat.l...@drexel.edu wrote: No, because Bradford is based on the increase in absorbance when the dye moves from a hydrophilic environment to a hydrophobic one (like the protein interior, or like the interior of a micelle). When detergents are present in excess of their CMC, the change in absorbance from partitioning into the micelles is generally large compared to any signal due to protein binding; plus preparing a perfectly matched blank solution is challenging when dealing with protein-detergent solutions. I second Michael's recommendation--BCA works well. On 14 Feb 2014, at 1:45 AM, Niks wrote: Dear All, May be a stupid question. But if we take buffer with detergent as control (Blank), would not the difference in ODs using any of the methods used e.g. Bradford assay, gives protein concentration? Regards Nishant On Thu, Feb 13, 2014 at 9:09 PM, Roger Rowlett rrowl...@colgate.eduwrote: Your basic choices for protein assays are: 1. Alkaline copper methods (e.g., Biuret and micro-biuret) 2. alkaline copper + molybdate methods (e.g., Lowry, BCA assays) 3. Hydrophobic dye methods (e.g. Bradford) 4. UV methods (e.g., A280, A230, A210, etc.) Method 1 is least sensitive to amino acid composition, but is also has highest detection limits. Thiols interfere. Method 2 is very idiosyncratic with amino acid composition, and also subject to interference by thiols. Method 3 is not usable in detergent solutions. Method 4 has many inteferences as most everything absorbs in the far UV region. If you have some special protein cofactors, metals, chromophores, etc. these can be exploited for better measurements. For ecample metalloproteins are easy to quantify by ICP-OES or TXRF if they are reasonably pure. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote: Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my membrane protein. My purified membrane protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%). After reading the friendly manuals and searching online, I've learned that detergents interferes with assays like Bradford but can't find good descriptions of what works best. For now, I am trying to estimate concentration from absorbance at 280nm and using molar extinction coefficients based on aromatic amino acids, but again suspect detergent interference. I would like to know what other folks working on membrane proteins are doing. Thanks very much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- The most difficult phase of life is not when No one understands you;It is when you don't understand yourself -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Trouble cleaving SUMO tag off of membrane protein
Hi Everyone, After several attempts to cleave the SUMO tag off my membrane protein under various conditions (different reducing agents, enzyme-to-substrate ratios, etc.) and after reading the manual and troubleshooting guide, I'm reaching out to the ccp4bb community. Experiment: I dialyze the mixture of SUMO-membrane protein and Ulp1 Express protease against buffer containing 20mM Tris pH 7, 150mM NaCl, 1mM DTT or 2mM beta-mercaptoethanol and 0.02% DDM at 4C overnight (14-18 hours). I am currently using an enzyme-to-substrate molar ratio of 1-to-15-20. Problem: With buffer containing 1mM DTT, I get about 50% tag-cleaved protein and 50% tagged protein. With buffer containing 2mM bME, I get about 30% tag-cleaved protein and 70% tagged protein. Couple of things: (1) Side-by-side cleavage of a SUMO-tagged control soluble protein by the same batch of Ulp1 works to 100% completion. (2) Detergent (0.02% DDM) did not interfere at all with cleavage of the SUMO-tagged control soluble protein. (3) I cannot set up the cleavage reaction at 30C or 37C and must stick with 4C, a protocol that I have used successfully in the past with SUMO-tagged soluble proteins. Although membrane proteins supposedly form a protein-detergent complex, I wonder if some of my protein is in micelles and if the random orientation of my SUMO-tagged protein in micelles may be the cause for incomplete digestion. I've also suspected that some of my membrane protein may be misfolded and oligomeric/aggregated, making the cleavage site inaccessible to the protease. But suppose the above explanations are not the problem in my case and that it's a technical issue and I am missing something very simple. Therefore, I am planning to set up more reactions ramping up the ratio of enzyme-to-substrate, increasing amount of bME (I prefer bME over DTT since I need to rebind the cleaved mixture to His-affinity resin) and decreasing the NaCl concentration to 100mM or lower (although 250mM NaCl did not interfere with cleavage of control protein). Have folks working with SUMO-tagged membrane protein encountered similar problems? I am purifying membrane protein from 30L bacterial culture and the yields are not all that great. So, if possible, I'd like to get the cleavage reaction to completion so that I don't have to suffer a 50% loss of protein at this step. I have a construct for my membrane protein without a SUMO tag and the expression is abysmal. Thanks very much for your time and suggestions! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Determining concentration of membrane protein
Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my membrane protein. My purified membrane protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%). After reading the friendly manuals and searching online, I've learned that detergents interferes with assays like Bradford but can't find good descriptions of what works best. For now, I am trying to estimate concentration from absorbance at 280nm and using molar extinction coefficients based on aromatic amino acids, but again suspect detergent interference. I would like to know what other folks working on membrane proteins are doing. Thanks very much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Sister CCPs
Hi Folks, Like many other folks who read, benefit and hopefully contribute to this bulletin board, I am required to stay abreast of the latest cloning, heterologous membrane protein expression, purification, biochemistry, crystallization, and structure solution techniques, as a matter of sheer necessity. So much of my time in the lab everyday is spent banging against one of these walls, so to speak, that it only makes sense to reach out to this extremely diverse and interdisciplinary community in times of genuine need. As is clear from many of the posts (but maybe not all), a lot of people seem to have done their homework (RTFM, tried several things, etc.) and are clearly stuck at the time of posting to the bulletin board. Consider my case, which I'm sure is also the predicament of many many folks who post here. I am the sole structural biologist in a lab in a department of Neurology where I am surrounded by biochemists and cell biologists whose idea of a large-scale culture is 250mL. I can read all I want, use my common sense and brain of limited capacity all I want, and can talk to myself all I want, but in the end, when I am badly stuck, this is not an approach I find scientifically healthy or productive. A couple of other things: Many rules of thumbs for soluble protein constructs, expression, purification and crystallization do not automatically carry over to membrane proteins or large macromolecular complexes or the remaining unsolved mysteries of challenging soluble proteins. I'd rather just delete emails where the subject line clearly indicates my lack of immediate interest in the topic than run the risk of splitting this bulletin boards into many smaller parts. The learning impact from the collective experience of the diverse members of this single historic bulletin board will always be orders of magnitude larger the sum of its individual superspecialized parts. To me, it seems natural that the ccp4bb has, over time, morphed in good ways not originally conceived or intended. If anything, this tide speaks to the collective scientific impact that a global and diverse group of researchers from various interrelated disciplines can have on one other. Many thanks to the amazing members of the ccp4bb! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University On Thu, Feb 13, 2014 at 10:05 AM, Eugene Valkov eugene.val...@gmail.comwrote: I absolutely agree with Juergen. Leaving aside methods developers, who are a completely different breed, there is no such thing as a crystallographer sitting in a dark room solving structures all day. If there are, these are anachronisms destined for evolutionary demise. More and more cell biologists, immunologists and all other kinds of biologists are having a go at doing structural work with their molecules of interest themselves without involving the professionals. Typically, they learn on the job and they need advice with all kinds of things ranging from cloning and protein preps through to issues with tetartohedrally-twinned data and interpreting their structures. So, a modern structural biologist is one who is equipped for the wet lab and has some idea of how to go about solving structures. CCP4BB is a wonderful resource that is great for both the quality of the advice offered to those that seek it and for the variety of topics that are addressed in the scope of structural biology. I have learnt greatly from reading posts from very skilled and knowledgeable scientists at this forum and then implemented these insights into my own research. I am very grateful for this. In short, please do not discourage your colleagues, particularly very junior ones, from posting to the CCP4BB. Some of the questions may appear quaint or irrelevant but it is easy to simply ignore topics that are of no interest! Eugene On 13 February 2014 14:41, Bosch, Juergen jubo...@jhsph.edu wrote: Let me pick up Eleanor's comment: is there something like a crystallographer today ? I mean in the true sense ? I think as a crystallographer you won't be able to survive the next decade, you need to diversify your toolset of techniques as pointed out in this article http://www.nature.com/naturejobs/science/articles/10.1038/nj7485-711a And I'm not quite sure how software developers see themselves, as I would argue they are typically maybe not doing so much wet lab stuff related to crystallography (I may be wrong here) but rather code these days. What type of crystallographer is a software developer ? I think like our beloved crystals we come in different flavors. And we need to train the next generation of students with that perspective in mind. Just my two cents on a snowy day (30cm over night) Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry
Re: [ccp4bb] Determining concentration of membrane protein
Hi Everyone, Thanks very much for your helpful responses and suggestions. I will use the BCA assay. Cheers, Raji On Thu, Feb 13, 2014 at 11:27 AM, R. M. Garavito rmgarav...@gmail.comwrote: Roger, While I agree with your list, the BCA assay does not use molybdate (as we make it from scratch with bicinchoninic acid, sodium carbonate, sodium bicarbonate, sodium tartrate, and cupric sulfate pentahydrate). For membrane proteins, I prefer the BCA assay until the protein is pure enough to use A280. Cheers, 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 Feb 13, 2014, at 10:39 AM, Roger Rowlett rrowl...@colgate.edu wrote: Your basic choices for protein assays are: 1. Alkaline copper methods (e.g., Biuret and micro-biuret) 2. alkaline copper + molybdate methods (e.g., Lowry, BCA assays) 3. Hydrophobic dye methods (e.g. Bradford) 4. UV methods (e.g., A280, A230, A210, etc.) Method 1 is least sensitive to amino acid composition, but is also has highest detection limits. Thiols interfere. Method 2 is very idiosyncratic with amino acid composition, and also subject to interference by thiols. Method 3 is not usable in detergent solutions. Method 4 has many inteferences as most everything absorbs in the far UV region. If you have some special protein cofactors, metals, chromophores, etc. these can be exploited for better measurements. For ecample metalloproteins are easy to quantify by ICP-OES or TXRF if they are reasonably pure. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote: Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my membrane protein. My purified membrane protein is in 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%). After reading the friendly manuals and searching online, I've learned that detergents interferes with assays like Bradford but can't find good descriptions of what works best. For now, I am trying to estimate concentration from absorbance at 280nm and using molar extinction coefficients based on aromatic amino acids, but again suspect detergent interference. I would like to know what other folks working on membrane proteins are doing. Thanks very much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] A question on protein-DNA complex crystallization
Hello There! Here are a couple of quick thoughts in response to your question, none of which will fully resolve your concern: (1) Wash your crystals, crush them, add native gel loading dye and run a native gel. First stain with ethidium bromide, then stain with coomassie blue. If you stain for both DNA and protein, that suggests you may have protein-DNA complex in the crystal. I know a lot of people use dyes like Izit (I don't use this approach!) that are used to stain crystals to detect protein. (2) Capture a few diffraction images, if possible. Generally there will be a DNA fibre diffraction pattern on the diffraction image for a crystal containing DNA but obviously that doesn't suggest presence or absence of protein. Is the unit cell estimate much larger than can be expected for DNA alone (unless you are unlucky enough that your DNA fragment will pack in a way to form a large unit cell)? Ultimately, the sure shot way is to get decent enough crystals to collect datasets and solve the structure to discover if the electron density has room to build a model with both protein and DNA. Cheers and good luck! Raji On Thu, Jan 9, 2014 at 9:13 AM, Acoot Brett acootbr...@yahoo.com wrote: Dear All, I am now working on the crystallization of a complex of protein-16 bp DNA by co-crystallization. In the screening very small needle-like crystal occurs. If not salt crystal, is there a method to know it is not the crystal of the DNA? Cheers, Acoot -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Membrane fractionation
Hi Theresa, To put Bert's comment in different words, ultracentrifugation is another purification step. As much as ultracentrifugation needs additional time and resuspending membrane is a real pain, I find that it really does separate my membrane protein from a lot of other soluble E.coli contaminants, proteolytic products, etc. Also, if I skipped ultracentrifugation and simply solubilized the supernatant from the cell lysate (I guess that's what you meant by whole cell), I worry that my yields might be compromised because of incomplete solubilization of all of the membrane protein in whatever volume and percent of detergent I experimented with. Many protocols recommend a second step of ultracentrifugation to get rid of *unsolubilized junk* after the first ultracentrifugation, membrane resuspension and detergent solubilization steps. I skip the second round of ultracentrifugation for one of my membrane proteins because the pellet from the second round is invisible and the protein is pure and functional after purification. Good luck! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University On Mon, Jan 6, 2014 at 5:03 PM, Bert Van-Den-Berg bert.van-den-b...@newcastle.ac.uk wrote: No real need, some folks do it like that. However, you'll need to fish out your protein from more junk and you may have more proteolytic degradation. Also, you will need more detergent compared to membrane-only extraction, especially when using mild detergents. bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Theresa Hsu [theresah...@live.com] Sent: Monday, January 06, 2014 9:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Membrane fractionation Dear crystallographers When we purify membrane proteins, we often fractionate the E. coli cell membrane by ultracentrifugation. Is there a need for this step instead of solubilizing the whole cell with detergents? Thank you. Theresa
Re: [ccp4bb] PDB structure validation
Hi Debasish, Randy's update sounds fantastic! Absolutely fantastic because I suspect many of us have gone through exactly what you described here, despite running many other kinds of validations! In the meantime, the PDB gave me two options as a workaround. One was to either email them the updated coordinates that they would then match with your PDB ID or to simply restart a new session and upload the updated coordinates (the old session gets deleted at the server at some point). Just that you will have the joy of filling out the submission/validation form one more time with option 2. Cheers, Raji On Thu, Oct 31, 2013 at 12:06 PM, Debasish Chattopadhyay debas...@uab.eduwrote: Thanks Randy. That would be fantastic. ** ** *From:* Randy Read [mailto:rj...@cam.ac.uk] *Sent:* Thursday, October 31, 2013 11:02 AM *To:* Debasish Chattopadhyay *Cc:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] PDB structure validation ** ** Yes, the intention at the PDB sites is to make available a standalone validation server that will run exactly the same validation tests that have been introduced for recent depositions. My understanding is that this server is currently being tested and will be rolled out to the community sometime in the new year once they're confident that it is stable. ** ** The motivation is exactly as you say: to save time for both the depositors and the annotators, and to improve the quality of the deposited structures! ** ** Best wishes, ** ** Randy Read ** ** On 31 Oct 2013, at 15:25, Debasish Chattopadhyay debas...@uab.edu wrote: I was wondering if there is a way to generate a PDB validation report before depositing the coordinates so that one can go back and make necessary corrections to the file before deposition. It will save a lot time and perhaps would improve the quality of deposited structures. Debasish Chattopadhyay University of Alabama at Birmingham CBSE-250 1025 18th Street South, Birmingham, Al-35294 USA Ph: (205)934-0124; Fax: (205)934-0480 ** ** -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk ** ** -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] About molecular replacement
Dear Dhanasekaran, There are many examples of molecular replacement failing even in cases where the model and target structure share 100% sequence identity. These examples illuminate several factors that MR search strategies are sensitive to, including percent sequence identity and related parameters like RMSD from target, the impact of loops, small domain movements etc. So if you want to learn in more depth about what makes a good model and what makes or breaks an MR search, perhaps some googling combined with a bit of obsessive reading might help. I actually found a ton of thoroughly helpful articles and reviews for MR on the web without too much sweat. Best wishes, Raji On Thu, Sep 12, 2013 at 5:21 PM, Dhanasekaran Varudharasu dhana...@gmail.com wrote: Dear crystallographers, I have solved a structure of a glucose binding protein of CE4 family. When I try to solve the structure using the same CE4 family enzyme as search model, it failed for many case. Finally, I solved the with a same family enzyme used as search model. As soon as I solved the structure, I superposed my final refined model with structures of CE4 family enzymes which did not produce the good molecular replacement solution for my enzyme. I found that all are having (Beta/alpha)7 fold and superpose very well with my model. Whereas, some loop region are not superpose very well. My doubt is why molecular replacement failed thought over-all fold is same?. -- *Dhanasekaran Varudharasu* Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] What kind of reflection data to deposit to PDB
Hi Folks, Sorry for the non-ccp4 post. I am trying to determine what is the best form of unmerged reflection data to deposit to the PDB. I have single wavelength anomalous data for my structure and I have two flavors of scaled files from the same exact set of diffraction images: (1) data indexed and scaled in p1, and (2) data indexed in p222, scaled in Scalepack using the no merge original index option and converted to .mtz since the unit cell in the header of the output .sca file was missing. The space group for the dataset is p212121. Please could you let me know what might be the best approach. Many thanks and cheers, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Xtal formed during purification
