Re: [ccp4bb] Rant: B vs TLS, anisou, and PDB headers
In the end, we're solving all these structures because we believe (or at least hope) that they'll be useful for understanding biology. That means that biologists should be able to understand what we deposit. When I've tried to teach undergraduates what to make of structural models, I find I can give (most of) them an intuitive feel for what B tells or doesn't tell them. I don't have time to even bring up TLS parameters. The file that gets downloaded from the databank and displayed by pymol or PDBviewer needs to be as simple as possible while still being true. Exactly how those Bs were derived should be included in the file, but in a way that the non-specialist users can get by without reading it all (since they most certainly won't ;-) ). Phoebe At 03:56 PM 3/29/2008, you wrote: I believe the simplest and most honest thing to deposit are the parameters of your model, viz the TLS parameters and the residual B factors. Derived quantities should be calculated as and when you need them. --- 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_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] Model ensemble for x-ray crystallography
Didn't that trick very successfully lower the R-factors of the completely wrong models that led to the Great Pentaretraction? Unless you have stunningly high resolution, beware. Phoebe At 10:13 AM 3/28/2008, you wrote: Some time ago I've heard about the idea of proposing an ensemble of models (as in NMR), instead of a single model for x-ray crystallography structures. If I remember correctly, this idea has been published somewhere. Can anyone tell me what article is that? Lucas Abra sua conta no Yahoo! Mail, o único sem limite de espaço para armazenamento! http://br.mail.yahoo.com/ --- 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_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] crashing-out protein eluted from Nickel column
An added benefit of EDTA is that it inhibits some proteases - for one of our wimpier proteins, spiking each fraction collector tube with a little EDTA before running the Ni column really helped reduce keep the sample in one piece. Phoebe At 01:18 PM 2/19/2008, Sophia Tsai wrote: Hi, Agreed on this. I used to have issues with aggregation due to the Nickel being stripped off the column (happens with elution in imidazole). Adding 10mM EDTA to the elution immediately AFTER it has come out of the column will chelate the Ni++ and prevents aggregation (at least in my case). Afterwards, I use gel filtration to remove the Ni++, as well. Hope that helps! Sophia On Mon, Feb 18, 2008 at 4:48 AM, Ngo Duc Tri mailto:[EMAIL PROTECTED][EMAIL PROTECTED] wrote: Hi, I used another way to deal with this problem. You can try to elute your protein with the buffer containing 50mM EDTA (You need at least 10CV to elute completely). Then use gel filtration to remove the Ni. I applied this method with two proteins and it showed good results. Good luck! TriNgo --- 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_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] an over refined structure
Rotational near-crystallographic ncs is easy to handle this way, but what about translational pseudo-symmetry (or should that be pseudo-translational symmetry)? In such cases one whole set of spots is systematically weaker than the other set. Then what is the theoretically correct way to calculate Rfree? Write one's own code to sort the spots into two piles? Phoebe At 01:05 PM 2/8/2008, Axel Brunger wrote: In such cases, we always define the test set first in the high-symmetry space group choice. Then, if it is warranted to lower the crystallographic symmetry and replace with NCS symmetry, we expand the test set to the lower symmetry space group. In other words, the test set itself will be invariant upon applying any of the crystallographic or NCS operators, so will be maximally free in these cases. It is then also possible to directly compare the free R between the high and low crystallographic space group choices. Our recent Neuroligin structure is such an example (Arac et al., Neuron 56, 992-, 2007). Axel On Feb 8, 2008, at 10:48 AM, Ronald E Stenkamp wrote: I've looked at about 10 cases where structures have been refined in lower symmetry space groups. When you make the NCS operators into crystallographic operators, you don't change the refinement much, at least in terms of structural changes. That's the case whether NCS restraints have been applied or not. In the cases I've re-done, changing the refinement program and dealing with test set choices makes some difference in the R and Rfree values. One effect of changing the space group is whether you realize the copies of the molecule in the lower symmetry asymmetric unit are identical or not. (Where identical means crystallographically identical, i.e., in the same packing environments, subject to all the caveats about accuracy, precision, thermal motion, etc). Another effect of going to higher symmetry space groups of course has to do with explaining the experimental data with simpler and smaller mathematical models (Occam's razor or the Principle of Parsimony). Ron Axel T. Brunger Investigator, Howard Hughes Medical Institute Professor of Molecular and Cellular Physiology Stanford University Web:http://atb.slac.stanford.eduhttp://atb.slac.stanford.edu Email: mailto:[EMAIL PROTECTED][EMAIL PROTECTED] Phone: +1 650-736-1031 Fax:+1 650-745-1463 --- 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_Alphabetically.php?faculty_id=123 http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] Codon Optimized Expression
