Re: [ccp4bb] Strange density in solvent channel and high Rfree
I second Phoebe's suggestion. Looks like another molecule to me. If you are doing molecular replacement you may need to get creative about trimming. When we had a case like that it wasn't until the third person worked on it that we go a solution because he trimmed the model differently than the first two who tried. Kendall On Mar 15, 2013, at 3:29 PM, Phoebe A. Rice pr...@uchicago.edumailto:pr...@uchicago.edu wrote: What happens if you solvent-flatten/flip/massage that map, but tell the software the solvent content much lower than what you think it is now? Maybe you'll find another copy of the molecule? From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Andrey Nascimento [andreynascime...@gmail.commailto:andreynascime...@gmail.com] Sent: Friday, March 15, 2013 1:39 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Strange density in solvent channel and high Rfree Dear all, I have collected a good quality dataset of a protein with 64% of solvent in P 2 21 21 space group at 1.7A resolution with good statistical parameters (values for last shell: Rmerge=0.202; I/Isig.=4.4; Complet.=93% Redun.=2.4, the overall values are better than last shell). The structure solution with molecular replacement goes well, the map quality at the protein chain is very good, but in the final of refinement, after addition of a lot of waters and other solvent molecules, TLS refinement, etc. ... the Rfree is a quite high yet, considering this resolution (1.77A).(Rfree= 0.29966 and Rfactor= 0.25534). Moreover, I reprocess the data in a lower symmetry space group (P21), but I got the same problem, and I tried all possible space groups for P222, but with other screw axis I can not even solve the structure. A strange thing in the structure are the large solvent channels with a lot of electron density positive peaks!? I usually did not see too many peaks in the solvent channel like this. This peaks are the only reason for these high R's in refinement that I can find. But, why are there too many peaks in the solvent channel??? I put a .pdf file (ccp4bb_maps.pdf) with some more information and map figures in this link: https://dl.dropbox.com/u/16221126/ccp4bb_maps.pdf Do someone have an explanation or solution for this? Cheers, Andrey
Re: [ccp4bb] vitrification vs freezing
I completely agree with Quyen. One of the many definitions of freeze is to make extremely cold. It is grammatically correct to say freezing your crystals, especially since, as you point out, everyone reading it knows exactly what you did, and which definition of freeze you were referring too. It is completely unambiguous in my opinion, and it's how people normally talk about it. I wouldn't go into the lab and say did you cryo-cool those crystals yet? or check out this nice crystal. Its ready for vitrification. Best, Kendall On Nov 16, 2012, at 11:28 AM, Quyen Hoang qqho...@gmail.com wrote: I enjoyed following this thread. Because English is not my first language, I was hoping to learn the official definitions of these terms. In my opinion, all the variations proposed so far are fine - I don't see problems with using them. For me, when I see flash frozen in liquid nitrogen or flash frozen in nitrogen stream I get unambiguous mental images of how the crystals were prepared. When I hear a policeman yelling freeze while pointing a gun (no personal experience here), there is no ambiguity that I should stop moving (and won't get confused with cooling myself such that the water in my body would form hexagonal ice). When I hear that a person is frozen by Parkinson's disease, there is no ambiguity that his/her muscle had become rigid. I think that I will continue to use flash frozen in liquid nitrogen or flash frozen in nitrogen stream and I hope that I would not need to explain to reviewers what that means. Quyen On Nov 16, 2012, at 10:48 AM, Ganesh Natrajan ganesh.natra...@ibs.fr wrote: Hi, Maybe we could just state the obvious, ie, that the crystals were 'Cryo-preserved' in liquid N2. Cheers Ganesh Le 16/11/12 16:27, Enrico Stura a écrit : As a referee I also dislike the word freezing but only if improperly used: The crystals were frozen in LN2 is not acceptable because it is the outside liquor that is rapidly cooled to cryogenic temperatures. But the use of freezing used as the opposite of melting is fine and does not imply a crystalline state. Ice is not always crystalline either: http://en.wikipedia.org/wiki/Amorphous_ice
Re: [ccp4bb] Ligand fitting into density
Sometimes you can distinguish disorder from the other possibilities if you have enough structures with the same protein. We have several examples where part of the ligand is not visible in the maps, but there is clear distortion of the ligand binding pocket to accommodate the missing piece. for example, we have one with a ligand that has an extended linker. You can see a big whole in the protein where the linker leaves the pocket, but no density for the tether. Kendall On Apr 11, 2012, at 8:09 AM, Fischmann, Thierry wrote: Dipankar, Herman's message describes the most common case, by far. In addition to his excellent post there are 4 other possibilities which I can think of of why, in general, the e- density may be missing for part of a ligand: - the compound solution are never 100% pure. One impurity may be the compound you're trying to soak but missing a specific moiety. This would be a result of the chemical synthesis: one of the steps would be incomplete and the impurity was not separated at a later stage. The impurity is what you'll see in the electron density if it happens to bind significantly more tightly than the intact ligand. Sometimes this possibility can be excluded just from the chemical synthesis (unless the purity of some of the starting reagents is questionable). Or you can check the inhibitor structure by Mass spec. - compound is not stable in the soak conditions. For instance it may not be stable at, say, in water, or in acidic conditions, or exposed to visible light, etc. - cleavage by the protein: on occasion the protein may be able to cleave the ligand. This is usually observed if the ligand is a substrate (say 3rd phosphoryl missing in a soak with ATP) or a close relative of the true substrate. - cleavage by X-ray: the compound gets degraded during data collection, fast enough that a significant part of the data set is collected with the compound with the missing piece. Thierry From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com Sent: Wednesday, April 11, 2012 5:39 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Ligand fitting into density Dear Dipankar, I have had a case where I had soaked the same compound in Trypsin (2.2 Å) and in Factor Xa (2.0 Å). In Trypsin, one six-membered ring was completely invisible, despite good resolution and phases, whereas this