Dear Zhizhi, I worked with a postdoc a while ago who also got crystals when he concentrated his purified protein in the final step of purification. It might be worth testing whether your crystals are of diffraction quality or not. In my colleague's case, the rapidly growing crystals did not diffract at all but after he optimized his buffer conditions to prevent those non-diffracting crystals and screened for optimal crystallization conditions, he got hits from the screens that diffracted to 1.8 Ang. Cheers and good luck! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University On Fri, Aug 9, 2013 at 4:20 PM, Tomas Malinauskas tomas.malinaus...@gmail.com wrote: Dear Zhizhi, we had a case like that. I would switch to slightly different buffer (e.g. different pH) so crystals do not appear overnight, and then do crystallisation screening. I bet you will have many hits in different conditions, likely with bigger crystals. Good luck! Best wishes, Tomas On Fri, Aug 9, 2013 at 8:31 PM, ZHIZHI WANG zzw...@u.washington.edu wrote: Hi all, After I purified my target protein by ion exchange, I left the fractions with high protein concentrations overnight @4C. Now I saw a lot of small needle crystals inside the EP tubes this morning. I wonder whether there is any technique or method to get bigger crystals from this? ZZ
Re: [ccp4bb] Off topic: Gel filtration of membrane protein
Hi Nazia, The DDM concentration you mention seems fine to me. I routinely run the FLPC at 4C with buffers containing 0.05% (1mM) DDM and 2% glycerol without issues. I have also used buffers with 5% glycerol in the past but I usually adjust the flow rate, as needed and when I know all else is good with the column and the machine. In addition to what Bert's comments and suggestions, here are a few more common culprits, which have to do with general FPLC and column maintenance and wear and tear: (1) Air bubbles in the system, which need to be manually purged out. (2) A Superdex200 column clogged with years of protein buildup (3) A clogged in-line filter (ideally should be replaced every couple of months (i) If your buffer is the problem, then flowing filtered water through the system without any columns hooked up should drop the pressure. (ii) If the inline filter is the problem, then the pressure will be higher than usual even in water or buffers without glycerol/urea/GuHCl and without any column attached. In that case, swap out the old one for a new inline filter. (ii) And if the column is the problem, the appropriate column-specific cleaning procedures are outlined in the manufacturer's manual. Also, in case you are not aware, suggestions and procedures for all the above can be found in the FPLC manual. Hope this helps. Raji On Fri, Jul 26, 2013 at 8:57 AM, Bert Van-Den-Berg bert.van-den-b...@newcastle.ac.uk wrote: The first thing is that you'll have to increase the salt concentration in your buffer. 5 mM is way too low and may cause non-specific binding of the protein to the resin. 100 mM is the minimum you should use. There is nothing in your buffer that will precipitate, so you should't have to worry about that. Running the columns at 4C and using 5% glycerol will result in a relatively high back pressure even for a new column, especially if you have the flow restrictor in place. But its hard to see how anything in your buffer could precipitate and clog things up. Are you storing the DDM at -20 and let it get to roomtemp before you weigh it out? If not I could see it getting hydrolysed over time and form alcohols which are poorly soluble. Bert -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Nazia Nasir Phd2009,ProteinCrystall.Lab [nazia.nasi...@nii.ac.in] *Sent:* Friday, July 26, 2013 1:45 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Off topic: Gel filtration of membrane protein We are trying to purify a membrane protein using different detergents (DDM, OG etc.). We have tried using 1mM DDM in 20mM Tris, 5mM NaCl and 5% glycerol buffer to purify the protein. however, we are facing problems in running the buffer in 16/60 Superdex 200 pg gel filytration coloumn using AKTA Explorer. The entire machine is in a cold cabinet. The buffer was also kept at constant slow stirring, thinking that it might be getting precipitated, which we are not able to see. Still the back pressure is very high and the in-line filter keeps clogging. We have filtered the buffer through a 0.2 micron filter, which too was very difficult. the Has anyone faced a similar proble? Or is there a way that buffers with detergents are supposed to be made? Or are there any particular coloumns meant for such runs. Thanks -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Off-topic post: Inverted DNA repeats vs direct repeats
Hi Everyone, One of the proteins I work with is a transcription factor that binds to two direct repeats in its cognate binding site. That my protein binds direct repeats is unlike most other members of its family that bind to inverted repeats. One of the reviewers of a paper that my colleagues and I submitted would like to know what the significance of having a direct repeat is vs. an inverted repeat is. So I dug around and learned the following about inverted repeats: (1) Inverted repeats can form intramolecular hairpin or cruciform structures. (2) Inverted repeats play diverse roles in biological processes (DNA replication, transcriptional regulation, translational control etc). (3) Inverted repeats can cause genome instability and genomic rearrangements. (4) Inverted repeats are associated with several human diseases. Is there anything known about whether one type of repeat or the other predominates in nature? Is anyone able to add anything more than the above about the particular significance of why nature may have chosen a direct repeat in the case of my protein instead of an inverted repeat? Also, any pointers towards relevant literature would be great. My searches thus far haven't yielded much more than what I've shared above. Many thanks! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Concentrating purified membrane protein
Thanks everyone for your responses. I definitely plan to save the flowthrough so we'll see what happens. My protein has a His tag and I did consider doing an affinity step for concentration except I do not want to have imidazole for some functional assays that I need to carry out with the protein. Just occurred to me that I could simply dialyze out the imidazole after the affinity step. Thanks again! Raji On Sat, Jul 13, 2013 at 8:47 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want to concentrate the protein from gel filtration fractions, which are in buffer containing 0.05% DDM (well above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane protein using a 100kDa MWCO concentrator but I am not sure if I can do the same without losing protein in the flowthrough. On the other hand, if use too low a MWCO for the concentrator, then I'm concerned that I may end up concentrating the DDM and end up with too much detergent in the final sample. Any tips about how to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Concentrating purified membrane protein
Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want to concentrate the protein from gel filtration fractions, which are in buffer containing 0.05% DDM (well above the CMC for DDM). My colleague was able to concentrate a 30kDa membrane protein using a 100kDa MWCO concentrator but I am not sure if I can do the same without losing protein in the flowthrough. On the other hand, if use too low a MWCO for the concentrator, then I'm concerned that I may end up concentrating the DDM and end up with too much detergent in the final sample. Any tips about how to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Detergent solubilization step (membrane proteins)
Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Detergent solubilization step (membrane proteins)
Thanks Jim and Bert for all your suggestions. I plan to increase time and/or amount of DDM and see what happens before switching detergents. I'll keep the suggestions about other detergents and 96-detergent screen in the back of my mind. Bert, I noted your suggestion of also using a larger amount of total detergent and will definitely try that. Jim, thanks for suggesting Elugent. Never heard about it before so great to know. Many thanks and cheers, Raji On Wed, Jul 10, 2013 at 5:23 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] off topic
Hi Careina, Yes it does. I have run ethidium bromide-containing agarose gels of samples containing poly(dI-dC) and they light up under UV, just like any other regular DNA does on agarose gels. Cheers, Raji On Fri, Jul 5, 2013 at 9:11 AM, Careina Edgooms careinaedgo...@yahoo.comwrote: Just a quick question, does anybody know if ethidium bromide binds to poly(dI-dC)? Careina -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Missing DNA density in Protein DNA complex structure
Dear Ashok, There are many questions underlying your questions. A couple of things to check right off the bat: (1) Do you actually know that your crystal still contains all of the DNA bp that you started with? Did you analyze the contents of your crystal by native PAGE, mass spec or other methods? (2) Yes, the number of base pairs do matter, especially if you have quasi-helical DNA stacking interactions that facilitate packing along one of the unit cell dimensions. For example, 12-bp is a little over a turn in contrast to 17-bp, which is a little more than 1.5 turns of a DNA B-form helix. (3) Are the crystal packing interactions in cases 1, 3, 4 and 5 similar? And, is there something unique about the packing in case 2, especially DNA-to-DNA packing? Make sure to display symmetry related molecules. That may explain why you can accommodate more molecules in the unit cell. (4) Compare the DNA sequences in cases 1-5 above and see if there is a pattern to the type(s) of nucleotides that are bound by protein in each case. It is hard to say more without knowing what the models look like but if your project is to investigate the DNA-protein interactions in more detail, the above-mentioned sorts of questions may be a place to start. Good luck! Raji On Sun, May 5, 2013 at 2:21 AM, ASHOK KUMAR Patel ashok...@gmail.comwrote: Hi all, I am working on a DNA binding protein (mol wt around 30 kDa), which binds to Duplex DNA in a non-specific sequence manner. The structure has been published with 12 base pair duplex DNA. I am trying to understand the DBD protein DNA interaction even more by choosing different lengths and sequences. In Co-crystallization I used 16, 18, 20 and 22 bases palindromic sequence random DNA bases (purchased from IDT), annealed and used in crystallization. I collected some diffraction data on NSLS recently at around 2.1 Å and 2.7 Å. But, when I did data processing, model building and refinement. I am getting strange results as depicted in the table.. S N a= b= c= α= β= γ= Space group No of molecules in asymmetric unit Length of DNA Used for crystallization Duplex DNA found in structure Resolution 1 38.67 61.43 76.77 90.00 104.17 90.00 P 1 21 1 1 12 base 12 base 2.0 Å 2 86.076 57.099 99.493 90.00 103.90 90.00 P 1 21 1 2 17 base 17 base 3.05 Å 3 37.855 61.668 76.601 90.00 102.24 90.00 P 1 21 1 1 *18base* *12 base* 2.1 Å 4 37.073 61.864 78.242 90.000 100.810 90.000 P 1 21 1 1 *20 base* *12 base* 2.7 Å 5 *20 base* *12 base* 3.1 My question and concerns are as: 1. How I am getting almost identical Cell parameters with different length of DNA (row 3 and 4) to the first row? 2. Why I am getting only 12 base duplex DNA instead of 18mer or 20 mer I used in crystallization. 3. Is anything has to do with ODD and EVEN duplex DNA. When odd 17 base duplex was used, it has 17 bases in the structure, while in all EVEN case of 18, 20 or 20, only 12 bases in the structure. 4. The complex having odd DNA length 17 has 2 molecules in ASU while all other has 1. Why only 12 mer DNA density in the complex? Why I am missing 6 or 8 bases in the density? How can we explain the missing DNA in the structure? I will appreciate any kind of explanation and suggestions. Thanks Ashok -- Ashok kumar patel Department of Biophysics Johns Hopkins University Baltimore, MD 21218 -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Off-topic: PDB statistics
Hi Folks, Does anyone know of an accurate way to mine the PDB for what percent of total X-ray structures deposited as on date were done using molecular replacement? I got hold of a pie chart for the same from my Google search for 2006 but I'd like to get hold of the most current statistics, if possible. The PDB has all kinds of statistics but not one with numbers or precent of X-ray structures deposited sorted by various phasing types or X-ray structure determination methods. For example, an Advanced Search on the PDB site pulls up the following: Total current structures by X-ray: 78960 48666 by MR 5139 by MAD 5672 by SAD 1172 by MIR 94 by MIR (when the word is completely spelled out) 75 by SIR 5 by SIR (when the word is completely spelled out) That leaves about 19,000 X-ray structures either solved by other phasing methods (seems unlikely) or somehow unaccounted for in the way I am searching. Maybe the way I am doing the searches is no good. Does someone have a better way to do this? Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Off-topic: PDB statistics
Thanks to everyone who responded. I appreciate your pointing out the caveats of such statistics mining expeditions. I remember (with no accuracy or precision) sitting in a lecture by Wayne Hendrickson years ago and watching him cringe at the notion of considering the simple case of difference Fourier (say, isomorphous protein and protein+ligand structures) as Molecular Replacement. Like you point out, Thierry, I believe he referred to these cases as Molecular Substitutions. And like you said, there's no consensus with the nomenclature. Phil, thanks much for the stats. Somewhat reassuring that they are somewhat similar to what I got from the PDB. Thanks to everyone! Raji On Mon, Apr 15, 2013 at 11:46 AM, Phil Jeffrey pjeff...@princeton.eduwrote: From my own db program: Number of entries in histogram: 711 Total number of instances : 78467 0 48249 0.6149 MOLECULAR REPLACEMENT 1 8557 0.1091 NULL 2 5632 0.0718 SAD 3 5128 0.0654 MAD 4 3600 0.0459 FOURIER SYNTHESIS 5 1762 0.0225 OTHER 6 1171 0.0149 MIR 7 511 0.0065 SIRAS 8 505 0.0064 DIFFERENCE FOURIER 9 392 0.0050 MIRAS 10 229 0.0029 AB INITIO 11 226 0.0029 MR 12 151 0.0019 RIGID BODY REFINEMENT 13 146 0.0019 ISOMORPHOUS REPLACEMENT 14 110 0.0014 AB INITIO PHASING 15 109 0.0014 MULTIPLE ISOMORPHOUS 1683 0.0011 N/A 1775 0.0010 SIR 1870 0.0009 RIGID BODY 1964 0.0008 DIRECT METHODS 2050 0.0006 RE-REFINEMENT USING 2137 0.0005 DIFFERENCE FOURIER PLUS 2236 0.0005 ISOMORPHOUS 2334 0.0004 REFINEMENT 2430 0.0004 MOLREP 2526 0.0003 SE-MET MAD PHASING 2625 0.0003 RIGID-BODY REFINEMENT 2724 0.0003 ISOMORPHOUS METHOD etc It's a very heterogeneous field, that REMARK 3 field, and the ones above are the most dominant entries (note the 8,557 that are NULL that are in fact crystal structures). At least in some versions of ADIT the guidance that RCSB gives about this field is very weak, which accounts for the variation. I'm interested in what ab initio phasing really means, but I've been too lazy to mine the actual entries for details. Phil Jeffrey Princeton On 4/15/13 9:48 AM, Raji Edayathumangalam wrote: Hi Folks, Does anyone know of an accurate way to mine the PDB for what percent of total X-ray structures deposited as on date were done using molecular replacement? I got hold of a pie chart for the same from my Google search for 2006 but I'd like to get hold of the most current statistics, if possible. The PDB has all kinds of statistics but not one with numbers or precent of X-ray structures deposited sorted by various phasing types or X-ray structure determination methods. For example, an Advanced Search on the PDB site pulls up the following: Total current structures by X-ray: 78960 48666 by MR 5139 by MAD 5672 by SAD 1172 by MIR 94 by MIR (when the word is completely spelled out) 75 by SIR 5 by SIR (when the word is completely spelled out) That leaves about 19,000 X-ray structures either solved by other phasing methods (seems unlikely) or somehow unaccounted for in the way I am searching. Maybe the way I am doing the searches is no good. Does someone have a better way to do this? Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] plate crystal optimization
I hate to take you back to the step of crystallization but have you tried seeding to improve the crystal morphology/volume? If tinkering around with cryoprotectants and mounting techniques does not help your case, seeding might be worth considering. In some of the cases I've seen, my labmate was able to transform plates into 3D crystals. Of course, if there's good reason one is getting plates in the first places, seeding may not help. Just to keep that in the back of your mind given the magical transformations I've witnessed. Good luck! Raji On Mon, Apr 1, 2013 at 11:19 AM, Phoebe A. Rice pr...@uchicago.edu wrote: Among many possible reasons, the streaking could be caused mechanical stress to the thin plates during mounting. Have you tried those loops that look like miniature tennis rackets? Phoebe -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Rakesh Chatterjee [rakesh.2665...@gmail.com] *Sent:* Monday, April 01, 2013 10:01 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] plate crystal optimization hello everyone, i have a protein which co-purifies with a ligand (small molecule ~ 810 Dalton). i didn't know what kind of ligand is it so i am planing to identify it. meanwhile my protein crystallized as plates and while diffracting i found that the crystal is showing a good diffraction pattern upto 2.6 Angstroms in home source Cu K-alpha. however when the crystal rotates between 90 degrees to 135 degrees, the spots become streaky. should i try different cryo-MPD/ PEG400 etc? to circuvent the problem i did some additive screen but was not of much help. any valuable suggestion willbe handfull. thanx in advance rakesh -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] delete subject
Ed, I very much agree with you. We've all had to learn that questions posted to ccp4bb and the ensuing discussions take on a life of their own. Once one posts a question on ccp4bb, there's no such thing as steering the direction of the discussion on the ccp4bb and there's no such thing as the equivalent of screaming Stop! Stop! Stop! on the ccp4bb. Also, I don't believe people simply woke up one day and posted irritating or mean comments to ccp4bb. Ed was spot on for why some folks reacted the way they did to the post so let's acknowledge that as well. I didn't get the impression that any of the replies suggested that students stop posting questions. There are many many students on this BB who are in small institutions without even the minimal help at arm's length and who get tons of help from posting questions to the ccp4bb. That situation is not all that distant in my own memory and I suspect for many other experts on this BB. But posting 10MB attachments and getting the entire ccp4bb community to crowdsource towards problem solving is all good, but only to a certain degree. It may be great to get things done quickly with the collective intellect of the ccp4bb but there comes a point when the correct answers may get fed back at such a rapid speed that if one doesn't go back and try to figure stuff out for oneself, including the reasons/theory/logic behind the answers/solutions that the community has posted, it may be to the detriment of one's own learning, especially if one is in the early stages of learning the subject matter. Cheers, Raji On Thu, Mar 28, 2013 at 9:09 AM, Ed Pozharski epozh...@umaryland.eduwrote: On Thu, 2013-03-28 at 12:15 +, Tom Van den Bergh wrote: I think this is a good time to end the discussion. As a general comment, discussions on boards like ccp4bb often digress and take direction different from you original intent. I may understand your desire to try to control the situation, but if people on this board feel that the questions of data sharing, student training, netiquette and proper choice of resolution cutoff are worthy of further discussion (that may not have much to do with specifics of your original request for assistance), it is their right too. What may have caused some extra grief is this unfortunate turn of phrase in your original post Could you try some refinement for me, because this is first structure that i need to solve as a student and i dont have too many experience with it. It goes a bit beyond the usual my R-values are too high what should I do question and may be instinctively construed as if you expect someone to actually do your work for you (I am sure that is not what you asked). So a bit of a vigorous reaction that you received likely results from misunderstanding your intent (albeit posting your data is very unusual and strengthens the impression) and perhaps misplaced feeling that you have abandoned attempts to resolve the problem independently too soon. I did *not* look at your data and therefore I may be completely wrong here, but it is my understanding that your actual issue was not realizing there could be more than one molecule in the asymmetric unit. More traditional route is to describe your situation in general terms and offer to provide data to those willing to take a closer look. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] How to calculate data collection strategy manually?