Rumor also has it that more than one bad codon in a row, particular near the beginning, can be extra-bad. For one small, A/T rich protein, we simply fixed a pair of bad Arg codons near the N-terminus at the same time as recloning it, by using the N-terminal cloning primer to do the mutagenesis. Combined with using codon-plus E coli, expression improved dramatically. Good luck! Phoebe Rice At 09:29 AM 2/1/2008, Looney, Andrea Lynn (Andrea Hevrdeys) wrote: Dear All, I am getting very little (not zero) expression of my protein. I am curious to know if it is worthwhile to codon optimize my gene, which is ~1200bp, for E.coli. Can you have too much of a good thing? Can codon optimizing overwhelm the cell or in some way cause death or lowered expression? Thank you all, Andrea L. Hevrdeys --- 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_Alphabetically.php?faculty_id=123 http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] differences between Rsym and Rmerge
High R merges with no reasonable excuse can certainly be a useful red flag during data processing (along with the % of observations rejected, which I've never had a reviewer request). Which brings up the point that one reasonable excuse is anisotropy - high Rs for merging random observations in the imaginary direction will be combined with lower Rs for merging decent data in the real direction. Is there any extant software that will calculate directionally-binned Rmerges? It would be useful both for re-assuring users that there isn't anything worse with their data, and for arguing with referees who don't read CCP4BB. Phoebe At 01:18 PM 1/18/2008, Mischa Machius wrote: OK, that brings us back to a more substantial question: is any of these R values actually suitable to judge the quality of a given dataset? Instead of introducing novel R factors, one could also simply ignore them altogether, make sure that the error models have been properly chosen and look at I/sigma(I) as the main criterion. [QUOTE ]If anyone then still wants to present low R factors, one can always divide by 2, if necessary. [/QUOTE] Best - MM On Jan 18, 2008, at 1:02 PM, Salameh, Mohd A., Ph.D. wrote: Thank you all, it was very, very helpful discussion. However, I collected crystal data and the Rmerge overall was very high around 0.17 at 2.6A resolution and I'm wondering what is the acceptable value (range) of R-merge that worth the time to continue processing! Very anxious to hear your thoughts. Thanks, M Mohammed A. Salameh, Ph.D. Mayo Clinic Cancer Center Griffin Cancer Research Building 4500 San Pablo Road Jacksonville, FL 32224 Tel:(904) 953-0046 Fax:(904) 953-0277 [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Chris Putnam Sent: Friday, January 18, 2008 1:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] differences between Rsym and Rmerge On Friday 18 January 2008 09:30:06 am Ethan A Merritt wrote: Rmerge is an average over replicate measurements of the intensity for identical [hkl]. Rsym is an average over the measurements for all symmetry equivalent reflections. In the presence of anomalous scattering, Rsym will be higher than Rmerge because the Bijvoet pairs, although symmetry related, do not have identical intensities. One might logically report two values for Rsym, one which averages over the Bijvoet-paired reflections and one which does not. This has been an eye-opening discussion for me. I've been really surprised that there's been such a diversity of opinion about what these common terms ought to refer to, and the fact that my understanding was wrong. I always thought that Rsym was an average over all symmetry equivalent reflections from the same crystal (including Bijvoet pairs) and Rmerge was properly restricted to cases of multi-crystal averaging. (My versions of Table 1's from single crystals have used Rsym rather than Rmerge.) I wonder if the problem here is that the terms have become overloaded (and hence non-specific). In that sense Rmerge is a particularly unfortunate name as every R that we're discussing is a really a merge of some sort or another. (In the most naive sense, Rmerge might be thought to be the R for whatever variation of reflection merging the experimenter chooses to do.) One possible solution would be to push the community towards a new set of terms with clearly defined meanings (and whose names would be used explicitly by new releases of MOSFLM, HKL2000, etc. and changes for new entries in the PDB). If new terms were to be adopted, they ought to specifically distinguish between single crystal and multi-crystal merging. I see three such R values that might be useful (I've arbitrarily chosen names to distinguish them from