ring was clearly visible in the Factor Xa structure. The electron density is shown in J.Med.Chem (2002)45:2749 figures 1A and 1E. It does happen that parts of a soaked compound are completely without electron density. In these cases I assume that this part is disordered and I refine the compound without the undefined parts, while in contrast to flexible surface residues, people look closely at bound compounds and use the structures e.g. to optimize scoring functions for docking programs. Leaving the undefined parts in the model in a guessed conformation would likely cause people to draw wrong conclusions. For the rest, if the inhibitor is well-defined in the electron density maps, I would not worry about the high B factors. They may even normalize once you leave out the undefined part. Best regards, Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar Manna Sent: Wednesday, April 11, 2012 11:11 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Ligand fitting into density Dear Crystallographers, The protein I am working with is having SG P3121, Structure is solved at 2.5A. the protein was soaked with compound, compound density is also looking prominent except one six membered ring. There is no density at all for the particular ring, but other parts of the compound is fitting well enough into the density. The B factor of the ligand is showing 100. How can I justify this issue. Asking for suggestions. Regards, Dipankar Manna This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com 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
Re: [ccp4bb] very informative - Trends in Data Fabrication
My intent with the troll joke was to give a humorous reminder that a little self promotion is ok, but a couple times a day is annoying. Orcus means troll, as in Internet troll, meaning one who subverts the intended use of the site and is annoying people. You have made a number of on topic posts that were very nice, but also a number that were clearly off topic and viewed as self promotion, with links to your consulting service. A couple times a day is a bit much. No one wants to be rude, so we try to humor you into toning it down a little. Compared to many Internet forums, this is likely one of the nicer responses you could expect. all the best, Kendall On Apr 3, 2012, at 8:22 PM, Kevin Jin kevin...@gmail.commailto:kevin...@gmail.com wrote: Thanks of your education. I got it. By the way, what does Orcus mean here? Regards, Kevin On Tue, Apr 3, 2012 at 5:11 PM, Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.commailto:hofkristall...@gmail.com wrote: Orcus, if you put yourself persistently into the face of guys who play hard, you need to learn to take a few hits and shake it off. Maybe a little retrospection on why your postings might perhaps possibly maybe perceived as somewhat self-promoting and ungracious could be helpful. The skill of presentation is at least as important in Science as being right. Best, BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kevin Jin Sent: Tuesday, April 03, 2012 3:34 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] very informative - Trends in Data Fabrication Dear All, Here may be another example for the importance of image storage. http://www.jinkai.org/DERA/DERA_1O0Y_3R12.html Regards, Kevin -- Kevin Jin Sharing knowledge each other is always very joyful.. Website: http://www.jinkai.org/
Re: [ccp4bb] very informative - Trends in Data Fabrication
James makes an important point. I've come to regret my joke as showing poor manners. I hesitate to add to more email that no one cares about, but I do think it is important to contribute the idea that the positive tone of this forum needs to be protected. I apologize, and suggest my comments should have been offered directly and off-line in order to be constructive and not off-putting to others who would want to contribute or ask questions. Kendall On Apr 3, 2012, at 10:01 PM, James Stroud xtald...@gmail.commailto:xtald...@gmail.com wrote: On Apr 3, 2012, at 7:19 PM, Katherine Sippel wrote: I would also consider looking into adding an RSS feed to your site so that those people interested in your articles can be informed without spamming the boards. Why continue to punish him? Adding an RSS feed means installing and configuring an RSS server. Aren't there rules against cruel and inhumane punishment? There are many free newsfeed disseminators. Twitter is the most famous. There are others, maybe better, so I'm not being a twittervangelist here. My point is this: free and easy is better than difficult. James
Re: [ccp4bb] one datum many data? [was Re: [ccp4bb] very informative - Trends in Data Fabrication]
My favorite part of the german humor link: Some German humorists such as Loriothttp://en.wikipedia.org/wiki/Vicco_von_B%C3%BClow use seriousness as means of humor. On Apr 2, 2012, at 1:38 PM, Bosch, Juergen wrote: Hm, last I checked my passport said German - still think I can make lots of fun of myself. Some Germans are epigenetically marked with humor-suppressor genes others not. Jürgen On Apr 2, 2012, at 11:03 AM, Gerard DVD Kleywegt wrote: Dear Manfred, Outside Germany, such excursions are called humour. If you are interested, here is the Wikipedia page for it: http://en.wikipedia.org/wiki/Humour --Gerard PS: It was on a Sunday so all levity was perpetrated in people's own time. Today we'll all be serious again and frown and tut-tut appropriately. On Mon, 2 Apr 2012, Manfred S. Weiss wrote: Dear all, I find this discussion most amazing. Here, we are dealing with the most serious issue that happened to Macromolecular Crystallography since the Alabama case, and the whole discussion is centered around singular and plural and Greek and Latin words and what not. In psychology such phenomenon is referred to as displacement activity. If you are interested, here is the MacMillon definition of it: http://www.macmillandictionary.com/dictionary/british/displacement-activity Cheers, Manfred On 01.04.2012 19:35, Gerard Bricogne wrote: On Sun, Apr 01, 2012 at 01:18:15PM -0400, David Schuller wrote: On 04/01/12 10:18, Gerard Bricogne wrote: Dear Paul, May I join the mostly silent chorus of Greek/Latin-aware grumps who wince when seeing data treated as singular when it is plural. When it are plural? Good nit-picking :-) . In my mind the quotes around data would have had the same effect as writing 'the word data', and referring to that word by the 'it'. So there is only one word, while its grammatical number is plural. At any rate, I heard a Nobel laureate use it incorrectly just two days ago. We shouldn't learn to write by imitating Nobel laureates, then. With best wishes, Gerard. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edumailto:schul...@cornell.edu -- Dr. Manfred. S. Weiss Helmholtz-Zentrum Berlin f?r Materialien und Energie Macromolecular Crystallography (HZB-MX) Albert-Einstein-Str. 15 D-12489 Berlin GERMANY Fon: +49-30-806213149 Fax: +49-30-806214975 Web: http://www.helmholtz-berlin.de/bessy-mx Email: mswe...