Yes, I highly highly recommend the Dauter (1999) paper that Harry has suggested. Also, check out: Dauter Z. Efficient use of synchrotron radiation for macromolecular diffraction data collection. Prog Biophys Mol Biol. 2005 Oct;89(2):153-72. Most everything you want to learn about data collection is in these two papers. Cheers, Raji On Tue, Mar 26, 2013 at 9:06 AM, Harry Powell ha...@mrc-lmb.cam.ac.ukwrote: Hi Saleem I can strongly recommend reading and digesting this paper (open access from the IUCr website, so you don't need a subscription) - Dauter, Z., Acta Cryst. (1999). D55, 1703-1717 - Data-collection strategies http://journals.iucr.org/d/issues/1999/10/00/ba0020/ba0020.pdf Once you've read it, the issues that you should take into account when devising your strategy shuld be apparent. Of course, all the best integration programs also have strategy options built into them as well if you decide not to do it manually after all On 26 Mar 2013, at 12:25, saleem raza wrote: How to calculate data collection strategy manually. I was wondering if any one can answer this, if we have to calculate data collection strategy manually? regards Saleem Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing) -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Fwd: [Err] Re: [ccp4bb] How to calculate data collection strategy manually?
Not sure why I keep getting this message from an email id in korea (see below). Are other members getting this email as well? Is someone someone at ccp4bb able to stop this id from spamming me everytime I send emails to the BB. Thanks. Raji -- Forwarded message -- From: spam_mas...@korea.ac.kr Date: 2013/3/26 Subject: [Err] Re: [ccp4bb] How to calculate data collection strategy manually? To: r...@brandeis.edu Transmit Report: yangr...@korea.ac.kr에게 메일 발송을 5번 시도했지만 실패하였습니다. (실패 이유 : 554 Transaction failed. 402 Local User Inbox Full ( yangr...@korea.ac.kr) 8,61440,240522(163.152.6.98)) 참고 실패 이유에 대한 설명 User unknown :메일을 수신할 사용자가 존재하지 않음 Socket connect fail:수신 메일 서버와 연결 실패 DATA write fail:수신 메일 서버로 메세지 송신 실패 DATA reponse fail :수신 메일 서버로부터 메세지 수신 실패 Final-Recipient: rfc822;yangr...@korea.ac.kr Diagnostic-Code: smtp; 554 error - Transaction failed. 402 Local User Inbox Full (yangr...@korea.ac.kr) 8,61440,240522(163.152.6.98) Action: failed Status: 4.0.0 -- Forwarded message -- From: Raji Edayathumangalam r...@brandeis.edu To: CCP4BB@JISCMAIL.AC.UK Cc: Date: Tue, 26 Mar 2013 10:17:35 -0400 Subject: Re: [ccp4bb] How to calculate data collection strategy manually? Yes, I highly highly recommend the Dauter (1999) paper that Harry has suggested. Also, check out: Dauter Z. Efficient use of synchrotron radiation for macromolecular diffraction data collection. Prog Biophys Mol Biol. 2005 Oct;89(2):153-72. Most everything you want to learn about data collection is in these two papers. Cheers, Raji On Tue, Mar 26, 2013 at 9:06 AM, Harry Powell ha...@mrc-lmb.cam.ac.ukwrote: Hi Saleem I can strongly recommend reading and digesting this paper (open access from the IUCr website, so you don't need a subscription) - Dauter, Z., Acta Cryst. (1999). D55, 1703-1717 - Data-collection strategies http://journals.iucr.org/d/issues/1999/10/00/ba0020/ba0020.pdf Once you've read it, the issues that you should take into account when devising your strategy shuld be apparent. Of course, all the best integration programs also have strategy options built into them as well if you decide not to do it manually after all On 26 Mar 2013, at 12:25, saleem raza wrote: How to calculate data collection strategy manually. I was wondering if any one can answer this, if we have to calculate data collection strategy manually? regards Saleem Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing) -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] molecular replacement problem.
Dear Appu, You want to be sure you have good reason to drop the space group from C222(1) to P2(1). There may be many reasons why your Rfree may not drop following refinement, especially if you only have one domain in your protein located and just in case there are more molecules to locate in the MR search. For C222(1) data, did Xtriage suggest any alternate space groups? Also, what program did you use for MR? If you used Phaser with your C222(1) data, did you ask to search for alternate space groups? Did you check for translational NCS? If you want me to take a look at your data, I'd be happy to look at your scaled data and MR results and try to help you out. If so, please email me off the bulletin board. Good luck! Raji On Sun, Mar 24, 2013 at 6:45 AM, vellieux frederic.velli...@ibs.fr wrote: Hello, Here we deal with symmetry and the unique part of reciprocal space (the reciprocal space asymmetric unit so to speak). C222(1) has eight asymmetric units (international tables, space group 20); P2(1) only has two. Assuming that Friedel's law does apply, then the minimum rotation range to collect a non-redundant data set (one observation per reflection) is 90 degrees, provided that the crystal is correctly and perfectly aligned. Normally with our current data collection methods where the crystal is randomly oriented, we would collect more than 90 degrees (180 degrees, or 360 degrees with the Pilatus detectors on an intense SR beamline where you cannot really check during data collection how well the crystal fares during exposure to the X-rays - shoot first, think later. The reciprocal space asymmetric unit in C222(1) is smaller. I assume that what you are doing is to take the reduced data set file (an MTZ file probably) and reduce the symmetry from C222(1) to P2(1). You will not cover the monoclinic reciprocal space asymmetric unit by doing so. The way to do it is to take the file from processing, before (crystallographic symmetry) merging of the equivalents, and perform the scaling and merging in the P2(1) space group. Or reprocess the data frames in P2(1) if you have lost the unmerged data file. Now of course this will still give you a poor completeness if you have used a strategy to optimize data collection in the orthorhombic space group (you won't have collected enough data then for good completeness in the monoclinic space group). I hope this is clear ! HTH, Fred. On 24/03/13 11:20, Appu kumar wrote: I run the phenix.xtriage to evaluate the twining but it suggest no twining. When i reindex from C2221 to P21, the completeness of data reduced from 95 % to 35% whereas the map is very good and Rwork and Rfree are 26/31 for 2.2 resolution. I do not understand why the completeness of data reduced so much on reindexing. please Can anyone explain this phenomenon. Thank you On 24 March 2013 13:30, Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de wrote: the p21 c2221 ambivalence can mean severe twinning (i had a similar case just now - try several crystals from the same condition) ! What do the twinning statistics suggest? cheers, Matthias - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - On 3/24/2013 7:46 AM, Appu kumar wrote: Thank you for the quick reply. After molecular replacement , i have done only few cycle of refinement in refmac. I have not done any solvent modification or NCS averaging. I have initially indexed the data in C2221 but Rfree was not decreasing so i reindexed the data in data in P121 space group keeping the Rfree flag of C2221. While analysing the symmetry mates , i found large space but no density. structure of Ligand binding domain is almost identical with 90% identity in sequence. I am stuck with this problem and don't know how to process further. Please give me your valuable suggestion. I will appreciate your effort. Thank you Appu On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote: Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high resolution cut-off were the data measured? What resolution limits were used for the MR search? How do the unit cell dimensions and space group in the two cases compare? I am guessing the ligand binding domain in your protein has the identical sequence to that of the published ligand binding domain that you use as a template in your MR search. In any case, here are a couple of my thoughts: (1) It might be worth setting up different runs of MR with different numbers for expected copies
Re: [ccp4bb] molecular replacement problem.
Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high resolution cut-off were the data measured? What resolution limits were used for the MR search? How do the unit cell dimensions and space group in the two cases compare? I am guessing the ligand binding domain in your protein has the identical sequence to that of the published ligand binding domain that you use as a template in your MR search. In any case, here are a couple of my thoughts: (1) It might be worth setting up different runs of MR with different numbers for expected copies (not just two copies but also one copy and three copies just in case you have one of the extreme cases of solvent content)? (2) If the MR solution is correct and there is physical room for a DNA binding domain in your lattice (check by displaying symmetry mates), perhaps the DNA binding domain is disordered. In that case (and if all attempts with current data fail), you may have to crystallize the protein in presence of DNA. Good luck! Raji On Sat, Mar 23, 2013 at 2:26 PM, Appu kumar appu.kum...@gmail.com wrote: Dear members, I am doing a molecular replacement of a transcription factor whose ligand binding structure(24000 Da) is available in PDB but not for the DNA binding(13000 Da). When i am searching for the two copies from ligand binding domain as a template model, i am getting very good solution but i am not getting any density for the DNA binding domain to build up in density. The space gorup is P 1 21 1 (4) and unit cell parameters are Unit Cell: 57.43 69.36 105.99 90.00 90.00 90.00. Please guide me how to get the complete model structure. Table below show the matthews statistics For estimated molecular weight 37000. Nmol/asym Matthews Coeff %solvent P(2.20) P(tot) _ 1 5.7178.46 0.00 0.01 2 2.8556.91 0.62 0.70 3 1.9035.37 0.37 0.29 4 1.4313.82 0.00 0.00 _ The phaser molecular replacement gives the following table. istogram of relative frequencies of VM values -- Frequency of most common VM value normalized to 1 VM values plotted in increments of 1/VM (0.02) --- relative frequency --- 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 ||||||||||| 10.00 - 8.33 - 7.14 - 6.25 - 5.56 - 5.00 - 4.55 - 4.17 - 3.85 -- 3.57 --- 3.33 -- 3.12 -- 2.94 (COMPOSITION*1) 2.78 --- 2.63 2.50 - 2.38 2.27 -- 2.17 --- 2.08 -- 2.00 -- 1.92 --- 1.85 --- 1.79 --- 1.72 - 1.67 - 1.61 - 1.56 - 1.52 - 1.47 * (COMPOSITION*2) 1.43 - 1.39 - 1.35 - 1.32 - 1.28 - 1.25 - $TABLE : Cell Content Analysis: $SCATTER :N*Composition vs Probability:0|3x0|1:1,2: $$ N*Composition Probability $$ loggraph $$ 1 0.306066 2 0.00141804 $$ Most probable VM for resolution = 2.27817 Most probable MW of protein in asu for resolution = 92664.2 Thank a lot in advance -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Oligomerization state
Dear Theresa, Although I haven't yet done the experiment myself, I am told that microscale thermophoresis (MST) is another useful method for oligomerization measurements, especially since it is designed to work for membrane proteins in presence of detergents etc. If you want to quickly learn more about microscale thermophoresis, you can google the technique for further information and literature. If you end up wanting to try MST experiments and don't have much of a handle on the method yet, contact me off the bulletin board and I'd be happy to give you a few pointers. As far as Thermofluor goes, some of the detergents work better than others in Thermofluor experiments so you may have it to determine the issues in your case empirically. Good luck! Raji On Thu, Mar 21, 2013 at 2:10 PM, Theresa Hsu theresah...@live.com wrote: Dear all I have a His-tagged membrane protein with unknown oligomerization state. But I am worried that tag addition may induce different state than in native and affect its crystallizability. Is there a single method that can determine the oligomerization state with nearly 100% accuracy? I have use of AUC and SAXS but there seems to be ambiguity about detergent and lipid effects. Is Thermofluor a right method? Does oligomerization require special assembly proteins, which will mean that tag cleavage is not useful to obtain native state? Thank you. Theresa -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Need specific molecular replacement test cases
Hello Everyone, I am looking for two specific test cases (below) and appreciate anyone pointing me to known structures/examples for the same. (1) For a successful case of molecular replacement in which the search model has an overall sequence identity to the target in the twilight zone or worse (25% or less). (2) A case in which the search and target models share 80-100% sequence identity but where conformational changes in the target relative to the search model prevented a successful MR solution. Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] How to slow down crystallization? Need hep!