each other and the older terms): Rhkl - R of identical hkl's Rrot - R of symmetry-related hkls, but not Bijvoet pairs (rot coming from the concept that all symmetry-related reflections can be found via rotations in reciprocal space and the fact that sym has already been used) RBijvoet - R of symmetry-related and Bijvoet-related hkls (including reflections related by both rotations and an inversion center in reciprocal space) Rhkl,multi - multi-crystal version of Rhkl Rrot,multi - muti-crystal version of Rrot RBijvoet,multi - multi-crystal version of RBijvoet The downside of adopting new names is that it makes the previous literature obsolete, but I wonder if the older terms were ambiguous enough that that's not such a problem. -- Christopher Putnam, Ph.D. Assistant Investigator Ludwig Institute For Cancer Research Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214
Re: [ccp4bb] SUMMARY: PEG MW vs. cryoprotectivity
Many thanks to all who replied. The answers were remarkably varied - see below. My own two bits worth - vitrification of mother liquor doesn't always lead to a nice, low-mosaicity, ice-free crystal freeze in our hands, although we're not willing to sacrifice a statistically significant number of crystals to figure out why. Most of our crystals are annoyingly fragile, which makes it hard to disentangle the effects of bad cryoprotection from those of mechanical damage. Phoebe From: [EMAIL PROTECTED] PEG 4000 has worked for us at high concentration (35-40%) depending on what else is in there. The same goes for PEG 3000. You can try increasing the concentration of the PEG in the reservoir gradually (over the course of many days) from the % they are grown at up to 25-40%. Hopefully this will not crack your crystals. From: Anastassis Perrakis [EMAIL PROTECTED] in my experience peg 4k reduces the amount of glycerol that you need but cant act as cryo on its own. From: Juergen Bosch [EMAIL PROTECTED] the larger the worse for cryo. But PEG4000 40% freezes well. PEG8000 needs some addition of smaller PEGs/Glycerol etc. From: Kevin Jude [EMAIL PROTECTED] I have used 23% PEG 3350/5% glycerol as a cryoprotectant (JACS 2006 p 3011). The PEG on its own didn't work at that concentration. It would be enough to test this by making up the solutions and shooting empty loops, like Elspeth Garman did for glycerol. From: Ezra Peisach [EMAIL PROTECTED] I have seen discussions in the past... The easiest thing to do is test it yourself. Try freezing a small loop of high concentrations of PEG. If it forms a clear glass it is worth pursuing... From: Jan Abendroth [EMAIL PROTECTED] 20% PEG 2000 just worked fine, 35% PEG 3350 seems ok too. also depends on the size of the loop. From: Edwin Pozharski [EMAIL PROTECTED] I have used pegmme2000 as cryoprotectant in the past (some 45% of it), and it worked fine. Indeed, PEG4K s included in Hampton's kit. From: Buz Barstow [EMAIL PROTECTED] In our experience with freezing protein crystals under high pressure, we've found that mid weight PEGs do help a little to enhance the cryo- protective effect of high pressure, although are not terrifically effective, especially when at a low concentration of around 5%. From: Li Sheng [EMAIL PROTECTED] I used 40% w/v PEG 4000 as cryoprotectant. From: Moody, Dr P.C.E. [EMAIL PROTECTED] my experience is that anything over PEG 600 is likely not to be a reliable cryoprotectant, and 400 is the maximum safe size.can't comment on the commercial kits, my cynical nature would suggest that testing may not be an important part of the product pipelinePeter From: Remy Loris [EMAIL PROTECTED] PEG 4000 is a very good cryoprotectant in the range of 30-35%. If your crystallization condition includes PEG4000, it is a good idea for a first trial to find a good cryo condition raise the PEG4000 concentration to 30-35%. Some of the Hampton ctrystal screen conditions (and other commercial kits as well) that contain PEG4000 do even not need further addition of cryoprotectant (even if in the corresponding Hampton cryo screen they are diluted with glycerol, one of the most horible cryoprotectants in common use) Lower MW PEGS can be useful as well, but the required concentrations will be higher. Please be aware that in quite a number of cases the ideal cryo solution can be very far away from the condition in which the protein was crystallized! From: gengxiang zhao [EMAIL PROTECTED] Before, I crystallized the complex of a protein with some small molecules. I use the PEG3350 as a precipatate. When I collect the dataset using X-ray detector. I only use 23% PEG3350 plus 5% PEG400 as a cryoprotectants. Fortunately, it has been successful. So, I think that the PEG3350 functions some cryo-protectants. From: Florian Schmitzberger [EMAIL PROTECTED] This might not be a direct answer to your question; I have found that PEG3350 at around 20-25 % (w/v) concentration (JSCG+ screen) - in combination with ~10 % (v/v) glycerol (which came from the protein sample buffer) was sufficient to cryoprotect crystals rather well; without further soaking or handling being necessary. From: Jennifer Cash [EMAIL PROTECTED] In our recent experience, larger PEGs work similarly to smaller PEGs as far as vitrification goes. Additionally, transferring crystals to a solution of increased PEG concentration (as compared to mother liquor) can substantially reduce the amount of cryo needed for freezing and can be more gentle on crystals than just using a high concentration of cryo. Here is a reference that tests cryoprotective ability of PEG 2000 2. J. Appl. Cryst. (2006) 39, 244-251 At 03:55 PM 12/4/2007, [EMAIL PROTECTED] wrote: We've been having a discussion in the lab about whether or not middle-sized PEGs such as 4000 can be expected to serve as cryoprotectants (and if not, why certain commercial kits are