@helmholtz-berlin.demailto:mswe...@helmholtz-berlin.de Helmholtz-Zentrum Berlin f?r Materialien und Energie GmbH Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren e.V. Aufsichtsrat: Vorsitzender Prof. Dr. Dr. h.c. mult. Joachim Treusch, stv. Vorsitzende Dr. Beatrix Vierkorn-Rudolph Gesch?ftsf?hrerin: Prof. Dr. Anke Rita Kaysser-Pyzalla Sitz Berlin, AG Charlottenburg, 89 HRB 5583 Postadresse: Hahn-Meitner-Platz 1 D-14109 Berlin http://www.helmholtz-berlin.dehttp://www.helmholtz-berlin.de/ Best wishes, --Gerard ** Gerard J. Kleywegt http://xray.bmc.uu.se/gerard mailto:ger...@xray.bmc.uu.se ** The opinions in this message are fictional. Any similarity to actual opinions, living or dead, is purely coincidental. ** Little known gastromathematical curiosity: let z be the radius and a the thickness of a pizza. Then the volume of that pizza is equal to pi*z*z*a ! ** .. 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/
Re: [ccp4bb] very informative - Trends in Data Fabrication
What is the single Latin word for troll? Kendall On Apr 1, 2012, at 3:06 PM, Kevin Jin kevin...@gmail.commailto:kevin...@gmail.com wrote: “I hope and believe that this is not the case. Even basically-trained crystallographers should be able to calculate andinterpret difference maps of the kind described by Bernhard. And with the EDS and PDB_REDO server, one does not even need to know how to make generate a difference map...” You are right! Actually, I am not an experienced protein crystallographer. I have learnt a lot from CCP4BB. I may have paid too much attention to bonding angle and bond length, like in small molecule. This may be an example to share with you. When I worked on those nitroreductase complexed with FMN in 2009 (?), I always observed that the flavin ring presented a strange geometry after refinement. Indeed, I had used the definition of FMN from CCP4 library all the time. In some cases, the methyl group at position of either 7a or 8a was bent off the aromatic ring, if the whole the rest of flavin was restrained in a flat plane. According to my limited knowledge from organic chemistry, carbon of 7 and 8 on the flavin ring is sp2 hybridized in a coplanar manner. How could those methyl groups be bent as sp3 hybridization? Any chemistry behind? With increased resolution (1.6 ~ 1.8 Ang), I observed that the electron density map was a bent along the N5-N10 axis. The bend angle was around ~16 degree. Again, I questioned myself why it was bent? Should this be correct? According to my limited knowledge in chemistry, N10 should be sp3 configuration even if FMN is in its oxidization form, in which the flavin ring should be bent. A quick “google” immediately gave me a link to a very nice paper published by David W. Rodgers in 2002. http://www.jbc.org/content/277/13/11513.full.pdf+html According to this paper, Yes! “In the oxidized enzyme, the flavin ring system adopts a strongly bent (16°) conformation, and the bend increases (25°) in the reduced form of the enzyme,…” When I reported this in the group meeting, I was laughed and told that this is just a model bias. It was over interpreted. Nobody has such sharp vision on electron density map. If this was correct, why nobody could find this and report to CCP4 within last 7 years? Eventually, a senior team member emailed to CCP4 about this issue. Since then, the definition of FMN was updated, according to my suggestion. I was asked “how did you find it?”……. “why you believed you are so right?” I really don’t how to answer. Je pense donc je suis Kevin On Sun, Apr 1, 2012 at 8:09 AM, Paul Emsley paul.ems...@bioch.ox.ac.ukmailto:paul.ems...@bioch.ox.ac.uk wrote: On 31/03/12 23:08, Kevin Jin wrote: I really wish PDB could have some people to review those important structures, like paper reviewer. So do the wwPDB, I would imagine. But they can't just magic funding and positions into existence... If the coordinate is downloaded for modeling and docking, people may not check the density and model by themself. However this is not the worst case, since the original data was fabricated. 1. All of data was correct and real, Hmmm... It will be very difficult for people to check the density and coordinated if he/she is not a well-trained crystallographer. I hope and believe that this is not the case. Even basically-trained crystallographers should be able to calculate and interpret difference maps of the kind described by Bernhard. And with the EDS and PDB_REDO server, one does not even need to know how to make generate a difference map... Paul. -- Kevin Jin Sharing knowledge each other is always very joyful.. Website: http://www.jinkai.org/
Re: [ccp4bb] recommendations_on_purification
I suspect that sometimes the protein chaperones the tag, which is solvent exposed some fraction of the time. Try very slow loading or batch binding. Kendall Nettles On Mar 26, 2012, at 8:15 AM, Petros Giastas peg...@pasteur.grmailto:peg...@pasteur.gr wrote: Dear all, I am expressing a 6xHis tagged secreted protein in a fermentor in P. pastoris, using the standard minimal medium described in the invitrogen manual (plus PTM1). Following collection of the culture medium, I am having problems with purification of the protein as only a small fraction (~10%) binds to the Ni-NTA beads even after extensive buffer exchange (when expressed in full BMGY media this is not observed). Could this be attributed to metal ions still present in my sample? Is it likely to be due to poor protein quality in this medium? Or any other suggestions? Thanks in advance Petros
Re: [ccp4bb] off topic: problematic protein
HI Savvas, We recently had a protein that showed two overlapping peaks on the disposable fast flow Q columns, so we decided to see if we could resolve them with a higher resolution Q media. It ended up having 7 distinct peaks, only one of which was free of contaminants. We have also noticed that the presence of heat shock protein bound to our favorite protein is highly dependent on the induction time/temp, and also varies between bacterial strains. It is also effected by the media. Yo might try osmotic shock or other additives in the media. We have also had luck by adding 1-3 molar urea in the lysis buffer. We get lots more protein out, with better purity, but some of it crashes, which I think is purifying out the misfolded protein. Lastly, you might try a fusion protein to something that has chaperone activity, like MBP, which may mask the binding epitopes for the other proteins. Best regards, Kendall Nettles On Apr 19, 2011, at 3:48 PM, Savvas Savvides wrote: Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non-micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html
Re: [ccp4bb] OT: Covalent modification of Cys by reducing agents?