Hi Lei Fang, I cannot tell what technique you are using for crystallization. Also, many details like protein concentration, temperature of crystallization etc. are missing in your email so it's hard to guess what's going on. In any case, here are my thoughts. I used to get a shower of needles from hanging drops at 19C but a lot fewer and bigger crystals when I set up sitting drops under mineral oil at 19C. The crystals took lot longer to grow (two weeks as opposed to four days using hanging drop) so bear that in mind. My guess is that you may be able to find protocols if you just do a quick web search. There are many other ways to optimize crystal growth and size including playing with temperature of crystallization (lowering to 4C), amount of precipitant vs protein, drop size, crystallization technique, seeding by dilution etc. Hope that helps. Raji On Mon, Feb 25, 2013 at 11:02 AM, lei feng spartanfeng...@hotmail.comwrote: Hello everyone, I need your suggestion for slowing down crystallization for my protein my protein got hit in PEG/ION #5 ( 0.2 M MgCl2, 20% PEG 3350, pH 5.9), but it crystallize too fast. In 1 hr I can see tons of tiny needles. Can anyone give me some suggestion on how to slow down the process? I used lower conc. of potein, lower conc. of PEG ( 10%), it helped a little bit, giving me small rod crystal. but no improvement after that. Thank you very much for your suggestions -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Thrombin cleavage of membrane protein with fusion tag
Hi Folks, Sorry this isn't a non-ccp4 post. I am working with a membrane protein for which I am finally able to scale up expression. I am now also able to partially purify my protein from a medium-scale (12-18L) bacterial culture using a two-step tandem affinity purification protocol (Talon followed by amylose affinity steps). As the next purification step, I am about to set up a pilot thrombin cleavage experiment to separate my protein from the His.MBP fusion tag (see below). The construct that I am working with is as follows: His.MBP--ThrombinSite--Membrane Protein There is only one theoretical thrombin cleavage site in the entire fusion protein i.e., at the desired cleavage site with no theoretical secondary sites. I would like to try cleavage both at 4C and around 25C from 4h to overnight but I also have to balance the trials with the material I must generate for the endless permutations and combinations one can try. Each sensible pilot experiment is going to use up partially purified protein from 6-12L preps. FYI. All purification buffers contain DDM and I haven't yet done extensive detergent screens. Please could I ask the community to share tips/suggestions about large-scale thrombin cleavage experiments with their favorite membrane proteins. Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag
Dear Pascal and Toufic, Many thanks to both of you for the pointers. Toufic, I actually poked around online and bought exactly the same kit that you are suggesting, especially because the beads are reusable. So your experience is reassuring. So I'll find out soon. I also plan to play around with the detergent type and concentration among other things. I have no plans to take the shortcut but it's nice to have at least one other human being to talk to on these matters. I appreciate your responses :) Many thanks! Raji On Wed, Feb 20, 2013 at 1:15 PM, Toufic El Arnaout elarn...@tcd.ie wrote: Hi Raji, I addition to the tips from Pascal, I would like to say that for a memb protein I worked on with a his-tag separated by a thrombin site, I used thrombin cross linked to agarose from Sigma (1 mL). The beads can be collected, washed and reequilibrated, making it ready for use so many times. In my case 1 mL of this thrombin was enough to cleave up to 5-10 mg of target protein, at 4 °C for 16 h (with slight rotation in a 15 mL tube, total volume up to 10 mL). In FC-12 there was a little better cleavage than in DDM. My opinion is that how thrombin (or most other proteases) will cleave may mostly depend on your protein/fusion type/protein-micelle complex structure/access to the site... You just have to try. Best wishes. toufic On Wed, Feb 20, 2013 at 5:15 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, Sorry this isn't a non-ccp4 post. I am working with a membrane protein for which I am finally able to scale up expression. I am now also able to partially purify my protein from a medium-scale (12-18L) bacterial culture using a two-step tandem affinity purification protocol (Talon followed by amylose affinity steps). As the next purification step, I am about to set up a pilot thrombin cleavage experiment to separate my protein from the His.MBP fusion tag (see below). The construct that I am working with is as follows: His.MBP--ThrombinSite--Membrane Protein There is only one theoretical thrombin cleavage site in the entire fusion protein i.e., at the desired cleavage site with no theoretical secondary sites. I would like to try cleavage both at 4C and around 25C from 4h to overnight but I also have to balance the trials with the material I must generate for the endless permutations and combinations one can try. Each sensible pilot experiment is going to use up partially purified protein from 6-12L preps. FYI. All purification buffers contain DDM and I haven't yet done extensive detergent screens. Please could I ask the community to share tips/suggestions about large-scale thrombin cleavage experiments with their favorite membrane proteins. Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- ** Toufic El Arnaout Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2 Tel.: +353 85 83 40 157 ** -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] protein crystals or salt crystals
Like Nat points out, I suspect they are phosphate crystals. I have seen those before. And I agree 100% with Frank, for once. Why would I risk making any guesses no matter how salt like my crystals look after all the time it took me to clone, express, purify and crystallize my precious protein. It takes MUCH less time than all of that slog to stick the crystal on a beam and here are a few possible scenarios: (1) Clear diffraction spots spaced planets apart in case of salt (2) Protein like diffraction (3) Inconclusive no diffraction situation, which could indicate a million things including the possibility that your cryoprotectant was sub-optimal for data collection done using flash cryocooled/flash frozen crystals in a stream of gaseous nitrogen. Note of caution: I may take more time to plan the diffraction test (room temperature, cryoprotectant etc.) if I only ever got one precious crystal and was never able to reproduce the crystallization. Cheers, Raji On Fri, Feb 8, 2013 at 8:18 AM, Ed. Pozharski epozh...@umaryland.eduwrote: Patrick, Something related: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Conditions_prone_to_salt_crystallization Truth be told, we recently had a major breakthrough with the peg/fluoride condition I came to consider a useless salt crystal generator. So tables like these are undoubtedly useful but do not reduce workload. :) This is also a rather interesting finding http://www.google.com/url?sa=tsource=webcd=12ved=0CDEQFjABOAourl=http%3A%2F%2Fwww.aseanbiotechnology.info%2FAbstract%2F21021153.pdfei=N_kUUYfkBafV0gG09ICoAgusg=AFQjCNE7C-m6IkLPUay9gEEM60yJF46ZQg Basically, presence of protein may induce salt crustallization. To me, this means that diffraction pattern is the best indicator. Frank already said exactly that, of course. Cheers, Ed. Original message From: Patrick Shaw Stewart patr...@douglas.co.uk Date: To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein crystals or salt crystals Good morning Frank On a related idea, do you typically use a limited number of buffers (buffer plus salt) for the final purification step of your proteins? If so, do you have a chart of where salt crystals may appear in the screens that you use most often? Could you put that chart on your web site to help the community? People could pick one of your standard buffer mixes to make their lives easier later on. Best wishes Patrick On 8 February 2013 07:18, Frank von Delft frank.vonde...@sgc.ox.ac.ukwrote: Test the diffraction - that's the only way. But given the other junk in the drop, chances are they're salt. (And don't post 5Mb attachments, please.) On 07/02/2013 22:24, amro selem wrote: Hallo my colleagues. i hope every one doing ok . i did screening since two weeks . i noticed today this crystals. i don`t know either it salt or protein crystal . my protein has zero tryptophan so i could distinguish by UV camera. the condition was conditions: 0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM. best regards Amr -- 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 -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] need some suggestions for crystallization
My suggestions would be to look up citations for thaumatin and glucose isomerase. If I remember correctly, both of them form well diffracting crystals within a short period of time. I think you can also buy the purified protein from a vendor. Perhaps you could also try the good old lysozyme. Cheers, Raji On Mon, Feb 4, 2013 at 11:03 AM, David Roberts drobe...@depauw.edu wrote: So, I know I say this every time I post on this board, but here it goes again. I'm at an undergrad only school, and every 2 years I teach a class in protein crystallography. This year I'm being super ambitious, and I'm going to take a class of 16 to the synchrotron for data collection. It's just an 8 hour thing, to show them the entire process. I'm hoping that we can collect 5-6 good data sets while there. I would like them to grow their own crystals, and go collect data. Then we'd come back and actually do a molecular replacement (pretty easy/standard really). Just to get a feel for how it works. The protein I do research on is not one that I would push on this, as the crystals are hard to grow, they are very soft, and the data just isn't the best (resolution issues). I do have a few that will work on my proteins, but I was thinking of having others in the class grow up classic proteins for data collection. Obviously lysozyme is one, but I was wondering what other standard bulletproof conditions are out there. Can you all suggest some protein crystallization conditions (along with cryo conditions) for some commercially available proteins? I'm looking to get 6-8 different ones (and we'll just take them and see how it goes). I wouldn't mind knowing unit cell parameters as well (just a citation works, I can have them figure it out). I have about 7 weeks to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Problems in scaling up expression
Hi James, Just a thought or two after reading your notes. I don't exactly know what KRX cells are but I suspect (based on your mention of rhamnose) that they are cells in which T7 RNAP levels are under the control of a rhamnose promoter. If that is so, it would be important to add rhamnose during cell growth to prevent an leaky expression of your protein, which may be toxic to E. coli, as opposed to adding it during the stage of induction along with IPTG like you describe. Just to rephrase, add inoculum, antibiotics and rhamnose to TB, grow to the OD you desire, then add IPTG to induce protein expression. Often, toxicity effects are felt in larger cultures and not seen in 15mL or 40mL cultures. Also, I have found that there are certain cases when it is better to use Luria Broth as opposed to TB. But that is not my primary concern in your case. My two cents! Raji On Tue, Jan 15, 2013 at 9:48 AM, Murray, James W j.w.mur...@imperial.ac.ukwrote: Dear All. A question on protein expression. We have been doing small scale test expressions in 15ml of terrific broth+kanamycin using E.coli KRX cells in falcon tubes. On reaching OD600 of ~0.6 we induced with rhamnose+IPTG and expressed for 4 hours at 37 C. There is a big band corresponding to our protein in the soluble fraction and not much in the insoluble fraction. However, on scaling up to 1 L TB cultures (with same concentrations of kanamycin and inducing with same concs of rhamnose+IPTG) we don't get strong over expression. Has anyone else experienced problems when scaling up expression? (and more importantly, solved them?) best wishes James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Today ...
Merry/Happy Christmas, Happy Your Favourite Festival/Celebration and Happy New Year! Raji On Thu, Dec 20, 2012 at 9:44 AM, case c...@biomaps.rutgers.edu wrote: On Thu, Dec 20, 2012, Phil Evans wrote: … is 20.12.2012 No, it's actually 12.20.2012, kind of a palindrome. Happy Christmas everyone! And Merry Christmas to those on the left side of the pond! ...dac -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] vitrification vs freezing
Hi Sebastiano, Elspeth Garman howls bloody murder everytime someone says they froze their crystals. I think her issue is with the description of the process of successfully flashcooling crystals in the presence of cryoprotectants as freezing. Freezing technically is understood to imply the formation of hexagonal ice while what one really means is the successful solidification of water in a random orientation (vitrification) and the prevention of the hexagonal ice. Semantics semantics! I'd stick with flashcooled or something along those lines. Raji On Thu, Nov 15, 2012 at 12:13 PM, Sebastiano Pasqualato sebastiano.pasqual...@gmail.com wrote: Hi folks, I have recently received a comment on a paper, in which referee #1 (excellent referee, btw!) commented like this: crystals were vitrified rather than frozen. These were crystals grew in ca. 2.5 M sodium malonate, directly dip in liquid nitrogen prior to data collection at 100 K. We stated in the methods section that crystals were frozen in liquid nitrogen, as I always did. After a little googling it looks like I've always been wrong, and what we are always doing is doing is actually vitrifying the crystals. Should I always use this statement, from now on, or are there english/physics subtleties that I'm not grasping? Thanks a lot, ciao, s -- Sebastiano Pasqualato, PhD Crystallography Unit Department of Experimental Oncology European Institute of Oncology IFOM-IEO Campus via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5167 fax +39 02 9437 5990 please note the change in email address! sebastiano.pasqual...@ieo.eu -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] usefulness of cacodylate?
Hi Frank, I worked with protein purification buffers and crystallization buffers containing 20mM potassium cacodylate for five years or so. And yes, the only precaution I used was gloves while weighing the chemical and while making buffers etc. And not just me, but all of my former colleagues worked with cacodylate. Then, there's those nasty chemicals for heavy atom soaks like mercury and tantalum compounds that are equally hazardous. And... many other chemicals we used on a daily basis in the lab but we don't suspect as much as we should. Nucleosome core particles stubbornly refuse to crystallize if you leave out cacodylate, not just from the crystallization buffers but also from the protein purification buffers. Yes, there are folks who accidentally left it out and never gotten crystals of nucleosomes. I don't rule out that someday someone may very well be able to substitute cacodylate for some other chemical and successfully crystallize nucleosomes. It might be hard to interpret the kind of studies you suggest and here's why, in my opinion. Even if one showed that there was no need for cacodylate for, say, a 1000 different proteins, I would definitely not exclude it from a crystallization screen for my favorite protein because we have not gotten to that point in crystallography where one can predict crystallization conditions for a new macromolecule with great accuracy. In my opinion, it's all relative. There are probably more chances of me being killed by a reckless bicyclist in Boston/Cambridge than by cacodylate. ;-) Cheerios! Raji On Fri, Nov 9, 2012 at 7:26 AM, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: Hi all - Anybody know a) how hazardous is cacodylate? b) does it really matter for crystallization screens? It seems by far the most hazardous component of the standard screens; this 2011 paper seems to think so (bizarrely, I can't access it from Oxford): http://onlinelibrary.wiley.**com/doi/10./j.1365-2818.** 1977.tb01136.x/abstracthttp://onlinelibrary.wiley.com/doi/10./j.1365-2818.1977.tb01136.x/abstract and this is site says lethal dose is 0.5-5g/kg: http://cameochemicals.noaa.**gov/chemical/4468http://cameochemicals.noaa.gov/chemical/4468 meaning 2ml of a 0.1M solution contains 1/10th lethal dose...? (Someone should check my maths...) [Coarse screens come mixed 2ml per condition.] Has anybody done careful experiments that showed it really mattered for a given crystal -- or even an entire screen? So I'm inclined to toss it out entirely rather than make crystallization screening a hazardous activity. (We're being subjected to a safety review.) Thoughts welcome. phx -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Resuspension of bacterial cell pellets
Hello Everyone, Sorry for this rather naive and non-CCP4 question but I am very curious. My rule of thumb is to resuspend bacterial cell pellets in about 1-2% of the original culture volume for a wet weight of about 3g of bacterial pellet per L of culture volume. For example, Typically, the total volume of my resuspension for a 6L-bacterial cell pellet is around 60-70mL or about 40mL, if I really try to minimize the volume of buffer. Every protocol I have read over the years seems to indicate something similar. In troubleshooting one of my colleague's protein preps, I found that she is resuspending 6L of cell pellet with a total of pellet+buffer volume of 5mL. In practice, I would not physically be able to resuspend a 6L pellet in 5mL (3g pellet/L culture) without making a very viscous and lumpy soup. My suspicion is that such small volumes are a source of some of her issues, including a high number of impurities in her elution from affinity columns. I'm curious to hear what other folks do and recommend. Cheers, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Resuspension of bacterial cell pellets
Hi Folks, Thanks for your responses. To clarify, I have looked into any fluctuations in cell pellet volumes (autoinduction, cell lysis, toxicity) and this isn't such a case. My colleague's cell pellet weights are the standard 3g or so/L and that's why I strongly suspect the resuspension volumes to be an issue. Thanks. Raji On Thu, Oct 25, 2012 at 8:15 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hello Everyone, Sorry for this rather naive and non-CCP4 question but I am very curious. My rule of thumb is to resuspend bacterial cell pellets in about 1-2% of the original culture volume for a wet weight of about 3g of bacterial pellet per L of culture volume. For example, Typically, the total volume of my resuspension for a 6L-bacterial cell pellet is around 60-70mL or about 40mL, if I really try to minimize the volume of buffer. Every protocol I have read over the years seems to indicate something similar. In troubleshooting one of my colleague's protein preps, I found that she is resuspending 6L of cell pellet with a total of pellet+buffer volume of 5mL. In practice, I would not physically be able to resuspend a 6L pellet in 5mL (3g pellet/L culture) without making a very viscous and lumpy soup. My suspicion is that such small volumes are a source of some of her issues, including a high number of impurities in her elution from affinity columns. I'm curious to hear what other folks do and recommend. Cheers, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Resuspension of bacterial cell pellets
Thanks for the confirmation. Raji On Thu, Oct 25, 2012 at 10:44 AM, Rob Gillespie robert.gilles...@duke.eduwrote: Put me down at another person who re-suspends bacterial cell pellets in 4-5 volumes of buffer. On Thu, Oct 25, 2012 at 9:15 AM, Opher Gileadi opher.gile...@sgc.ox.ac.uk wrote: It makes sense to use a fixed ratio of resuspension buffer to cell weight; we weigh the pellets after centrifugation, then suspend in at least 4-5 volumes (ml/gr) of buffer. -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Plate crystals
Hi Jahan, Since I can't tell what you tried in terms of improving/optimizing crystallization, here are some methods that my some colleagues and I have had good luck with, in addition to what Gopal has suggested. (1) Sitting drop technique under oil (2) Varying ratios of protein to precipitant (3) Seeding with several serial dilutions of the seeded material Since you write that you have high mosaicity, perhaps it is also worth double-checking your crystal cryoprotectant and flashcooling conditions. Like Elspeth Garman likes to remind everyone, cryoprotectant and crycooling conditions that mitigate ice formation do not automatically ensure the best diffraction conditions, which often need further optimization. Good luck! Raji On Mon, Oct 15, 2012 at 9:30 PM, Parthasarathy, Gopal parth...@merck.comwrote: During optimization, have you tried Hampton's additive screen? Gopal From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jahan Alikhajeh [ja...@graduate.org] Sent: Monday, October 15, 2012 6:01 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Plate crystals Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan 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. -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Professor Dame Louise Johnson