[ccp4bb] PEG MW vs. cryoprotectivity
We've been having a discussion in the lab about whether or not middle-sized PEGs such as 4000 can be expected to serve as cryoprotectants (and if not, why certain commercial kits are formulated the way they are). Can anybody shed some light / references on the question of the size of PEGs vs. their ability to help in freezing? 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_Alphabetically.php?faculty_id=123 http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] protein degradation?
Some proteases are metal-dependent, and inhibitors for those aren't Ni-column-compatible. We (meaning my students) found that it helps to (1) work very quickly and (2) put EDTA into the fraction collector tubes before eluting from the Ni column. At 06:22 AM 11/4/2007, Vijay Kumar wrote: Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay --- 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_Alphabetically.php?faculty_id=123 http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] PDB Crazy or Me?
I don't see a concise list of changes there? At 07:34 AM 10/9/2007, Eleanor Dodson wrote: Did you go to this web site? http://remediation.wwpdb.org/downloads.html It has the new dictionary stuff Eleanor James Stroud wrote: Hello All, I noticed some things different about the PDB today causing me to rub my eyes vigorously and to put my nose right on my monitor in disbelief: * - ' for nucleic acid sugars O#P - OP# for the phosphate oxygens C5A - C7 for thymidine exocyclic methyl (didn't it used to be C5A?) And who knows what else. In fact, my real question *is* who knows what else? I looked at the RCSB site and googled PDB changes, etc, but really couldn't find the low-down. Another question is how long before it all changes again? I noticed some threads here with PDB in the title recently, but didn't read them because I thought they discussed esoteric and philosophical issues that do not affect us common folk. James -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com --- 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_Alphabetically.php?faculty_id=123 http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] solving structure of which 70% is known
Hi, - I think someone already pointed out that you should try P6522. - check that it isn't really a twinned P61 or P65 with 2 per asymmetric unit - buy DNA with BrdU and some more with IdU, and/or grow your protein in SeMet - at your resolution, the more real phase info the better! Phoebe At 06:30 PM 8/29/2007, you wrote: Hello, I am trying to solve a multi-protein DNA complex structure from a 3.6 A native data set. The target structure is a dimer (95 aa in each monomer) in complex with DNA( 15 base pairs) plus a second protein of 131 aa. The data has been scaled to P6(1)22 sp. gr. and one target structure is expected to be in the asymmetric unit that corresponds to 68% solvent content. 70 % of the target structure is known in two parts(two different pdb structures previously solved contributing 55% and 15% of the target structure). I tried with molrep and phaser considering the first part(55%) as the search model but it turned out to no good solution which all clashes with symmetry related copies. If I assume the sp. gr. is not the case what else I can try. Any suggestion is well appreciated. Thanks... Raja --- 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/index.html http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
[ccp4bb] water water everywhere
While we're still on the subject of good model-building habits and reviewing pitfalls, I've been shocked to download a couple of structures recently that seem to have solvent channels chock full of allegedly ordered water (many layers deep, and not exactly at 0.5A resolution). To any new students out there: making Rfree go down a bit by putting a water in every unexplained blob is NOT the same as building a good model! I'm afraid I reviewed one of these (sans coordinates) ... so sorry to the community ... it wasn't obvious from table 1. 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/index.html http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] nature cb3 response