We see BME adducts in all of our estrogen receptor structures, though we don't always put them in the models. Sometimes we only see one or two atoms of the adduct, and in others it is completely ordered. We only see it on the solvent accessible cysteines. We do it on purpose. We used to treat the protein with iodoacetic acid to generate uniform modification of the cysteines, but then we realized we could get then same homogeneity with 20-50mM BME. Kendall Nettles On Apr 15, 2011, at 4:09 PM, Michael Thompson mi...@chem.ucla.edu wrote: Hi All, I was wondering if anyone knew whether or not it is possible for reducing agents with thiol groups, such as DTT or beta-mercaptoethanol (BME), to form covalent S-S bonds with Cys residues, particularly solvent-exposed Cys? I have some puzzling biochemical results, and in the absence of a structure (thus far), I was wondering if this might be something to try to control for. I have never heard of this happening (or seen a structure where there was density for this type of adduct), but I can't really think of a good reason for why this wouldn't happen. Especially for something like BME, where the molecule is very much like the Cys sidechain and seems to me like it should have similar reactivity. The only thing I can think of is if there is a kinetic effect taking place. Perhaps the rate of diffusion of these small molecules is much faster that the formation of the S-S bond? Does anyone know whether or not this is possible, and why it does or does not happen? Thanks, Mike -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] Reproducing crystals.
You might also try to control the degree of oxidation using the microwave, and setting up trials after different numbers of cycles of heating. Kendall On Apr 12, 2011, at 12:41 PM, Jim Pflugrath wrote: Frances Jurnak published a paper in 1986 on PEG impurities and purification. As I recall, it turns out that different manufacturers put different additives in PEGs as preservatives. These are generally anti-oxidants. PEGs do get oxidized. I suggest you heat up your new PEG solutions to say 80 deg C and cool them down, then use them. Let us know what happens. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jun Yong Ha Sent: Tuesday, April 12, 2011 6:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reproducing crystals. Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
Re: [ccp4bb] kinase purification
There is a paper from John Kuriyan on co-expressing a phosphatase with c-Src kinase domain to enable bacterial expression of homogenous protein ( PMID: 16260764). Also look at work from E. Goldsmith (PMID: 16829129) Lastly, I would suggest general approaches, such as: varying the ends of the DNA construct; checking different buffers (EDTA, reducing agents, glycerol, pH, ligands) with use DLS or analytical gel filtration to check for aggregation. Also try low temp induction, or different fusion proteins. Maybe this is a good thing. With our favorite protein we often get aggregation of half the protein. We assume this is the misfolded protein. We pellet this and have dozens of structures using the supernatant. So maybe your aggregation is a feature and not a bug. Kendall Nettles On Mar 29, 2011, at 8:10 PM, Neeraj Kapoor wrote: Hi All, I am trying to express a kinase but unfortunately there is aggregation happening as the protein is purified over a column. SInce I am new to the field of kinase expression and purification, I was wondering if someone could provide me with a couple of good references that can hit the ground running for me. I would also very much appreciate any helpful suggestions that anyone might have. thanks Neeraj
Re: [ccp4bb] Deposition of riding H: R-factor is overrated
Very interesting discussion. I wonder if the inexperienced user of PDB really exists? I don't know anyone off-hand who would really make use of information from hydrogen positions but not understand the issues. Although I hear they have been sighted in the Everglades http://en.wikipedia.org/wiki/Skunk_ape Kendall
[ccp4bb] Postdoctoral position at the Scripps Research Institute
Please bring this position to the attention of anyone in your lab who might be interested. Thanks, Kendall The Scripps Research Institute Palm Beach Co, Florida Post-doctoral Position: Nuclear Receptor Signaling and Structure-Based Drug Design The Scripps Research Institute has established a major science center in Palm Beach County, Florida, focusing on biomedical research, technology development and drug design. A post-doctoral fellowship position is available to study nuclear receptor signaling and structure based drug design in the laboratory of Associate Professor Kendall Nettles. Candidates should have a PhD degree and experience in protein crystallography and/or a good background in molecular biology and protein biochemistry. The laboratory is equipped with robots for high throughput cloning, protein purification, solution mixing and crystallization, and automated crystal visualization. The institute has a home source, and dedicated time at SSRL and APS. We use a variety of structural and molecular approaches to understand the connections between small molecule ligand, receptor structure and function, and endocrine physiology. Ongoing projects include investigation of the structural basis for tissue and pathway specific signaling through the estrogen and glucocorticoid receptors, signal transduction across the RXR heterodimer interface, and structure-based drug design. The fellow will interact closely with the Drug Discovery, Advanced Technology, and Genomics groups at Scripps Florida to take advantage of other automated technologies, including robots for small molecule screening and transfection. Please send curriculum vitae, a brief statement describing research experience and scientific interests and the names of at least two references to: Kendall W. Nettles, PhD Associate Professor, Department of Cancer Biology The Scripps Research Institute 130 Scripps Way Jupiter Fl 33458 Email: knett...@scripps.edu
[ccp4bb] Postdoctoral position at the Scripps Research Institute
Please bring this position to the attention of anyone in your lab who might be interested. Thanks, Kendall The Scripps Research Institute Palm Beach Co, Florida Post-doctoral Position: Nuclear Receptor Signaling and Structure-Based Drug Design The Scripps Research Institute has established a major science center in Palm Beach County, Florida, focusing on biomedical research, technology development and drug design. A post-doctoral fellowship position is available to study nuclear receptor signaling and structure based drug design in the laboratory of Associate Professor Kendall Nettles. Candidates should have a PhD degree and experience in protein crystallography and/or a good background in molecular biology and protein biochemistry. The laboratory is equipped with robots for high throughput cloning, protein purification, solution mixing and crystallization, and automated crystal visualization. The institute has a home source, and dedicated time at SSRL and APS. We use a variety of structural and molecular approaches to understand the connections between small molecule ligand, receptor structure and function, and endocrine physiology. Ongoing projects include investigation of the structural basis for tissue and pathway specific signaling through the estrogen and glucocorticoid receptors, signal transduction across the RXR heterodimer interface, and structure-based drug design. The fellow will interact closely with the Drug Discovery, Advanced Technology, and Genomics groups at Scripps Florida to take advantage of other automated technologies, including robots for small molecule screening and transfection. Please send curriculum vitae, a brief statement describing research experience and scientific interests and the names of at least two references to: Kendall W. Nettles, PhD Associate Professor, Department of Cancer Biology The Scripps Research Institute 130 Scripps Way Jupiter Fl 33458 Email: knett...@scripps.edu
Re: [ccp4bb] Moving copies to be close to one unit cell.