I am very sorry to hear the sad news. Like innumerable others, I am very grateful to have had the opportunity to meet The Prof. Johnson of the classic Blundell Johnson crystallography textbook. I met Prof. Johnson at Diamond a few years ago and immediately saw in her an inspiring, graceful and sophisticated lady and scientist. Soon after, I had the opportunity to interact a lot with Prof. Johnson to help coordinate her trip to Brandeis for the 2010 Petsko-Ringe anniversary symposium. Unfortunately, sudden illness kept her from traveling for that occasion. I would also like to acknowledge that Prof. Johnson offered me her generous help and support on a few other occasions. My condolences to her family, friends and colleagues. Raji On Tue, Oct 2, 2012 at 1:03 PM, Gloria Borgstahl gborgst...@gmail.comwrote: This indeed is sad news for today. I just wanted to note that Professor Johnson's early papers on time-resolved crystallography truly inspired me to continue in crystallography, influenced my decision for my first postdoctoral position and to push the limits. I still have the carefully highlighted photocopies (yes used a photocopier and a real bound journal in gradual school) in my filing cabinet next to my office. My condolences to those close to her and her family. Gloria On Tue, Oct 2, 2012 at 6:40 AM, elizabeth.d...@diamond.ac.uk wrote: It is with great sadness that I would like to inform the crystallographic community of the death of one of the great pioneers of the field, Professor Dame Louise Johnson. ** ** Those of us who had the privilege to work alongside her benefitted greatly from her vision for extending technique and instrumentation such that increasingly complex problems could be successfully solved and found her quiet determination to succeed inspirational. ** ** Dr. Liz Duke Diamond Light Source Harwell Science and Innovation Campus Chilton, Didcot Oxon OX11 0DE UK ** ** Tel. +44 (0) 1235 778057 Mob. +44 (0)7920 138148 ** ** -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Detergent and protein oligomerization
Hi Everyone, Sorry for the non-CCP4 post. I have a very basic question about detergents, critical micelle concentration and behavior on gel filtration. A 33kDa membrane protein was purified by gel filtration in a buffer containing 0.4%(w/v) beta-NG (CMC: 6.5mM) and 0.046%(w/v)LDAO (CMC: 0.14mM). So the concentrations of beta-NG and LDAO in the gel-filtration buffer are ~2X and ~14X that of the CMCs of the respective detergents. The elution volume of the protein peak (plus detergent) on Superdex200 corresponds to a molecular mass of 100kDa. I think that the 100kDa mass above includes contributions from both the protein as well as the detergent micelles. If this is correct, is it then accurate to try to glean the oligomerization state of the protein (and conclude that it is a trimer or tetramer) without taking into account detergent micellar mass and its influence on elution volume? How should one interpret the 100kDa mass estimate from the gel filtration? Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Einstein would be proud
Hi Folks, Sorry for a non-CCP4 post but I simply couldn't resist. Here's a video that's simply out of this world! This video was made by an undergraduate student at Northeastern and it just won him a trip to outer space. Yes, outer space! Not only did the student write the script and make the video but he also did all the chalk art and composed and played the music in the video. The narration is slow but totally funky! Well worth the fifteen minutes, in my opinion... http://www.northeastern.edu/insolution/other/2012/04/rocket-man/ Please DO NOT miss it if you like science or art or music or imagination or philosophy or some vague combination of the five... Enjoy! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] weird--No protein expression using pET30a
Hi Gerry, In one case, I had a mutation in the ribosome binding site sequence. If you haven't already checked, it might be worth checking whether the 5'- and 3'- junctions close adjacent to the ORF are all correct to make sure the transcriptional and translational elements do not somehow contain mutations. On occasion, figuring out what is wrong with the current construct might send one on a endless wild goose chase. So if a bunch of ideas with the current construct fail and you still want to try and express in the pET system before moving on to another system, I recommend the pET Expresso system. No need to ligate etc. Add clean PCR product + vector + cells and you directly screen for clones that are made by homologous recombination in bacteria. Cheers, Raji On Sat, Mar 17, 2012 at 2:26 AM, Jerry McCully for-crystallizai...@hotmail.com wrote: Dear ALL; Recently, one of my colleagues cloned a gene (200aa) into pET30a vectors with either a N-ter or C-ter His6 tag. The correct reading frame was confirmed by sequencing. However, it is weird that there was no protein expression either in the soluble fraction or as inclusion bodies. Could anyone give some instruction? Thanks a lot and have a nice weekend, Jerry -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Human multi-pass integral membrane protein crystal structures
Dear fellow CCP4bb-ers, Does anyone know how many crystal structures have been published to date for unique human multi-pass alpha-helical integral membrane proteins? For example, if there are multiple structures of the same membrane proteins with different ligands, I do not wish to count them as unique for my purposes. I have already tried to search for this information on Stephen White's database, on Caffrey's page, in the PDB database, Google etc. but my searches either return no results or incomplete results. I am looking for a specific subset of membrane proteins that satisfy all following criteria: human + multi-pass + alpha-helical + integral membrane protein. If anyone can provide the answer, that would be very helpful. I need this information for a fellowship application. Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Aggregated protein for crystallization
Hi Folks, As crazy as it sounds, if you have crystallized and managed to solve the structure of a protein from aggregated protein, please could you share your experience. After many constructs, many many expression schemes and after the usual rigmarole of optimization that is also often discussed on ccp4bb (buffers, glycerol, salt concentrations, pH, detergent, additives etc.), I now have a decently expressing truncated construct for my protein (80 kDa) that is pure but aggregated (elutes in the void volume from a Superdex200 column). I am tempted to make a boatload of aggregated protein and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Fwd: HR3699, Research Works Act
If you agree, please signing the petition below. You need to register on the link below before you can sign this petition. Registration and signing the petition took about a minute or two. Cheers, Raji -- Forwarded message -- From: Seth Darst da...@mail.rockefeller.edu Date: Tue, Feb 14, 2012 at 12:40 PM Subject: HR3699, Research Works Act To: Rep. Caroline Maloney has not backed off in her attempt to put forward the interests of Elsevier and other academic publishers. If you oppose this measure, please sign this petition on the official 'we the people' White House web site. It needs 23,000 signatures before February 22nd and only 1100 so far. Please forward far and wide. Oppose HR3699, the Research Works Act HR 3699, the Research Works Act will be detrimental to the free flow of scientific information that was created using Federal funds. It is an attempt to put federally funded scientific information behind pay-walls, and confer the ownership of the information to a private entity. This is an affront to open government and open access to information created using public funds. This link gets you to the petition: https://wwws.whitehouse.gov/petitions#!/petition/oppose-hr3699-research-works-act/vKMhCX9k -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]
Hi Fred, If I understand you correctly, you are concentrating your 50uL reaction to 4uL and then transforming all of it. If that is correct, that is WAY too much DNA, especially based on what I see on your gel. Too much DNA inihibits transformations and that is a very common mistake. Assuming I am reading you correctly. Simply transform say 2.5uL of the PCR reaction (post DpnI) directly into cells and that is likely to work. You could also try, say a bunch of transformations with 1uL, 2.5uL and 5uL. Good luck and I can help you out further to get your mutants! Raji On Fri, Feb 3, 2012 at 12:13 PM, Fred ccp4bb.l...@gmail.com wrote: Dear CCP4users biologists, I'm trying to make a single aa mutant of a 5.7 kb non commercial vector with the Agilent's Quick Change Site-Directed Mutagenesis Kit. I have strictly followed the instructions manual, however, I could not be able to transform bacterial cells with my PCR product. I can observe the amplified PCR product before and after DpnI digestion (see image in http://ompldr.org/vY2x3aw), but cannot get any colony on LB plates. I'm using very fresh super competent cells so that I've got dozens of colonies with 60 ng of the parental/non-mutated vector as positive control. The bands in the referenced image corresponds to 2.5 microL of a 50 microL reaction volume. I usually concentrate it to 4 microL before transformation. Also, I've already optimized the primer's temperature annealing (best is 62 oC) and I've increased the extension time up to 9 min. Is there anything else I can try? Any help is appreciated! Regards. Fred P.S.: Agilent's e-mail support is not working. P.P.S.: this might not be of other's interest, address the answers, please, to my e-mail only. -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] THANK YOU: Crack-resistant tubes for centrifugation
Hello Everyone, Many many thanks to all the folks who responded to my question with very good suggestions. Here's a very quick and dirty summary of the various tubes and rotors that people use without any issues: (1) 50ml Nalgene tubes for an SS-34 rotor (2) Shape-matched new Fiberlite rotors (3) Nalgene round-bottom centrifuge tubes (re-usable) (4) Beckman ultracentrifuge tubes (re-usable) (5) 50 ml Falcon tubes (red cap) (6) 50 ml Corning tubes in F13S-14x50cy rotor (7) Polyethylene tubes work but polycarbonate do not, for some folks It seems Fiberlite rotors were a common suggestion and a bunch of folks suggested that breakage may have AS MUCH to do with centrifuge and shape-complementarity (understandably) as much as with the centrifuge tubes. Many thanks for your time and help. Go CCP4BB! Raji -- Forwarded message -- From: Raji Edayathumangalam r...@brandeis.edu Date: Tue, Jan 31, 2012 at 11:59 AM Subject: Crack-resistant tubes for centrifugation To: CCP4BB@JISCMAIL.AC.UK CCP4BB@jiscmail.ac.uk Hi Folks, Are you any favorite brands out there for crack-resistant 50mL centrifugation tubes. It seems we are having recurring episodes of Falcon and Corning tubes cracking even at 9,000 rpm, which is the maximum speed possible with our rotor. I have used Falcon tubes for years in the past without problems and I want to be able to spin down bacterial lysates without a mess. Any suggestions for tubes that have worked well in your experience? Thanks, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Crack-resistant tubes for centrifugation
Hi Folks, Are you any favorite brands out there for crack-resistant 50mL centrifugation tubes. It seems we are having recurring episodes of Falcon and Corning tubes cracking even at 9,000 rpm, which is the maximum speed possible with our rotor. I have used Falcon tubes for years in the past without problems and I want to be able to spin down bacterial lysates without a mess. Any suggestions for tubes that have worked well in your experience? Thanks, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Crack-resistant tubes for centrifugation
9000 rpm translates to 13,000 g on this centrifuge/rotor. I am not referring to pelleting bacterial cells. My question is about centrifuging bacterial lysates and some recommendations for sturdy tubes. Thanks. Raji On Tue, Jan 31, 2012 at 12:02 PM, Bosch, Juergen jubo...@jhsph.edu wrote: To how many g does your 9000 rpm translate ? Perhaps that's the problem ? 10 minutes @ 5000xg for pelleting cells is more than enough in my opinion. Jürgen On Jan 31, 2012, at 11:59 AM, Raji Edayathumangalam wrote: Hi Folks, Are you any favorite brands out there for crack-resistant 50mL centrifugation tubes. It seems we are having recurring episodes of Falcon and Corning tubes cracking even at 9,000 rpm, which is the maximum speed possible with our rotor. I have used Falcon tubes for years in the past without problems and I want to be able to spin down bacterial lysates without a mess. Any suggestions for tubes that have worked well in your experience? Thanks, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University .. 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://web.mac.com/bosch_lab/ -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] TCA or acetone precipitation of proteins
Hi Everyone, Sorry for the naive and non-CCP4 question. Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye with SDS and all. Cheers and thanks. Raji -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Coot File Save Coordinates
Hi Folks, Apologies for a non-CCP4 question. I am trying to write our coordinates following SSM superposition using the File Save Coordinates option in Coot. But if I click the Select Filename button, nothing happens. I thought I would get an option to pick a filename and specify what I want my output coordinate filename to be called. But that isn't happening. Also, clicking on the molecule on the graphics screen (as someone pointed out in a previous post) doesn't help either. I was able to run this very identical routine several times recently so not sure what just happened now! Haven't upgraded Coot or anything. Am using Coot 0.6.2-pre-1 (revision 3468) [with guile 1.8.7 embedded] [with python 2.7.1 embedded]. Help? Thanks. Raji -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Coot File Save Coordinates
Thanks Mischa and Juergen. That was probably my most ridiculous post to the CCP4BB!! I found the pop-up dialog box hiding behind all my zillion windows. Now why the pop-up window would not pop up actively on top of all other windows is a question for another time. Nevertheless, your replies helped :) Raji On Mon, Aug 15, 2011 at 3:56 PM, Bosch, Juergen jubo...@jhsph.edu wrote: Have you moved your primary window away ? I mean just in case the pop up window opened behind the actual scene window. Jürgen On Aug 15, 2011, at 3:54 PM, Raji Edayathumangalam wrote: Hi Folks, Apologies for a non-CCP4 question. I am trying to write our coordinates following SSM superposition using the File Save Coordinates option in Coot. But if I click the Select Filename button, nothing happens. I thought I would get an option to pick a filename and specify what I want my output coordinate filename to be called. But that isn't happening. Also, clicking on the molecule on the graphics screen (as someone pointed out in a previous post) doesn't help either. I was able to run this very identical routine several times recently so not sure what just happened now! Haven't upgraded Coot or anything. Am using Coot 0.6.2-pre-1 (revision 3468) [with guile 1.8.7 embedded] [with python 2.7.1 embedded]. Help? Thanks. Raji -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Coot File Save Coordinates
Thanks for pointing that out. -Raji On Mon, Aug 15, 2011 at 4:29 PM, Antony Oliver antony.oli...@sussex.ac.ukwrote: If you're running Coot on a Mac - it's also unfortunately a well-documented feature, something to do with Apple's implementation of X11. Sent from my iPhone On 15 Aug 2011, at 21:25, Raji Edayathumangalam r...@brandeis.edu wrote: Thanks Mischa and Juergen. That was probably my most ridiculous post to the CCP4BB!! I found the pop-up dialog box hiding behind all my zillion windows. Now why the pop-up window would not pop up actively on top of all other windows is a question for another time. Nevertheless, your replies helped :) Raji On Mon, Aug 15, 2011 at 3:56 PM, Bosch, Juergen jubo...@jhsph.edu jubo...@jhsph.edu wrote: Have you moved your primary window away ? I mean just in case the pop up window opened behind the actual scene window. Jürgen On Aug 15, 2011, at 3:54 PM, Raji Edayathumangalam wrote: Hi Folks, Apologies for a non-CCP4 question. I am trying to write our coordinates following SSM superposition using the File Save Coordinates option in Coot. But if I click the Select Filename button, nothing happens. I thought I would get an option to pick a filename and specify what I want my output coordinate filename to be called. But that isn't happening. Also, clicking on the molecule on the graphics screen (as someone pointed out in a previous post) doesn't help either. I was able to run this very identical routine several times recently so not sure what just happened now! Haven't upgraded Coot or anything. Am using Coot 0.6.2-pre-1 (revision 3468) [with guile 1.8.7 embedded] [with python 2.7.1 embedded]. Help? Thanks. Raji -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/http://web.mac.com/bosch_lab/ -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Pymol question
Hi Christopher, I saw your script and there's many ways to the same destination. Here's how my brain thinks: load A.pdb hide everything, A show cartoon, A [cmds for whatever else I like: fancy helices etc. etc.] Option 1: show sticks, A and resi 197-199 Option 2: And if you hate that the backbone CA sticks out in an ugly manner say for resi 197 on the cartoon, then do: hide sticks, A and resi 197 and resn CA (...or even just...) hide sticks, A and and resn CA (made hide CA you don't want...) Hope this helps. Raji On Fri, Jul 15, 2011 at 9:52 AM, Christopher Browning christopher.brown...@epfl.ch wrote: Hi, I'm a little stuck with a Pymol script. I'd like to represent my protein with fancy helices and B-sheets, and in a looped domain I want to show a few residues, but only the side chains. In my script I tried using set cartoon_side_chain_helper, on, HISA on the selection of residues I want to show, but I still get the rest of the side chain displayed. In the script window I can see that it loads this command, but it does not seem to lead to what I want. I'm using Pymol 1.4, so I suppose it is recent enough for this script to work. Below is a section of my script. Help will be gratefully appreciated. Chris B xxx SCRIPT xxx load gpV-trimer.pdb, gpVabc center gpVabc #util.ss gpVabc hide everything, gpVabc create VA, (gpVabc//A/6:211/) alter VA//A/45:47/, ss='S' #show ribbon, A #set ribbon_width=8, ecr20e set cartoon_fancy_helices=1, VA show cartoon, VA #set cartoon_transparency=0.7, VA #set cartoon_rect_length=0.75, VA color salmon, VA #hide everything, (VA//A/128:211/) create HISA, (gpVabc//A/197,199/) show sticks, HISA set cartoon_side_chain_helper=1, HISA color gray40, (HISA and elem C) color red, (HISA and elem O) color blue, (HISA and elem N) color yellow, (HISA and elem S) set stick_radius=0.20, HISA #hide everything, HISA -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40 -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Potential Space Group Issue
Oops sorry for the slippery fingers. I meant h00, 0k0 and 00l in my original email and NOT 00h, 00k, 00l. Note the correction especially if you are a first-year graduate student trying to learn stuff from these emails :) Raji On Thu, Jul 7, 2011 at 11:30 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hello Everyone, I have a 3.1 Ang dataset for which I'd like to get to the bottom of what the correct space group is. The current unit cell in p212121 is 98.123 101.095 211.20190.000 90.00090.000 I fed the reflection data into Xtriage to look for twinning and pseudotranslational NCS and there is no indication for either issue in the Xtriage output. Also, all odd 00h, 00k, 00l reflections are systematically absent as they should be for p212121. However, my colleague who is also working on the same dataset recently reprocessed the data in P21. Here's the cell in p21: 98.010 100.940 210.470 90.00 90.04 90.00 p21 I am not sure if BETA=90.04 is significant enough to treat as p21 (0.04% deviation of beta angle from ideal lattice for p212121). I don't think so but I could be wrong. Could someone please clarify? Also, what kind of twinning and twinning operators can relate a p212121 cell to a p21 cell with almost identical unit cell parameters as that of the p212121 cell and leave all systematic absences intact? Thanks much. Raji --- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Potential Space Group Issue
Hello Everyone, I have a 3.1 Ang dataset for which I'd like to get to the bottom of what the correct space group is. The current unit cell in p212121 is 98.123 101.095 211.20190.000 90.00090.000 I fed the reflection data into Xtriage to look for twinning and pseudotranslational NCS and there is no indication for either issue in the Xtriage output. Also, all odd 00h, 00k, 00l reflections are systematically absent as they should be for p212121. However, my colleague who is also working on the same dataset recently reprocessed the data in P21. Here's the cell in p21: 98.010 100.940 210.470 90.00 90.04 90.00 p21 I am not sure if BETA=90.04 is significant enough to treat as p21 (0.04% deviation of beta angle from ideal lattice for p212121). I don't think so but I could be wrong. Could someone please clarify? Also, what kind of twinning and twinning operators can relate a p212121 cell to a p21 cell with almost identical unit cell parameters as that of the p212121 cell and leave all systematic absences intact? Thanks much. Raji --- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] immobilized DNA resin
Hi Alex, Most DNA-binding proteins has decent affinity to heparin columns. Have you tried purifying your protein over heparin columns? Cheers, Raji On Sat, Apr 9, 2011 at 8:44 PM, Alexandra Deaconescu deac...@brandeis.eduwrote: Hello ccp4 enthusiasts: I am afraid this is a non-ccp4 related question. Can anyone recommend an immobilized dsDNA chromatographic resin for purification of DNA-binding proteins? GE seems to have something - I was wondering if people have other recommendations? In the age of GST and His tags etc., these are not very much used, but I do not have a tag in this case... Thanks a lot, Alex -- --- Raji Edayathumangalam Research Fellow in Neurology, Harvard Medical School Postdoctoral Fellow, Center for Neurologic Diseases, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?