A comment from my collaborator's student suggests a partial answer. This afternoon he happened to say but of course the reviewers will look at the model, I just deposited it!. He was shocked to find that hold for pub means that even reviewers can't access the data. Can that be changed? It would take a bit of coordination between journals and the PDB, but I think the student is right - it is rather shocking that the data is sitting there nicely deposited but the reviewers can't review it. Phoebe Rice At 05:33 PM 8/16/2007, Bernhard Rupp wrote: Ok, enough political (in)correctness. Irrespective of fabricated or not, I think this points to a general problem of commercial journals and their review process, as it seems that selling (.com) hot stuff induces an extraordinary capability of denial. The comment, as someone noted, does not address the allegations at all. This is reminiscent of my dealings with Nature in two related cases: They ignore or stonewall until the dispute is ended with an irrelevant comment. In one case, Axel B later proved with the correct structure that what we had commented on earlier was entirely correct. In the second case, the comment (by some of the leading experts, not just by me nobody) was rejected with no recourse based on another non-fact-addressing author comment and not published at all. Compare this to a similar case, when the Jacs editor (.org --) contacted me on its own accord to check for a related problem, leading to retraction of the paper after the editor (a scientist himself) evaluated facts and response. It also seems to depend on the handling Nature editor. I have made maps of several structures from data unhesitantly provided by the editor when I had reason to ask for them during review. Those were also responsive to a mini-table-1-comment I sent on cb3, but I did not hear from the editor assigned to cb3. This time again, the review completely failed (table 1 and comment issues), and the editorial process failed as well, because the response is not adequate. If someone - as tentatively and tactfully it may have been phrased - accused me of faking data they'd eat shit until hell freezes over It is as simple as that: Extraordinary claim (super structure, bizarre stats and properties) requires extraordinary proof. This rule has not been followed, which reflects poorly on the scientific process in this case. I also note that in no case known to me, persons involved in irregularities have ever appeared as frequent (or at all) communicators on the ccp4bb. As long as grant review and tenure committees rely on automated bibliometrics and impact factors (and who knows who) to decide academic careers and funding, the big journals will remain the winners. The system has become self-perpetuating. Back to grant writing now. Need to get that paper out to nature... Cheers, br PS: it is pointless flaming me. I am the messenger only. -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Bernhard Rupp Sent: Thursday, August 16, 2007 2:03 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] nature cb3 comment pdf thxthxthx to all the day and night owls for the many copies The winners have been selected, no more entries needed. thx again br -Original Message- From: Miriam Hirshberg [mailto:[EMAIL PROTECTED] On Behalf Of Miriam Hirshberg Sent: Thursday, August 16, 2007 1:58 PM To: Bernhard Rupp Subject: Re: [ccp4bb] nature cb3 comment pdf attached, Miri On Thu, 16 Aug 2007, Bernhard Rupp wrote: my nature web connection just died for good (probably a preventive measure..) Could someone kindly email me the pdfs of the comment and response? Thx br - Bernhard Rupp 001 (925) 209-7429 +43 (676) 571-0536 [EMAIL PROTECTED] [EMAIL PROTECTED] http://www.ruppweb.org/ - People can be divided in three classes: The few who make things happen The many who watch things happen And the overwhelming majority who have no idea what is happening. - --- 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/index.html http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] domains missing
Hi, Did you do rigid body refinement or regular minimization? If over half your structure is missing, anything besides rigid body refinement will probably just bias the phases toward nothing in the rest of the asymmetric unit. I've had correct molrep solutions with R-factors that high, so there is hope. But depending on how you refined your partial model, you could have a completely wrong solution with that R-factor as well. How sure are you that your solution is really correct? If you take the rigid-body-refined-only model (to minimize phase bias), and delete something you know should be well-ordered (a fat side chain if you've got good data, a helix if you've got lousy data), then make an Fo-Fc map, does it reappear? If not, forget all the statistics and start over with your searches. If yes, can you see any sign of the other domains in that map, that solvent modification might help bring up from the land of noise? Phoebe Rice At 02:38 PM 8/13/2007, you wrote: Hi, ccp4 community, I am solving my protein (300 aa) structure using molecular replacement. The space group is P622. There is only one molecule in the ASU. The protein is supported to have three domains. We have solved the domain 1 (120aa) structure; therefore we tried to use it as a model to solve the new structure. MolRep and Phaser can find the domain 1. We refined the model and the R and Rfree factor was around 50%. When we used Coot to see the model and map, we found that the other two domains are missing. In the map, there is a big 'hole' and little extra density (with 6-fold) inside. It is a big surprise to us. Is there any possibility that R is around 50 if the other two domains are missing? Any suggests to figure out what happen to my structure? Thanks a lot! Mousheng Wu --- 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/index.html http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] resolution vs ramachandran