If using Coot, you can also merge molecules on the original and all symmetry related pdb files that you saved, which will automatically renumber the chains for you. Kendall Nettles
Re: [ccp4bb] protein-progesterone or estrogen complexes
Are you talking about the respective ligand with progesterone or estrogen receptor ligand binding domains? That has been done many times. Larger pieces of the receptor have proven more difficult. Adding 10uM compound in the fermentation media is the traditional route, because a significant portion of the receptor misfolds in bacteria otherwise. If you do want to add compounds to concentrated protein, don't worry about compound solubility. We have solved over 20 ER LBD structures with different compounds by diluting 100mM stocks in 100% ethanol or DMSO to 1mM. Most of the compound crashes out, but gets soaked up by the protein. The next day we microcentrifuge and set up trials with supernatant. We used to carbamylate free cysteines with iodoacetic acid, but switched to high MBE (10-50mM), which gives adducts. You'll also want the LxxLL peptide at 3-5 fold excess. With ER LBD, almost all our agonist conformation structures are in PEG3350. Also, we never did get a structure with estradiol; the receptor crystallized readily with genistein. See this paper for purification details. http://www.nature.com/nchembio/journal/v4/n4/abs/nchembio.76.html Regards, Kendall. On 4/1/09 12:59 PM, KUMARASWAMI MUTHIAH megun...@hotmail.com wrote: Anybody tried to cocrystallize the protein-progesterone or estrogen complexes, if so how do you go about the solubility of these compounds? Progesterone is only soluble in 50% chloroform or 100% DMSO and the dilution of this stock is not possible as chloroform falls out of solution. Lots of papers out there used soaking with progesterone or expressed the protein in the presence of progesterone. Any suggestions would be appreciated. Thanks Rediscover Hotmail®: Now available on your iPhone or BlackBerry Check it out. http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Mobi le1_042009
Re: [ccp4bb] Ligand binding in multiple conformation
Hi Mariah, We have had one case of this, with two partially overlapping conformations of a ligand, which is not yet published. Model in both ligand conformations. Then edit the PDB to give them the same Chain ID, but with alternative conformations for each atom, or each one that is different. You might try giving 0.5 occupancy to each for REFMAC refinement. In Phenix you can also refine the occupancy. Here is an example for the CAA atom of our drug. ATOM 7090 CAAADRG E 1 9.312 2.643 8.223 0.50 42.57 C ATOM 7091 CAABDRG E 1 12.707 2.133 -0.186 0.50 36.22 C Regards, Kendall -- Kendall W. Nettles, PhD Asssociate Professor Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458 On 1/12/09 6:52 PM, protein.chemist protein.chemist pp73...@gmail.com wrote: Hi, I had a question about flexibility in ligand binding in an enzyme active site. Is it possible for a substrate/product analogue to bind in more than one conformation in the active site. Since the ligand/enzyme interactions are very specific I am a little confused about this. Also which program would you use if you have to refine with alternate ligand conformation. Please mention if you have ever come across any paper that explains such a phenomena. Thanks a lot. Mariah
Re: [ccp4bb] O/T: can a protein which dimerizes in solution crystallize as a monomer?
There are a number of examples of nuclear receptor heterodimers, where crystallization of the individual partner, such as PPAR or LXR, crystallizes as a homodimer, even though these species do not exist in solution. There are also many examples of dimers showing one molecule per asymmetric unit, but the physiological dimer is apparent in the crystal packing. Kendall Nettles On 12/11/08 11:09 AM, Santarsiero, Bernard D. [EMAIL PROTECTED] wrote: In parallel with the discussion around this off-CCP4-topic, are they any good examples of the opposite case, where the protein is a monomer in solution (as evident from light scattering, MW determination through centrifugation, EPR, etc.) but crystallizes as a dimer or higher multimer? Bernie Santarsiero
Re: [ccp4bb] CCP4MG can't start