Hampton Research actually sells bent loops. At least used to. Raji On Tue, Apr 5, 2011 at 8:52 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: For plate crystals with the long axis normal to the plate surface (anecdotally, this is usually the case), you can use bent loops. Bent loops can be made using tweezers by folding over the loop onto the stem and crimping with the tweezers. When the loop relaxes a bit, it will leave a ~90deg-bent loop, so the crystal can sit on the loop and be shot edge-on in the beam. It takes a bit of time to get it right, but it worked well for me one time. JPK On Tue, Apr 5, 2011 at 7:29 AM, Jürgen Bosch jubo...@jhsph.edu wrote: What do you consider long ? 200, 300 ? 600 A ? Before shooting try to run strategy or xplan. Move the detector back to first reliably be able to determine your cell. Then double your estimated mosaicity and see what strategy suggests. If you don't get many overlaps (5%) then try a closer distance. Don't rotate 1degrees but take 1/2 of the mosaicity. Obviously you want to make good use of the detector area so adjust the edges to where your crystal really diffracts. And if that resolution leads to too many overlaps then limit your resolution and get first a good datasets home. You then can play with 2theta for a higher resolution dataset. Another obvious thing to do and you don't mention what reduction program you use is to let XDS sort your problem out. Unless you collected to high resolution without being cautious XDS could help. If not, well then you had your experience and now should know better. SSRL has options to collect 450 A cells to 3A without much hassle. That was my largest cell so far. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Apr 5, 2011, at 1:05, dengzq1987 dengzq1...@gmail.com wrote: hello all, does anyone have the experience of Collecting Data from Long Unit Cell Axes ? I have a crystal that diffracts to about 4 A. in some direction the spots overlap. we can't use the data to index .we think it is because that there is a long unit cell axes. so is there any method to solve this problem? best wishes. 2011-04-05 dengzq1987 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- --- Raji Edayathumangalam Research Fellow in Neurology, Harvard Medical School Postdoctoral Fellow, Center for Neurologic Diseases, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
[ccp4bb] Protein melting temperatures
Hi Folks, Sorry for the pre-xtallo question; pre-xtallo right now, but hoping to take my protein the xtallo way one of these days! I am currently performing Thermofluor assays with my protein and the results show that the Tm is ~45C. I am looking for some examples of proteins and their melting temperatures so that I can gauge where my protein falls in the spectrum of unstable-to-stably folded. For example, the melting temperature of some forms of lysozyme is 73.8C (very stable, I suppose). Just need a sense for whether my protein is considered unstable or somewhat stable. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University
[ccp4bb] Petsko-Ringe Symposium on June 18-19, 2010
Dear CCP4bbers, On September 4, 1980, Prof. Dagmar Ringe and Prof. Greg Petsko of MIT embarked on a scientific collaboration, and the scientific community has never been the same. Now, 30 years later, the members of the Petsko-Ringe lab are holding a symposium on June 18 and 19, 2010 at Brandeis University in honor of their combined lifetimes of achievement. The symposium and celebration is titled, From Sequence to Consequence: Celebrating 30 Years of Science with Dagmar Ringe and Greg Petsko. Read more about the symposium at: http://www.bio.brandeis.edu/PRSymposium2010/ Please post your congratulatory messages for Greg Petsko and Dagmar Ringe on our online Message Board at: http://prsymposium2010.blogspot.com/2010/05/celebration-of-dynamic-duo.html With Warm Regards, Members of the Petsko-Ringe Lab --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University
[ccp4bb] Symposium Celebration of Greg Petsko and Dagmar Ringe
Dear CCP4bbers, On September 4, 1980, Prof. Dagmar Ringe and Prof. Greg Petsko of MIT entered into an official scientific collaboration, and the scientific community has never been the same. Greg and Dagmar have jointly trained over 120 graduate students and postdocs and countless undergraduates in the past 29 years, and many of you might have worked with a student, postdoc, PhD advisor or postdoc mentor who previously either trained in or collaborated with the Petsko-Ringe lab. Now, 30 years later, the members of the Petsko-Ringe lab are holding a symposium at Brandeis University in honor of their combined lifetimes of achievement. The symposium and celebration is titled, From Sequence to Consequence: Celebrating 30 Years of Science with Dagmar Ringe and Greg Petsko. Read more about the symposium at: http://www.bio.brandeis.edu/PRSymposium2010/ While the symposium is a lab reunion of sorts for all the former and current students, postdocs, staff and collaborators of the Petsko- Ringe lab, we invite everyone from the structural community to take part in the celebration by posting congratulatory messages for Greg Petsko and Dagmar Ringe on our online Message Board. Please post your HEARTY GREETINGS for Greg and Dagmar at: http://prsymposium2010.blogspot.com/2010/05/celebration-of-dynamic-duo.html Please could you circulate this email to colleagues who do not subscribe to CCP4BB. With Warm Regards, Members of the Petsko-Ringe Lab --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University
Re: [ccp4bb] Expression of large proteins in E. coli
Hey Nick, Short answer is that there are no magical tricks, as you probably already expect to hear. Seems there's a lot that you are trying already. I have some limited experience with some 125kDa, 100kDa proteins. 1. I've had some luck with the E. coli C41, C43 strains. Especially if your proteins are inherently toxic to E. coli. There are commercial vendors who sell these strains with the plus or minus pLysS options. 2. Try moving the tags to the other terminus N- to C- or vice versa. Can't do that for SUMO in any easy manner. 3. Try MBP tag as well 4. Try Studier's autoinduction protocol 5. Try expression with chaperone kit, trigger factor (Takara) 6. You don't mention whether the protein is human etc., but you may have to move to yeast or insect cells, in the worst case. DISCLAIMER: I am not paid by Takara to mention their kit. Good luck! Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard Medical School Brandeis University On Jan 13, 2010, at 4:53 PM, n...@silvaggi.com wrote: Hello All, I apologize for the non-CCP4-related query. I have been working for several weeks now trying, with limited success, to express some very large proteins (ranging from ~100 to 180 kDa) from pET15b in E. coli. Limited success means I have expressed enough soluble protein to see on a gel, but not enough to purify. I have tried the obvious tweaks - changing strains (BL21, BL21-star, Rosetta, pLysS), screening induction temperature (16 to 37C), [IPTG] (0.3-1.0mM). I am in the process of subcloning into vectors for (1) SUMO fusion and (2) periplasmic expression (pET26b). I get the sense from digging through the literature that high level expression of large proteins depends mostly on the individual protein and I will ultimately have to look for homologs. But, this is my first experience expressing such large proteins and I am curious to know if anyone out there has some magical trick they wouldn't mind sharing. Thanks in advance, Nick - Nicholas R. Silvaggi, Ph.D. University of Wisconsin-Milwaukee Department of Chemistry and Biochemistry 3210 North Cramer Street Milwaukee, WI 53211 Phone: 414-229-2647 Email: silva...@uwm.edu
Re: [ccp4bb] align DNA structures
Hi Mike, By 'align', if you mean superimposition, lsqman will do the job. Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard Medical School Brandeis University On Oct 21, 2009, at 11:06 AM, Mike England wrote: Hi all, I will highly appreciate your help regarding following: How to align two DNA structures in Pymol or Coot or any other softwares? ( I tried regular align in Pymol, but it doesn't work for DNA; it works great for protein structures.) Thanks a lot in advance ! Mike
Re: [ccp4bb] Weird expression behavior
Hi Ezra and others, Just thought I'd let you know that I have noticed that one or two of those E. coli proteins that love to bind Ni-affinity resin do not bind to Cobalt resin. Of course, there is always a price to pay (for the Co resin, in this case) :)! Cheers, Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard Medical School Brandeis University On Sep 2, 2009, at 7:55 AM, Ezra Peisach wrote: A little tangental You mentioned lowering the temperature - how low... Stratagene markets a call line - Arctic Express - that adds chaperones that are more active at lower temps. I know someone who overcame inclusion body problems by expression at 16 using these cells. (I know someone else who regularly induces o/n at 16C w/ regular cells). The only draw back is that these cells produce a protein that binds to NiNTA resin... Good luck...
Re: [ccp4bb] problems of co-crystallization of protein-DNA complex
Couple of things, Ru Heng. 1. What buffer conditions is your protein in? Is it similar to the buffer you describe as using to dissolve your DNA in? In general, you can even get away with dissolving and annealing the oligos in just Tris etc. 2. Play with buffer conditions, particularly NaCl concentrations. 3. Tweak the protein and DNA ratios. For nucleosomes, we always got white precipitate if we did not always titrate the DNA to protein ratios for every individual prep, I believe optimization of the above parameters would help with the white precipitate formation. Hope that helps. Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard Medical School Brandeis University On Aug 13, 2009, at 12:56 AM, ruheng wrote: Dear CCP4bbers, I am now working on a DNA binding protein and the purity of the protein is quite good, however the results of DLS showed that the protein aggregates terribly in quite a lot of different buffer conditions I tried and still no crystals can be obtained. So I am going to co-crystallize the protein in complex with DNA. I synthesized the oligonucleotides varying different numbers of basepairs to determine the optimal length which can bound to my protein by EMSA. I dissoved the oligos in the buffer containing 50mM Tris-HCl, 100mM NaCl, 10mM MgCl2 and 1mM DTT, pH 7.9 and then annealed the DNA into the double stranded form at a final concentration of 50uM. When I performed the EMSA experiment, I mixed the purified protein with the dsDNA at the molecular ratio approximately 1:1, but white precipitate was generated as I mixed them. Does anyone have this kinds of experience when working on DNA binding proteins and co-crystallizing the protein-DNA complex? Any suggestions from yours will be appreciated. Thank you all. Ru Heng 搜索本应是快乐的，不是么? 快乐搜索，有问必应！微软隆重推出! 立即试用！
Re: [ccp4bb] How to express a 95KD FAD protein
Hi Wei Yong, Sounds like a tricky situation. A couple of things come to mind: 1) Have you tried expression from a synthetic gene? Sometimes the mRNA is unstable and improving mRNA stability through optimization (synthetic gene) helps. 2) Are you able to look at either human isoforms or orthologs from other species? 3) Have you tried expressing with an N-terminal SUMO tag? I have seen expression with an N-terminal SUMO tag in some cases where expression was previously undetected. 4) Have you tried cell-free expression? 5) You might also consider insect cell expression or expression from mammalian cells 6) Do you get enough protein in some cases that you might try to attempt some refolding from inclusion bodies? Just a couple of avenues to think along. Good luck! Raji On Jun 29, 2009, at 5:54 PM, Yong, Wei wrote: Dear all, I know that there are a lot of experts having experience in expressing a big protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding protein (Human protein). I tried very hard but have a bad luck over the past 1.5 years. I list what I did briefly so far. I am looking forward to getting suggestions from you. Thanks a ton in advance. E.Coli 1. Express full-length in pET28a (N-6xhis) No expression 2. Express full-length in pET21b (no- tag)Inclusion body 3. N and C terminus truncated construct Inclusion body (tagged and non-tagged) 4. Co-expression with GroEL/ S Inclusion body 5. Co-expression with with cofactors Inclusion body 6. In pMAL vector Not sure Yeast: (No tag) 7. In pPICZb vector in Pichia No expression 8. In GHB30 vector in Saccharomyces cerevisiae No expression I also tried to use different ways to break cells, use detergent, express at low temperature (down to 15 degree), modify IPTG concentration and so on. I do not know what else I can try. Please give me suggestions. Thank you very much. Best wishes Wei Yong
Re: [ccp4bb] desktop models
I've seen some very pretty 3D models in optical crystal glass made and sold by http://www.luminorum.com/ Cheers, Raji Disclaimer: I have nothing to do with this company. On May 5, 2009, at 2:41 PM, Christopher Rife wrote: Hi, I am looking to have a model produced from a PDB, i.e. something that we can put on the desk for everyone to admire :) Googling for such a thing is rather challenging, so I'm curious if anyone has suggestions as to where I might get something like that made. So far, I have found two options: 1. Molecular Dimensions: http://www.moleculardimensions.com/us/merchant.ihtml?new_id=223step=2 (etching in a glass block) 2. 3D Molecular Designs: http://www.3dmoleculardesigns.com/ (nylon models) I think #2 is more what we have in mind. Does anyone have any experience with their final product in terms of quality and durability? Thanks very much. Chris __ __ ___ This is an e-mail from Danisco and may contain confidential information. If you are not the intended recipient and you receive this e-mail by mistake, you are not allowed to use the information, to copy it or distribute it further. Please notify us and return it to Danisco by e-mail and delete all attachments. Thank you for your assistance. __ __ __
Re: [ccp4bb] problem in transformation of pqe 30 clone
One thing to check is whether there is too much DNA in the transformation reaction. This is sometimes a reason for failed transformations, be it DNA from regular minipreps, PCR DNA or ligation reactions etc. Raji On May 4, 2009, at 3:57 PM, b...@freesurf.fr wrote: This story is rather puzzling indeed, and it is difficult to see why in case of toxicity one would have transformation problems with that one strain in particular. Also, I am wondering how likely it is that a combination of toxicity and leaky expression would lead to transformation problems in the first place. I have quite a bit of experience with the expression of extremely toxic proteins/peptides and I usually find that most of the popular E. coli strains are quite capable of somehow getting rid of the gene or its expression if they really need to, while retaining full antibiotic resistance (the upshot of which is a normal transformation efficiency but zero expression upon induction). My advice would be to first check very carefully for (extremely) trivial reasons for the failed transformation. Best wishes, Sebastiaan Werten. - Original Message - From: Cynthia Kinsland To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, May 04, 2009 4:52 PM Subject: Re: [ccp4bb] problem in transformation of pqe 30 clone Using pQE30, any E. coli is an expression host. Because it uses the T5 promoter, you don't need an E. coli strain carrying the T7 RNA polymerase (so, you don't need a DE3 strain). As noted by Artem, you are most likely having a leaky expression problem. However, it is odd that DH5a will transform if this is the case since it does not carry the lacIq mutation. XL1-Blue does, which is why it was suggested below. You could try expression right in DH5a, since you have the plasmid there. Your transformation difficulties seem strange. Using this vector, DH5a should not transform any more stably than the other strains you tried.