Hi Bill, I commented out the export DISPLAY lines, but it didn't help. What did fix it was reinstalling x11 from the OSX 10.5 CD. Any ideas? I've attached the launch script as a text file. Thanks! Kendall On 7/25/08 12:27 PM, William G. Scott [EMAIL PROTECTED] wrote: Hi Kendall: I haven't used CCP4Mg^2+ but if it is launched by a shell script that dumbly sets the DISPLAY variable, then on 10.5 this is equivalent to a suicide directive, since the new X11 (as of 10.5) sets the DISPLAY variable using launchd. If this is the case, hack that line out of the script or put in a conditional test for the operating system version. The latest X11 for 10.5.4 is worth getting too, as it has a lot of improvements: http://xquartz.macosforge.org/trac/wiki/X112.3.0 Bill William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ On Jul 25, 2008, at 9:18 AM, Kendall Nettles wrote: CCPMG is launching X11 right before it quits. Could it relate to the version of X11? Coot was not working with the version that updates with the OSX, so I had to install the Xquartz version to get Coot to work. Kendall On 7/25/08 10:53 AM, Jendrek [EMAIL PROTECTED] wrote: Hi, I've had the same problem and discovered that in order to start CCP4MG the X11 must be closed. So, just quit your X11 and then start again. It worked fine for me. Andrzej Kendall Nettles wrote: I have tried installing the newest version of CCP4MG 1.1.1, the closedirfix version, and QtMG 1.99.0. In each case, the program does not start. I did get a problem report for pyton, shown below. Mac ppc dual 2.7 GHz, OSX 10.5.4 X11= XQuartz 2.3.0(xorg-server 1.4.2-apple5) Any suggestions would be greatly appreciated! Kendall ccp4mg.txt Description: video/flv
[ccp4bb] CCP4MG can't start
I have tried installing the newest version of CCP4MG 1.1.1, the closedirfix version, and QtMG 1.99.0. In each case, the program does not start. I did get a problem report for pyton, shown below. Mac ppc dual 2.7 GHz, OSX 10.5.4 X11= XQuartz 2.3.0(xorg-server 1.4.2-apple5) Any suggestions would be greatly appreciated! Kendall -- Kendall W. Nettles, PhD Assistant Professor Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458 office 561-799-8851 fax 561-799-8805 cell 561-306-7566 Problem report for python: Process: python [189] Path: /Applications/ccp4mg.app/Contents/MacOS/../ccp4mg-1.1.1/bin/..//pythondist// bin/python Identifier: python Version: ??? (???) Code Type: PPC (Native) Parent Process: sh [160] Date/Time: 2008-07-25 09:12:14.642 -0400 OS Version: Mac OS X 10.5.4 (9E17) Report Version: 6 Exception Type: EXC_BAD_ACCESS (SIGBUS) Exception Codes: KERN_PROTECTION_FAILURE at 0x0020 Crashed Thread: 0 Thread 0 Crashed: 0 libSystem.B.dylib0x96594494 pthread_mutex_lock + 40 1 libSystem.B.dylib0x96592e68 free + 92 2 libSystem.B.dylib0x9662cad8 closedir + 52 3 libfont_cache.dylib 0x029ae724 LoadAllFreeTypeFonts() + 4052 (freetype_font.cc:1009) 4 libfont_cache.dylib 0x029a04b0 FontCache::LoadAllFonts() + 16 (font_info.cc:251) 5 _font_cache.so 0x03a72d70 _wrap_FontCache_LoadAllFonts + 48 (font_cache_wrap_py.cc:3913) 6 org.python.python0x0016f104 _PyEval_SliceIndex + 16528 7 org.python.python0x00171360 PyEval_EvalCodeEx + 2256 8 org.python.python0x001714b0 PyEval_EvalCode + 44 9 org.python.python0x0018d534 PyErr_Display + 1932 10 org.python.python0x0018f42c PyRun_SimpleFileExFlags + 424 11 org.python.python0x001987f0 Py_Main + 1988 12 python 0x257c start + 400 13 python 0x2424 start + 56 Thread 0 crashed with PPC Thread State 32: srr0: 0x96594494 srr1: 0xd030 dar: 0x0020 dsisr: 0x4000 r0: 0x4d555458 r1: 0xbfffce40 r2: 0xa0bfcab4 r3: 0x0020 r4: 0x r5: 0x r6: 0x80808080 r7: 0x r8: 0x r9: 0x029b32cc r10: 0x6004 r11: 0xa0bfd874 r12: 0x9659446c r13: 0xbfffcfb4 r14: 0x029ad764 r15: 0xbfffd15c r16: 0x090a65c0 r17: 0x r18: 0xbfffcf78 r19: 0x r20: 0xbfffcf4c r21: 0x r22: 0x0001 r23: 0x0001 r24: 0xbfffcf74 r25: 0xbfffd168 r26: 0xbfffd16c r27: 0xbfffd170 r28: 0x0020 r29: 0xa0bf8924 r30: 0x r31: 0x9659447c cr: 0x88248204 xer: 0xlr: 0x9659447c ctr: 0x9659446c vrsave: 0x Binary Images: 0x1000 -0x2fef +python ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/bin/python 0x51000 -0x53fff +_ssl.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/_ssl.so 0x9d000 -0x9eff7 +math.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/math.so 0xa1000 -0xa2fe7 +libmginterrupt.dylib ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/lib/libmginterrupt.dylib 0xaf000 -0xb5ff7 +_socket.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/_socket.so 0xbb000 -0xbcfff +time.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/time.so 0xc -0xc1ff2 +libXau.6.dylib ??? (???) bfcdfbb3063882f751c11f520b6ac773 /usr/X11/lib/libXau.6.dylib 0x104000 - 0x1c9ff7 org.python.python 2.3.5 a (2.3.5 a) 0d8cca187f8ff8c4eacf04b69a66f39b /System/Library/Frameworks/Python.framework/Versions/2.3/Python 0x288000 - 0x28bfff +strop.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/strop.so 0x2cf000 - 0x2dbffb +libXpm.4.dylib ??? (???) aaf5463bfc482f192b8c3d23c446991b /usr/X11R6/lib/libXpm.4.dylib 0x2df000 - 0x2edfff +libXext.6.dylib ??? (???) 92d500d9cfda747c5a1c9a3d43c16104 /usr/X11R6/lib/libXext.6.dylib 0x2f9000 - 0x2fcff7 +libXdmcp.6.dylib ??? (???) cecb0b212033df7f58df772aed52bf36 /usr/X11/lib/libXdmcp.6.dylib 0x68 - 0x685ff0 +libSM.6.dylib ??? (???) f79c5f0032ecbfca21c93c55e00b2072 /usr/X11R6/lib/libSM.6.dylib 0x689000 - 0x690ff3 +libXi.6.dylib ??? (???) 6e141ddf695eb5b9732d75fb861361f0 /usr/X11R6/lib/libXi.6.dylib 0x693000 - 0x695fff +binascii.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/binascii.so 0x6d8000 - 0x713feb +_opengl.so ??? (???)
Re: [ccp4bb] CCP4MG can't start
CCPMG is launching X11 right before it quits. Could it relate to the version of X11? Coot was not working with the version that updates with the OSX, so I had to install the Xquartz version to get Coot to work. Kendall On 7/25/08 10:53 AM, Jendrek [EMAIL PROTECTED] wrote: Hi, I've had the same problem and discovered that in order to start CCP4MG the X11 must be closed. So, just quit your X11 and then start again. It worked fine for me. Andrzej Kendall Nettles wrote: I have tried installing the newest version of CCP4MG 1.1.1, the closedirfix version, and QtMG 1.99.0. In each case, the program does not start. I did get a problem report for pyton, shown below. Mac ppc dual 2.7 GHz, OSX 10.5.4 X11= XQuartz 2.3.0(xorg-server 1.4.2-apple5) Any suggestions would be greatly appreciated! Kendall
[ccp4bb] References for ligand flipping
Hi, Thanks to everyone for suggestions on making disulfides. I do have another question. Can anyone suggest some references for structures showing that a small molecule ligand binds differently in closely related proteins? Thanks, Kendall -- Kendall W. Nettles, PhD Assistant Professor Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458
[ccp4bb] Engineering disulfide bonds
I'm trying to engineer a disulfide bond into a protein that has several other cysteines. My question is whether there is a crystallization friendly reducing agent that can be used to prevent oxidation of the free cysteines without breaking the disulfide? Also, can I expect 100% disulfide formation from standard bacterial expression (assuming good geometry of the cysteines)? Thanks, Kendall -- Kendall W. Nettles, PhD Assistant Professor Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458
Re: [ccp4bb] Zn fingers and Ni columns
Phoebe, We were able to purify the estrogen receptor DNA binding domain with a 6his-tag. After cutting off the tag and re-applying to the Ni-NTA matrix, the protein did not stick to the beads. We had some low resolution crystals that contained a second protein and DNA, which suggests to me that the zinc was bound in the protein, but we did not analyze it further. Kendall -- Kendall W. Nettles, PhD Assistant Professor Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458 office 561-799-8851 fax 561-799-8805 cell 561-306-7566 On 1/3/08 7:21 PM, [EMAIL PROTECTED] [EMAIL PROTECTED] wrote: This has probably been discussed before, so apologies in advance. We're eyeing a protein that has a probable C4 Zn finger in the middle. The collaborators who are nicely going to PCR it up want to know if we'd like it with or without a His tag. Is it a bad idea to co-mingle Zn-binders and Ni columns? Or is it likely to bind the column quite nicely without the tag? thanks, Phoebe -- - Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 fax 773 702 0439 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabe tically.php?faculty_id=123 http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
[ccp4bb] crystallization robot
We have been quite happy with our crystallization robot from Innovadyne. It transfers from deep well blocks to crystallization plates, and very reliably sets up 200nl + 200nl drops. We have tried smaller drops with decent success, which works better with certain plates. It can be easily programmed, has good support from the company, and has very inexpensive tips. I¹ve also used the Cartesian Honeybee extensively, which I found worked well, but required much more work in cleaning and daily maintenance, and did not do the large volume transfers. Kendall -- Kendall W. Nettles, PhD Assistant Professor Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458 office 561-799-8851 fax 561-799-8805 cell 561-306-7566
Re: [ccp4bb] insoluble ligand
Simon, We routinely obtain structures from protein solutions with a big pellet of ligand in the bottom of the tube. For co-crystallizations we add 1mM compound to a 0.3mM solution of the protein and incubate overnight. Many of the compounds are only soluble to 50micromolar, so we get a lot of precipitate. The next day, we spin the tube at high speed, and use the supernatant for crystallization trials. We have started from 100mM stocks in 100% DMSO or ethanol. This has worked for compounds ranging for picomolar to micromolar affinity, which surprised us, but it worked. Regards, Kendall On 12/11/07 11:55 AM, Yue Li [EMAIL PROTECTED] wrote: Hi all, I have one ligand which is insoluble in water, and I would like to co-crystallize it with my protein. Is there any other method except for dissolving it in DMSO ? Thanks Simon
[ccp4bb] Postdoctoral position at the Scripps Research Institute
Please bring this position to the attention of anyone in your lab who might be interested. Thanks, Kendall The Scripps Research Institute Palm Beach Florida Post-doctoral Position: Nuclear Receptor Signaling and Structure-Based Drug Design The Scripps Research Institute has established a major science center in Palm Beach County, Florida, focusing on biomedical research, technology development and drug design. Funding for facilities and initial staffing (some $800 million) is supported by the State of Florida via economic development funds as well as by the local county government. A post-doctoral fellowship position is available to study nuclear receptor signaling and structure based drug design in the laboratory of Assistant Professor Kendall Nettles. Candidates should have a PhD degree and experience in protein crystallography and/or a good background in molecular biology and protein biochemistry. The laboratory is equipped with robots for high throughput cloning, protein purification, solution mixing and crystallization, and automated crystal visualization. The institute is purchasing a home source, and has dedicated time at SSRL and APS. We use a variety of structural and molecular approaches to understand the connections between small molecule ligand, receptor structure and function, and endocrine physiology. Ongoing projects include investigation of the structural basis for tissue and pathway specific signaling through the estrogen and glucocorticoid receptors, signal transduction across the RXR heterodimer interface, and structure-based drug design. The fellow will interact closely with the Drug Discovery, Advanced Technology, and Genomics groups at Scripps Florida to take advantage of other automated technologies, including robots for small molecule screening and transfection. Please send curriculum vitae, a brief statement describing research experience and scientific interests and the names of at least two references to: Kendall W. Nettles, PhD Assistant Professor, Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458 Email: [EMAIL PROTECTED]
Re: [ccp4bb] His tag does not bind.
We have found that our His-MBP fusion doesn¹t bind well after we cut off the protein of interest, and are trying to remove it. We have to use very low salt, cold temp, and slow loading rates. You might also try batch instead of column loading. We have also had good luck adding 1-2M urea to uncut His-MBP-protein fusions that show poor binding to the Qiagen Ni-NTA. Kendall On 10/10/07 9:12 PM, changrui lu [EMAIL PROTECTED] wrote: Dear all, I am trying to express a 150 kd protein in E coli. I have it in two constructs, one with pmal-his and other with only his tag at N terminus. The full length protein can be detected both by sds and western using anti-his (190kd and 150kd respectively) but strangely neither binds to his-column very well. The majority of the full length comes through the column either at loading step or low salt wash step. The major species that gets trapped and eluted is the mbp-his truncation (~40kd). Some, though very little, full length protein did make it out the his column. The pmal-his construct does not bind amylose resin any better with majority flows right through. All purification are carried out under standard conditions as mentioned in the manuals. The protein is soluble and does not precipitate in the columns. I appreciate and ideas or explanations. Thanks in advance. Ray Cornell Univerisity
[ccp4bb] Crystallography short courses?