[ccp4bb] SUMMARY (Link two proteins into one polypeptide)
Hi Folks, First of all, thanks to all the people that responded. Many of you asked me for a summary of responses. So, below is a concise summary containing mainly the references that people pointed me to: Cheers, Raji --- ORIGINAL POST: Hi People, Could anyone point me to successful examples for two unrelated proteins that have been stitched together into one single polypeptide chain with flexible amino acids to create a functional chimera that was subsequently crystallized. I've looked up a few. I am particularly interested in understanding all the important considerations while designing a flexible linker even though many of these factors might be case dependent might be variable, obvious and commonsensical. Either way, I'd like to hear what folks have to say. Thanks very much. Raji --- SUMMARY OF RESPONSES: 1. VAMP7 longin domain (a SNARE protein) and its binding partner, Hrb. Cell. 2008 Sep 5;134(5):817-27 http://www.pubmedcentral.nih.gov/articlerender.fcgi? tool=pubmedpubmedid=18775314 - Lauren Jackson 2. To look at lysozyme engineering in the case of the GPCR. Also, to look at Bostjan Kobe's papers, who has been advocating crystallization of MBP and GST fusion proteins. - Boaz Shaanan 3. To check out the structure and method paper for a TrxA-RRM domain fusion. The design of the linker is discussed in the method paper. Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60. Corsini L, Hothorn M, Stier G, Rybin V, Scheffzek K, Gibson TJ, Sattler M. J Biol Chem. 2009 Jan 2;284(1):630-9. Epub 2008 Oct 29. Thioredoxin as a fusion tag for carrier-driven crystallization. Corsini L, Hothorn M, Scheffzek K, Sattler M, Stier G. Protein Sci. 2008 Dec;17(12):2070-9. -Michael Hothorn 4. Panne et al. Cell. 2007 Jun 15;129(6):-23. Nature. 2008 May 22;453(7194):489-4. - Jayakrishnan Nandakumar 5. MBP-linker-Target. It was one of our targets in Berkeley Structural Genomics Center. The linker was Gly4Ser. Both, the target with His-tag and the fusion one crystallized with similar resolution of around 2.3A - Vaheh Oganesyan 6. One example is by David Owen's group in Cambridge. Molecular basis for the sorting of the SNARE VAMP7 into endocytic clathrin-coated vesicles by the ArfGAP Hrb. Pryor PR, Jackson L, Gray SR, Edeling MA, Thompson A, Sanderson CM, Evans PR, Owen DJ, Luzio JP. Cell. 2008 Sep 5;134(5):817-27. - Bret Collins 7. http://www.pubmedcentral.nih.gov/articlerender.fcgi? tool=pubmedpubmedid=15159535 - Richard Kingston 8. Example of a domain-swapped chimera Biochem. J. (2006) 393, 767–777 - Selvaraj 9. See 1axk - Juergen-Joachim Mueller 10. James Stroud already posted to the BB. 11. What about single-chain Fv contructs of antibodies? PDB accession code 1DZB, for example. - Joe 12. Examples: GPCR's fused with T4 lysozyme. Fusion of cytochrome oxidase soluble part to Lac permease (Ron Kaback lab). - Vinothkumar 13. Structural basis for the attachment of a paramyxoviral polymerase to its template. Kingston RL, Hamel DJ, Gay LS, Dahlquist FW, Matthews BW. Proc Natl Acad Sci U S A. 2004 Jun 1;101(22):8301-6. - Anthony Addlagatta 14. Panne, D., Maniatis, T., and Harrison, S. C. (2004) Crystal structure of ATF-2/c-Jun and IRF-3 bound to the interferon-beta enhancer, EMBO J 23, 4384-4393. Panne D, Maniatis T, Harrison SC, (2007) An atomic model of the interferon-beta enhanceosome. Cell. 2007 Jun 15;129(6):-23. - Tiancen/Tony Hu 15. Smyth et al., Protein Sci (2003), 12, 1313-1322. - Ramanathan Natesh 16. Suggestion to use protease cleavage sites as linkers. - Artem Evdokimov 17. Smyth DR, Mrozkiewicz MK, McGrath WJ, Listwan P, Kobe B. Crystal structures of fusion proteins with large-affinity tags. Protein Sci. 2003 Jul;12(7): 1313-22. - Bostjan Kobe
[ccp4bb] Link two proteins into one polypeptide
Hi People, Could anyone point me to successful examples for two unrelated proteins that have been stitched together into one single polypeptide chain with flexible amino acids to create a functional chimera that was subsequently crystallized. I've looked up a few. I am particularly interested in understanding all the important considerations while designing a flexible linker even though many of these factors might be case dependent might be variable, obvious and commonsensical. Either way, I'd like to hear what folks have to say. Thanks very much. Raji
Re: [ccp4bb] Lowest resolution you can do MR with
If you look at the molecular replacement search parameters, you will find that the rotational and translational searches can be done at 4 Angstrom or lower values assigned to the 'high resolution' values. So the real worry in your case, in all likelihood, is not whether MR will work for 3.6 Ang resolution data. The greater worry is as to how much model bias gets introduced at that resolution and as to how well one can refine the model only based on the phases from molecular replacement. I can't tell from your description if you have DNA in your search model or not. But If you get a molecular replacement solution and if at the least, some bits of your DNA molecules are ordered, you will see big bloopers of density that will correspond to the phosphate molecules. The density for sugars and base-pairs may or may not be well defined in the initial stages. Raji On Mar 30, 2009, at 12:25 PM, Muthiah wrote: What is the lowest resolution one can try to do molecular replacement with? I have a 3.6 angstroms resolution data for a protein-DNA complex and wondering whether I can try MR to see the density for DNA.
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Some thoughts about SUMO tags and fusion tags in general. Fusion tags also follow the Garbage In, Garbage Out philosophy. Yes, if for many of the reasons already hashed out extensively on CCP4BB, one is dealing with lack of expression or miniscule expression, often tagging the protein with a fusion/cleavable tag does indeed bump up the expression and lead to 'improved solubility'. Sometimes, it's very important to ask: improved solubility of what though? Everything that Phoebe describes, namely the chaperone contamination, precipitation after cutting off tag etc., reeks of an intrinsically misfolded/unstable/unhappy protein. My experience-- and those of many others-- is that the fusion tag and fusion tag alone can only fix little in cases: 1) when one observes lots of degradation of the untagged protein, 2) where the untagged protein is made as an intrinsically misfolded/unstable protein. In these cases, the carrier protein then notoriously comes along for the ride in the soluble fraction with the fusion/cleavable tag, initially giving the impression of improved expression and improved solubility. Even then, one might even see multiple degradation products with the tagged expression product. Next, cleave the tag off in such a case and lo and behold! all protein precipitates and you are back to square one. I am not trying to discourage anyone from using fusion tags -- to improve expression, solubility, crystallization etc. We all know of many examples where fusion tags have worked wonders. I only caution that if your favourite protein is intrinsically misfolded in a particular expression system and then you have tried tagging a fusion/ cleavable tag onto the protein in the same expression system and you observe all that Phoebe describes, perhaps it is time to bang your head against a different wall now. In many difficult cases, I am unaware that a fusion tag actually aids in the proper folding of a carrier protein. I will not rule out this possibility but I do not know that this is the general rule. I have worked quite a bit with SUMO tags. As far as GST and SUMO tags are concerned, I banged my head against the GST-tag and SUMO- tag wall for my target protein for a frustrating while. I tried a His tag, then a GST tag, then a SUMO tag. All had exactly the same symptoms. In my case, clearly the problem lay with the carrier problem but I was never allowed to conclude so. Just my two cents, the worth of which will already have diminished by the time you have read this email. Raji On Feb 26, 2009, at 11:30 AM, Phoebe Rice wrote: We haven't tried SUMO, but had some frustrating results with GST fusions. They did improve expression and solubility - BUT in one case the target protein precipitated immediately when the tag was cleaved off, and resisted all attempts to bring it back to life. In another case, the fusion protein dragged chaperones into the prep that were nearly impossible to get rid of completely, thus ruining our ATPase assays. Is SUMO, being smaller, less likely to drag such crud along with it? Phoebe Original message Date: Wed, 25 Feb 2009 14:48:57 -0500 From: Mo Wong mowon...@gmail.com Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli To: CCP4BB@JISCMAIL.AC.UK Thanks to all who responded. Actually, this bulletin board is better for help with molecular biology than the molecular biology bulletin board I am subscribed to! On Tue, Feb 24, 2009 at 7:47 PM, Stephen Weeks stephen.we...@verizon.net wrote: Mo, Just to add my 50 cents, I didn't see any mention of the use of fusion proteins in your original post. GST, MBP or my personal, and completely biased, favourite SUMO (plus many more proteins) have been shown to enhance expression when fused to the amino terminus of a target protein. If you fear you have toxicity, simply tracking the OD600 pre and post induction normally tell you if this is happening. I've worked with proteins that basically baselined the cell growth upon induction and, as Artem stated, at least I knew my protein was being made albeit at very low levels. Stephen -- Stephen Weeks, Ph. D. Drexel University College of Medicine Department of Biochemistry and Molecular Biology Room 10102 New College Building 245 N. 15th St. Philadelphia, PA 19102 Phone: (+) 215-762-7316 Fax: (+) 215-762-4452 Mo Wong wrote: I thought I'd post this to the CCP4bb, as judging by previous posts, it seems I could get some useful insight into my problem... This is question has probably been asked by people for a long as molecular biology has been around, but hopefully my question isn't a complete rehash of other peoples: I am trying to express a human protein in bacteria where the only modified amino
[ccp4bb] Pymol: Zoom without Mouse
How does one zoom into the molecule in Pymol without a mouse and with just the Mac trackpad and keyboard? Have tried to look it up in the manual and on the web. No success finding it yet; I did figure it out once before but can't redo it now for the life of me. Need to know how to do it without the mouse. Thanks, Raji
[ccp4bb] Summary: Phylogenetic analysis??
Hi Everyone, Since some folks have been asking me, below is my original question and here's a summary of the responses. I had already googled a bunch of these links before posting to ccp4bb but still lots of useful info in responses, as always! Raji DNAStar will do this if you have a copy lying around. Gabriel Birrane T-coffee http://www.ebi.ac.uk/Tools/t-coffee/index.html Graeme Garvey HI Raji, ~15 years ago I remember using treetool on a solaris system to flip nodes of a tree to alter the groupings. I am not sure what the current tools are that people use, but it looks like the old sparc treetool package is archived on http://iubio.bio.indiana.edu/IUBio-Software+Data/molbio/unix/GDE/ treetool/ It looks like development stopped in 1995, there is a debeian/linux version -- http://packages.debian.org/unstable/science/treetool and it may also be included with GDE as part of the BIRCH system now -- http://home.cc.umanitoba.ca/~psgendb/treetool/treetool.html However, searching for treetool, I also found a list of other tree manipulation tools here http://evolution.genetics.washington.edu/phylip/ software.html#Interactive Perhaps one of the newer programs will work for you Mitchell Miller Some suggestions: 1)download your PSI-BLAST hits in FASTA format 2)align the sequences using PRANK http://www.ebi.ac.uk/goldman-srv/ prank/prank/ 3a)Use BEAST to evaluate the phylogeny http://beast.bio.ed.ac.uk/ and/or 3b)Use MrBayes to evaluate the phylogeny http://mrbayes.csit.fsu.edu/ 4)Use FigTree to examine the BEAST or MrBayes output tree http:// tree.bio.ed.ac.uk/software/figtree/ Starr Hazard Hi Raji..I am assuming you have looked at the Blast Link section on NCBI called BLINK..You can filter by Archaea , Eukarya , Prokarya in the least.. You can find a screencast video describing Blink at http://www.bioscreencast.com/html/tag/blink Or the actual documentation at http://www.ncbi.nlm.nih.gov/sutils/static/blinkhelp.html#SampleQuestions Its good to look for homologs this way Another good tree viewer and manipulator is jalview check it out or check out http://www.bioscreencast.com/html/tag/jalview Hope this helps Hari Jayaram I had to create the tree of life for a bioinformatics class recently. Download the ribosomal rna sequences for the species you're interested in from http://www.arb-silva.de/browser/, you can use their aligner or you can align them yourself (clustalw or pick your favorite), then I used raxml (doi:10.1093/bioinformatics/bti191 ) to make the trees and then you can visualize the trees in something like HyperTree (Mac). Don't know of any PC alternatives as it's not my main OS. HTH Francis Reyes = Original question: Hi Everyone, Could someone please tell me how to display the evolutionary/ phylogenetic tree of the homologs of my protein of interest. When I perform a PSI-BLAST search for my protein, I receive about 130 top hits for homologs. The NCBI or EBI tools that I've laid my hands on seem to only display a 'phylogenetic' tree based on the distance relationships between the protein sequences and that is not what I am after. I'd like to find a way to resort the results and redisplay a tree that say progresses from, say 'yeast to human'. What I have now is the organisms shown in some random order, say, rat followed by C. elegans followed by human. I am going bonkers trying to find a simple way to do what I want. Thanks and sorry for this Bioinformatics101-type question. Raji
[ccp4bb] Thanks: Phylogenetic analysis??
Thanks to all who responded. I was able to somewhat generate the info I needed. Raji
[ccp4bb] Phylogenetic analysis??
Hi Everyone, Could someone please tell me how to display the evolutionary/ phylogenetic tree of the homologs of my protein of interest. When I perform a PSI-BLAST search for my protein, I receive about 130 top hits for homologs. The NCBI or EBI tools that I've laid my hands on seem to only display a 'phylogenetic' tree based on the distance relationships between the protein sequences and that is not what I am after. I'd like to find a way to resort the results and redisplay a tree that say progresses from, say 'yeast to human'. What I have now is the organisms shown in some random order, say, rat followed by C. elegans followed by human. I am going bonkers trying to find a simple way to do what I want. Thanks and sorry for this Bioinformatics101-type question. Raji
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Hi, Many things can lead to your observation. Please outline all steps of your purification procedure as it is not clear what is done before and after the Ion Exchange steps. I am not sure if IEF in your emails refers to Isoelectric focusing, as the acronym is usually used?? Couple of suggestions: 1) Instead of contamination, you might just be seeing multiple bands due to 'aggregation' of your protein! Make sure you boil the sample prior to loading on gel and also that your loading dye contains SDS, bME/DTT. 2) We used to do entire purifications with inclusion body preps under denaturing conditions to prevent unwanted aggregation of partially folded or misfolded species. Not sure if denaturant is present all along. 3) If the problem is of contamination, try making the gradient shallow for the 35 –80% gradient step in Ion Exchange (increase to say, 20 cv or more). 4) If the problem is of contamination, try to add more steps to purification -- e.g., affinity step (if possible), anion exchange as well etc. 5) If IEF (in the sense I mean it) is what you did and it shows only 1-2 bands, the problem is likely (#1) outlined above. 6) If all else fails, cut out one or two of the bands from your gel and run a mass spec. An expensive way to find out that it is aggregation. Nevertheless Hope that helps. Raji On Sep 23, 2008, at 3:51 AM, Meg wrote: Dear All, This is with reference to the purification of our recombinant protein sample expressed in E.coli as inclusion bodies. After Solubilization refolding we perform the cation exchange chromatography of our protein sample using SP sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF results of the collected fractions. In addition to our protein of interest we are also getting high molecular weigh contaminants, which we cannot get rid of in IEX. Can anyone please guide me on a technique to get rid of these bands as even after gel filtration of samples few high mol wt contaminant bands are not separated from main proteins and sample gets diluted too. In cation IEX procedure is Column Sp Sepharose Fast flow packed in fineline 35 column packed bed volume 100 ml System AKTA FPLC Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc], washing to remove unbound materials 2 C.V. step elution 0-35% gradient – 1 C.V., 35 –80% gradient – 10 C.V. and 60 –100% gradient 1 C.V. Protein elutes at 40-50% gradient. Protein details: Our protein is stable at acidic pH and has a pI of 5.8 –6.3 and buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer containing 0.4 M NaCl. We get only one peak on AKTA but on running SDS page we get so many bands even IEF shows 1-2 bands at the most. How can we modify the method or what can be done to get rid of extra high mol wt bands. Any help will be deeply appreciated. SDS PAGE.JPGIEF.doc
Re: [ccp4bb] regarding cloning
Hi Vijay, I have heard of TOPO-TA cloning. Not sure what T/A cloning is. I have a couple to check based on your description: 1) Do you CIP-treat your vector? If not, that might be a step to add. Also, you could include a 'vector only' transformation control to determine how many colonies are obtained in the 'vector+insert' plate above background levels. 2) For NheI/BamHI, a sequential digestion (rather than a double digestion) is recommended. 3) Make sure the primers have sufficient extensions (6-8nt) outside of the RE site. 4) Make sure both enzymes work by doing single digestion controls with the vector. It's obviously hard to tell with the PCRT product 4) Titrate vector:insert ratios -- lower and higher than you mention 5)... Once ALL possibilities are exhausted and when nothing else works, I have seen people reorder the exact same primers and then things have worked like a charm! Hope that helps. Raji -Included Message-- Date: 1-sep-2008 03:06:29 -0400 From: vijay srivastava [EMAIL PROTECTED] Reply-To: [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] regarding cloning Hi, I am trying to clone a 1.2kb insert into a expression vector pET 23a through T/A cloning.#194; The restriction enzyme used is Nhe1(NEB) and BamH1 (NEB) in the forward and reverse primer recpectively. I was succesful#194; in subcloning (T/A vector) and getting my insert at 1.2kb after#194; double digestion and also the vector at 3.7kb ,for the ligation i am using the ratio of vector to insert is 1:3,1:2,getting the colony after the transformation but some how#194; when i used to confirm my clone through double digestion i am not getting my insert at the correct position.Some time in the gel only the size of the vector was there. Connect with friends all over the world. Get Yahoo! India Messenger at http://in.messenger.yahoo.com/?wm=n/ -End of Included Message--
Re: [ccp4bb] Anion binding sites in proteins