I am looking for information on short courses on data collection, processing, and refinement. Thanks, Kendall -- Kendall W. Nettles, PhD Assistant Professor Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458 office 561-799-8851 fax 561-799-8805 cell 561-306-7566
[ccp4bb] Problem installing coot with FINK
I installed Coot 0.3.3-3 from the Binary: Reading Package Lists... Building Dependency Tree... The following extra packages will be installed: coot-shlibs The following NEW packages will be installed: coot 1 packages upgraded, 1 newly installed, 0 to remove and 67 not upgraded. Need to get 0B/41.7MB of archives. After unpacking 178MB will be used. Do you want to continue? [Y/n] (Reading database ... 54975 files and directories currently installed.) Preparing to replace coot-shlibs 0.2-2 (using .../coot-shlibs_0.3.3-3_darwin-powerpc.deb) ... Unpacking replacement coot-shlibs ... Selecting previously deselected package coot. Unpacking coot (from .../coot_0.3.3-3_darwin-powerpc.deb) ... Setting up coot-shlibs (0.3.3-3) ... Setting up coot (0.3.3-3) ... and got the following error message when launching coot: $ coot dyld: Library not loaded: /sw/lib/libpng12.0.dylib Referenced from: /sw/bin/coot Reason: Incompatible library version: coot requires version 19.0.0 or later, but libpng12.0.dylib provides version 13.0.0 Trace/BPT trap Any ideas? Kendall
[ccp4bb] ccp4MG- selecting h-bonds
I have a question about how to show certain h-bonds with CCP4MG. I¹d like to show a specific bond between an Arg and Glu, but not other H-bonds made by the Arg. How can I do this? Thanks! Kendall
[ccp4bb] Maps look different from auto-mtz vs EDS vs FFT in Coot or CCP4MG.
I¹d like help in interpreting some mystery density in a structure. I¹m writing a paper about soaking the apo-estrogen receptor with different ligands. The apo structure is already released, as pdb code 2B23. The question is whether there is a mystery molecule in the pocket of the apo receptor. If you superimpose 3ERD, you can see where the ligand binds. The problem is that with some maps the pocket appears completely empty, and with others, there appears to be something there. Protein looks essentially identical with the different maps. We have used a few different approaches to identify the compound with LC-MS, and are pretty sure there is nothing there. For example, we can bind our protein to beads, soak it with estradiol, wash extensively, elute with organic solvent and find a great peak for estradiol, but nothing for the apo protein. We have also tried non-denaturing MS. If you look at the 2mFo-DFc map from EDS in Coot or CCP4mg, you see mystery density in the pocket. If I use the MTZ, you see density in Coot, but not CCP4MG. I then downloaded the structure factors from the PDB and made an MTZ. The map in CCP4MG shows some density, but much less that with the map from EDS. When I used FFT to make a 2mF1-1nF2 map, there is no mystery density in either CCP4MG or coot. I was ready to submit the manuscript with a picture of the mystery density, but now I¹m not sure if that is appropriate. Any suggestions, as far as how to interpret this mystery density would be greatly appreciated. Best Regards, Kendall -- Kendall W. Nettles, PhD Assistant Professor Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458 office 561-799-8851 fax 561-799-8805 cell 561-306-7566
Re: [ccp4bb] Crystallisation of a extremly soluble protein
Sabine, There are protocols to modify surface residues that can help with crystallization, and make the protein less soluble. Unfortunately, I¹m drawing a blank on the details. I remember someone in Andrzej Joachimiak¹s group was working on this as a rescue approach for the structural genomics pipeline, and it had been previously published by others. Have you looked at the protein with dynamic light scattering? Are their cysteines? Are you using reducing agents? Sometimes mutating cysteines to serines can help. How much purification have you done? Try ion exchange and gel filtration. Do you have more than one ligand? I think there is quite a lot of variability in how different ligands promote crystallization. Are you adding the ligand in excess? Try a few different molar ratios. If if has high affinity, you might want to try removing excess unbound ligand at the end. Good luck! Kendall On 2/22/07 9:08 AM, Schneider Sabine [EMAIL PROTECTED] wrote: Hi everyone, I am trying to crystallise an extremely soluble and charged protein. It is ~30kDa and has an estimated PI of 5.2 and theoretical charge over pH range 4-10 from + 24 to -29. It is still happy at a concentration of 190mg/ml and fully reconstituted with its ligand. I have tried high throughput crystallisation with 10 different screens from Nextal with concentrations of 60, 100 and 150mg/ml with no NaCl and NaCl concentrations of 100mM, 300mM and 1M in either Hepes pH 8 or Tris-HCl pH 7.5. The distribution of heavy precipitation, light crystalline precipitation and clear drops through out the screens locks like I am in the right concentration range around the 100mg/ml, but I am not getting any real hit. There are some drops with extreme phase separation. I also tried changing the temperature from 20C to 4C. I chased up a few conditions with this strong phase separation (or where I imagined little objects...) by manual screening and also adding additives like 3% Succrose, 50-200mM LiCl, 100mM EDTA, varying the PEGs (1500, 3350, 4000, 6000, 8000) as well as adding NaCl to the reservoir solution in sitting as well as hanging drop screens. But I am just getting nowhere - either just precipitation or the drop stays clear with the strong phase separation. I also re-cloned it with chopping of a few more residues on the N-term where according to a secondary structure prediction a helix starts and it is still very happy at high concentrations, but again nothing in the high-throughput screens. Has anyone any suggestions what else I could try? Thanks! Sabine This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation.