Hi Albert, Please refer to the following paper (and references therein) for a description of the four chloride located in the nucleosome structure.. 1: Davey CA, Sargent DF, Luger K, Maeder AW, Richmond TJ. Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution. J Mol Biol. 2002 Jun 21;319(5):1097-113. In the nucleosome structure, there are four locations where chloride ions are found. For example, one of the hydrated chlorides is found in a Van der Waal's cup of sorts formed by three amino acids (Met, Pro and Lys). See PDB id 1AOI, 1KX5 or 1S32. Hope that provides some info. Raji -Included Message-- Date: 10-jul-2008 11:47:59 -0400 From: Jacob Keller [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Anion binding sites in proteins I have been interested in this topic as well, so could any responses that are not also addressed to the BB be sent to me as well? Thanks very much, Jacob Keller *** 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: [EMAIL PROTECTED] *** - Original Message - From: Albert Guskov To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, July 10, 2008 3:52 AM Subject: [ccp4bb] Anion binding sites in proteins Dear all, can someone point me to something similar to Metal coordination sites in proteins (http://tanna.bch.ed.ac.uk), but describing anions (I'm mainly interested in chloride-binding sites)? Thank You, Albert -- Albert Guskov, Freie Universitaet Berlin Fachbereich Biologie, Chemie, Pharmazie Institut fur Chemie/Kristallographie -End of Included Message--
Re: [ccp4bb] Two off-topic questions
hloy cow! taht msut be vrey crortcet idened ! rjai -Included Message-- Date: 12-may-2008 14:15:03 -0400 From: Scapin, Giovanna [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Two off-topic questions Hello there, I don't usually do this, but the missing letter reminded me of thiseverything is relative! More Brain Stuff . . . From Cambridge University O lny srmat poelpe can raed tihs. cdnuolt blveiee taht I cluod aulaclty uesdnatnrd waht I was rdanieg. The phaonmneal pweor of the hmuan mnid, aoccdrnig to a rscheearch at Cmabrigde Uinervtisy, it deosn't mttaer in waht oredr the ltteers in a wrod are, the olny iprmoatnt tihng is taht the frist and lsat ltteer be in the rghit pclae. The rset can be a taotl mses and you can sitll raed it wouthit a porbelm. Tihs is bcuseae the huamn mnid deos not raed ervey lteter by istlef, but the wrod as a wlohe. Amzanig huh? yaeh and I awlyas tghuhot slpeling was ipmorantt! if you can raed tihs psas it on !! -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Partha Chakrabarti Sent: Monday, May 12, 2008 2:05 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Two off-topic questions Or, make sure that you don't post it on Monday, some people, sometimes are in very bad mood on Mondays, I am not going to explain why.. :P On Mon, May 12, 2008 at 5:57 PM, Yong, Wei [EMAIL PROTECTED] wrote: Sorry that I missed a letter. I wanted to extract mRNA from pig liver. Thank you, Frank, for having taught me a lesson. I will carefully check my emails before I send them to ccp4bb. I am looking forward to getting suggestions. Best wishes Wei Yong From: Frank von Delft [mailto:[EMAIL PROTECTED] Sent: Mon 5/12/2008 11:12 AM To: Yong, Wei Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Two off-topic questions only tried once). Does any body have ideas about how to extract mRNA from pig live (or animal tissue in general)? From pig live? Yeah sure, toss it in a blender -- you can tell whether it's alive by the squealing. You may need quite a large blender, though, and a crane to lift it, pigs are deceptively large. It's trickier to keep the pig alive even after extracting the mRNA. The subject was dealt with at some length as far back as the late 16th century by a well-known English dramatist, although he framed the problem in more general terms. But do you really need a pound of the stuff? phx. -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: [EMAIL PROTECTED] Phone: + 44 208 816 2515 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 (which may be known outside the United States as Merck Frosst, Merck Sharp Dohme or MSD and in Japan, as Banyu - 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. -End of Included Message--
Re: [ccp4bb] sa_omit_map
To me, it seems that your syntax for chain and residue definitions is incorrect. See the 'Tutorial' section on the CNS website, which gives some very nice examples. For example, if you want to select chain K and residues 2 and 15, chain L and residues 1, 2, and 14, and so on, here's pne possible syntax: ((segid K and resid 12) or (segid K and resid 15) or (segid L and resid 1) or (segid L and resid 2)blah blah) Make sure to keep track of the opening and closing parentheses. Hope that helps. Raji -Included Message-- Date: 2-may-2008 12:56:39 -0400 From: Raja Dey [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] sa_omit_map Hi, I am trying to omit some residues from 4 chains to calculate sa_omit_map.map. How I should declare the command in the inp file? See the error below. %CNSsolve-ERR: unrecognized command: ident ( store8 ) ( byresidue ( ( segid K and resid 2, 15 or segid L and resid 1, 2, 14 or segid C and resid 2, 3, 15 or segid D and resid 1, 2 ) Thanks for your help Raja Meet people who discuss and share your passions. Go to http://in.promos.yahoo.com/groups/bestofyahoo/ -End of Included Message--
[ccp4bb] Bacterial induction at 18C
Hi Folks, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks. Raji
Re: [ccp4bb] Bacterial induction at 18C
Thanks to everyone for all your suggestions. I am growing the cultures as we speak and have increased the temp to 22C and plan to harvest in about 6-8 hrs. Thanks for the Q7 rule. I read it before but I couldn't remember exactly and a quick-and-dirty Google and Pubmed search did not bring it up. Let me clarify what I mean by lysis. Here are my observations: a) At the time of harvest, the final OD is lower for protein A + protein B (on two plasmids) than that for the same cells expressing only protein A or protein B (all else being similar at the time of induction). b) When the cells are spun down, the supernatant is cloudy and the pellet is smaller for A+B. The supernatant is clear for A alone or B alone. I am not sure this is a result of phage contamination since I have two other 'controls' for the same batch of competent cells in the same shaker, one containing just plasmid A and the other with only plasmid B. And, this is reproducible. Yes, I also very much suspect that my proteins may be a culprit, even though I only mentioned the antibiotics. Will see what happens this time. Thanks very much for all the helpful suggestions. Raji -Included Message-- Date: 30-apr-2008 12:30:50 -0400 From: Guenter Fritz [EMAIL PROTECTED] To: [EMAIL PROTECTED] Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Bacterial induction at 18C Raji, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. Sounds more like a phage contamination. The phage becomes active as soon as the cells energy level decreases, e.g upon induction. We had once the same trouble. If it is a phage, autoclave everything and clean the lab thoroughly. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested growing cultures at 18C, say for 4-6h instead. Has anyone had reasonable protein expression levels by inducing cultures at 18C for 6h? From what I understand, the E. coli doubling time is manyfold longer than at 37C. But I thought I'd ask. Rule of the thumb is the Q10 rule, or in the case of e.coli it is a Q7 rule. Doubling decreases twofold when temperature eis decreased by 7 deg C. Godd luck, Guenter I am already playing with lowering and/or doing away with the antibiotics. Any suggestions wrt 18C? The protein is insoluble at 30C. Thanks. Raji -- *** Priv.Doz.Dr. Guenter Fritz Fachbereich Biologie Sektion Naturwissenschaften Universitaet Konstanz http://www.biologie.uni-konstanz.de/fritz Universitaetsstrasse 10 Postfach M665 D-78457 Konstanz e-mail: [EMAIL PROTECTED] Tel. Office: +49-(0)7531 88 3205 Tel. Lab : +49-(0)7531 88 3687 Fax: +49-(0)7531 88 2966 -End of Included Message--
Re: [ccp4bb] Co-expression plasmids
I do use the Duet vectors extensively and they work just fine. I have trouble with my protein complex, but that is solely related to my proteins. My lab mate has used these vectors very successfully. I have been using pRSF-Duet and pETDuet1 more than pCDFDuet1 or pACYCDuet1 for no specific reason except for a slight preference for the antibiotics. I avoid pACYCDuet1 (Cam resistance) since a lot of expression cells I use have plasmids with Cam resistance. Also, I recently heard about the chaperone co-expression from Takara and folks claim that this system works well. Also, some folks recommend pCOLD vectors (Takara). Might be worth looking into if you are shopping anyway. I don't think these are for co-expression but it is just as easy to stitch in a T7 promoter and RBS to incorporate additional genes. Hope that helps. Raji -Included Message-- Date: 2-apr-2008 09:56:09 -0400 From: Mark J. van Raaij [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Co-expression plasmids Dear All, Anyone have experience with the NovaGen Duet co-expression vectors? Or can recommend others? http://www.emdbiosciences.com/html/NVG/Duet_Spot.html Greetings, Mark Mark J. van Raaij Dpto de Bioqu#237;mica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/ -End of Included Message--
Re: [ccp4bb] Co-expression plasmids
I thought only I was having trouble with cloning into pETDuet1. But just to add to what someone just said Yes, I had hell with trying to get one of my ~2kb fragments into pETDuet1 (5.4kb). Could be a combination of vector size and insert size.. Who knows! Sometimes, I do what Tasos says: Transform two plasmids into cells, either sequentially or co-transform. I just play down the amount of each DNA to be used. Raji -Included Message-- Date: 2-apr-2008 09:56:09 -0400 From: Mark J. van Raaij [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Co-expression plasmids Dear All, Anyone have experience with the NovaGen Duet co-expression vectors? Or can recommend others? http://www.emdbiosciences.com/html/NVG/Duet_Spot.html Greetings, Mark Mark J. van Raaij Dpto de Bioqu#237;mica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/ -End of Included Message--
Re: [ccp4bb] Tough Low-Res MR
Absolutely try Phaser! See Phaser documents for all the nifty combinations. Multiple molecules in MR model; break down molecule by domains etc etc. You can trim down side chains, make hybrid models and what not. All easy to get up and going through the GUI. Once the GUI drove me insane because of some 'hyphen' mark in the file name; sometimes I just prefer to set up scripts. Run to the documentation and examples, which are both pretty darned good. NO! I don't get paid by Randy. However, given the low resolution and the ambiguities you describe, especially of the combination in the ASU and if the two subunits are closely related and of similar lengths, my hunch is that experimental phasing will become necessary at some point. Since you describe you have 2*AB in the ASU, first search for AB as search model. One way for internal check is to feed Phaser only Molecule A and ask to look for 4 molecules. Then, feed only Molecule B and look for 4 molecules. If all these results look the same, I'd be worried and I might think of SelMet-labeling one of the two molecules or something like that. Just some thoughts. Good luck and just download Phaser for starters. Raji -Included Message-- Date: 21-feb-2008 06:40:57 -0500 From: James Stroud [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Tough Low-Res MR Hello All, I have a tough ~3.5 #197; (pushing it) MR problem where I have a solution of sorts, but because I'm working with a heterodimer of two closely related subunits (with two such heterodimers in the ASU) I have a 2**2 possibilities for the arrangement of these subunits in the ASU. Each subunit is composed of two more-or-less independent domains. Basically I'm looking for the best possible software to disambiguate this problem. I usually use CNS for these problems, but I think I may have exhausted its capabilities. I've heard that there have been some advances in MR in recent years, but I haven't kept up with all of the software. Does anyone have suggestions for packages to try? James -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com -End of Included Message--
Re: [ccp4bb] secondary structure restraints
Hi Sean, Not an answer to your question but have you looked into model building with TEXTAL? I am not sure about 3.6Ang resolution data but it might be worth looking into. Raji -Included Message-- Date: 13-feb-2008 16:54:33 -0500 From: Sean Johnson [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] secondary structure restraints I am trying to build and refine a model into 3.6 angstrom Se-met phased maps. What is the best way to define secondary structure restraints for refinement? (hydrogen bonds? backbone torsion angles?) Are there any tools available to help me define restraints for a specified region, or do I have to define each restraint one at a time (which strikes me as a very tedious exercise). Any other thoughts on best practices for low-resolution model building and refinement would also be appreciated. Thanks, Sean -- Sean Johnson, PhD R. Gaurth Hansen Assistant Professor Utah State University Department of Chemistry and Biochemistry 0300 Old Main Hill Logan, UT 84322-0300 (435) 797-2089 (435) 797-3390 (fax) [EMAIL PROTECTED] -End of Included Message--
[ccp4bb] Solved: PDB file column-cut-paste issues
Thanks everyone for all your suggestions. What's the issue? Each residue has one CSV value (per residue value) and the PDB line contains many lines of B-values per residue. This would leave us with no easy one-to-one line correlation between the B-value column and the CSV column. From what I understand, that would rule out a simple cut-and- paste in nedit solution. Basically, the student wanted to colour the molecule by chemical shift. It took a couple of PhDs in the lab to figure out the solution. The SOLUTION: Many folks have apparently had the same issue before and therefore, someone (aka Warren Delano, I guess), wrote a script for precisely the same and posted an example script on the PymolWiki. All that was needed was a more extensive Google search :) Your suggestions and tips will be diligently used elsewhere. Cheers, Raji
[ccp4bb] PDB file column-cut-paste issues
Hi People, I post this on behalf of my colleague. My colleague has a file containing chemical-shift values for the 150 aa in his structure. He also has the PDB file for the crystal structure. Now, he would like to replace the B-factor column with the CS values to make some figures. It would be easy to yank out the column from the PDB file and paste the column containing the CS values. However, there is only one value per residue. I don't want to ask him to use Moleman to reset B-factor column per residue with the CS value and do this 150 times!! Also, the number of lines per amino-acid type is different!!! How do we do this in a less than manual way? Thanks. Raji
Re: [ccp4bb] Solved: PDB file column-cut-paste issues
Ooh..la la! Where were you 12 hrs ago when we were suffering brain damage! Cheers! Raji PS: Thanks! Oh dear - too late :-(. You can do it in Coot too! (The solution is on the Coot Wiki now) http://xanana.ucsc.edu/~wgscott/xtal/wiki/index.php/Coot#Example_Scheme_Script_7:_Applying_arbitrary_value_to_.22B.22_factor_column Paul. -End of Included Message--
Re: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)
Can the CCP4BB provide something like a website to upload pictures and then have the BB-ers just post the link in their email. Please! These attachments are clogging my inbox... Thanks much. Raji -Included Message-- Date: 31-jan-2008 03:58:44 -0500 From: Frank von Delft [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex) Actually, it's not book keeping, it's simple courtesy -- and not only on a BB: an attachment is lazy, and a large attachment is downright rude. I am routinely stuck with a slow connection (travelling), and others are *permanently* stuck with one. So please be nice... ;) phx. Anastassis Perrakis wrote: Dear all - Sorry to intervene on a 'book keeping' issue, but indeed over the last few months an increasing number of people (Jerry is not the first, so Jerry please do not take it personally) attach pictures etc. I think in a bb standard practice dictates to only use text - if illustrations are needed to explain the problem, you can put them in eg a web site. Some text like that was in the 'code of conduct' off ccp4bb in the past, but I could no longer find it. Thus apologies if I am wrong and policies have changed, but maybe the ccp4 crowd could tell us what is the suggested policy. And, if you really want to send an image please do bother to make it small. The initial posting had a 630k image, which it took me 1 min to make 20k and it still makes the point (attached so I can also violate the rules i am suggesting - I love inconsistency). Thanks, Tassos On Jan 30, 2008, at 20:11, Jerry McCully wrote: Dear All: Thanks a lot for the prompt reply on this topic of salt sensitive complex. Attached please find one ITC final figure done under 25mM Tris(pH8.0), 60mM NaCl. As mentioned before, the ionization of Tris will interfere with the ITC experiments. Therefore I am sure of my binding results. Can anyone give me some comments on this ITC experiment? Basically do these two proteins bind to each other? If so, how should I improve the ITC experiments to get a similar affinity shown by BIAcore(about 0.5uM)? Thanks again. Jerry Hi Jerry, Tris can cause problems, you are better off using something like HEPES, and HEPES should be ok at pH 8. (Buffers with an ethane-sulphonic acid group tend to be the best - those ending in 'ES', so MES, TES and HEPES) FYI, the error on your K is bigger than the actual measurement - 1.49x10^5 #177; 1.5x10^5. Signal to noise to is probably your enemy, which is making the curve fitting difficult. Changing buffer may help this - there may be some non-specific component to what you're seeing - increasing salt a bit or dropping in something like 5% glycerol may help with this. Would you be able to post a jpeg/pdf of the curve? Regards, David On 25/01/2008, Jerry McCully wrote: Dear All: Firstly I would like to thank many folks here for giving me great ideas several days ago. The following are some updates for this question. I did ITC experiments again using 25mMTris(pH8), 60mM NaCl(low salt condition). But things still turn out to be a little weird. I increased the concentration of both proteins(60uM in the cell and 1200uM in the syringe). At the end of the ITC, I saw a little of precipitation of both the proteins. Fortunately I can roughly fit the curve this time. However, the heat was still low, around 1Kcal/mole of per injectant. I am not sure about the fitting statistics. N 1.10 #177;0.17 K 1.49E5 #177;1.5E5 DH -893.5 #177;213 DS 20.7 Was the enthalpy was offset by the ionization of Tris buffer? Can I use Hepes buffer around pH8 to do ITC? Welcome any comments about the statistics and suggestions on how to improve the ITC experiments.have a nice weekend. Jerry Helping your favorite cause is as easy as instant messaging. You IM, we give. Learn more. http://im.live.com/Messenger/IM/Home/?source=text_hotmail_join Shed those extra pounds with MSN and The Biggest Loser! Learn more. http://biggestloser.msn.com/ test-ITC-012608.JPG -End of Included Message--
Re: [ccp4bb] protein expression problem
I wholly agree with the below. I am not sure how well E.coli can correctly fold snaky misfolded/unfolded protein that are chaperoned by folded tags! Not to rule out that tags do it sometimes... Folded by association for insoluble proteins has often not worked well for me. Sometimes, when it 'works' for me and my colleagues, removal of the tag leads to insoluble protein/aggregation etc. I am dealing with SUMO tagged proteins that have enhanced solubility but severe degradation issues. Screening for pH, buffers and all the good-old stuff folks have suggested here is a good approach. Sometimes autoinduction protocols, which keep from 'overexpression' of protein in the cell might be an approach to explore after the above tests. Good luck! Raji First of all, using a carrying protein (like GST, MBP) can be disconcerting. These proteins are very soluble and can solubilize an insoluble protein in testing condition. So you have something soluble but your protein of interest can be misfolded or can precipitate when the carrying protein was cleaved. So keep in mind that a soluble carried protein is not always a good protein.