Every modern structure should be above average by procheck criteria. For modest resolution structures, the availability of ncs restraints should have a big effect on one's Ramachandran expectations: I've found ncs restraints at 3-3.5A do wonders for the Ramachandran plot even when they don't do much for Rfree. Phoebe At 11:44 AM 8/3/2007, Edward Berry wrote: Procheck puts out such a correlation (% most favorable vs resolution) in the _04.ps file. For example look at page 7, first panel of the sample procheck output at: http://sb20.lbl.gov/SQR/procheck-2H88.pdf It appears that 83.5% would be well above average for a 3 A structure according to procheck statistics. (However I think most structures nowadays are above average by procheck statistics) Ed Xiaofei Jia wrote: Dear all, I am now preparing my structure for deposition. The crystal diffracts to 3.0 A. R: 0.20; R free: 0.25. What I am concerned with is the Ramachandran plot; 83.5% in core region, 15.8% in allowed, 0.5 % in general allowed,0.2% in disallowed region. The model fits in density pretty well and all the attempts to improve Ramachandran have not been successful after quite a few trials. I am wondering if 83.5 % in core region is acceptable with 3.0 A resolution data? Moreover, is there some numeric correlation between diffraction resolution and model Ramachandran? Thank you for your help. Xiaofei Moody friends. Drama queens. Your life? Nope! - their life, your story. Play Sims Stories at Yahoo! Games. http://sims.yahoo.com/ --- 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/index.html http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] Protein-DNA complex for crystallization
Dear Kumar, One often has to try duplexes with many different ends before getting decent crystals (e.g. 18 for one project in my lab, even more for others). Depending on your Kd, you might find that your complex falls apart during gel filtration. How long are your oligos? Gel purification sometimes helps us and sometimes doesn't, but longer ones ( 20-30nt) are more likely to benefit from it. Did you check the concentrations yourself before annealing? (my students get better results when they do). Can your oligos hairpin? If you have a dimer that binds a symmetric DNA site, having individual monomers bound to hairpinned oligos would certainly make a mess of your xtals. If you think you have an excess of one single strand, you could try annealing small amounts at several different ratios, and check how much is really duplex on a native agarose or acrylamide gel. Have you checked your prep for nuclease contamination? Just incubate some with a supercoiled plasmid and ~10mM Mg++ for a couple hours and see if the plasmid stays supercoiled. This is a beautifully sensitive assay because cleaving only 1 bond out of thousands will change the plasmid's mobility - but bear in mind it will only reveal endonucleases, not exonucleases. Finally, its always a good idea to pull up a pile of old papers and skim their methods sections for inspiration. Good luck! Phoebe Rice At 11:01 AM 7/16/2007, you wrote: Hi, I am trying to crystallize a protein-DNA complex. I purify the protein finally using gel filtration. I purchase single stranded complementary oligos (desalting from idtdna.com), mix them up and make DNA duplex by heating to 95 degree C and cooling to room temperature. I mix protein and DNA, concentrate and use it for crystallization. I am geting small crystals consistently under a specific condition. These crystals take up IZIT dye but are not well shaped. I am not able to improve the size and shape of the crystals substantially even after screening with additives (Hampton research). I suspect that purity of the duplex DNA (presence of unpaired oligos) is limiting the chances of obtaining better crystals. How can I purify the duplex DNA further? Are there better ways of making protein-DNA complex for crystallization? If I make the protein DNA complex and then do the gelfiltration, will the complex purified so be a better choice for crystallization? Thank you Kumar Dept. of Biochemistry, Cellular and Molecular Biology, Walters Life Science, # 406, University of Tennessee, TN, Knoxville, USA --- 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/index.html http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
[ccp4bb] Post Doctoral Position: Microfluidic Protein Crystallization, University of Chicago
Crystallization and structural biology of soluble and membrane proteins using microfluidics. This NIH-funded project at the University of Chicago aims to develop technologies for crystallization and structural analysis of proteins in nanoliter volumes (see PNAS 2006 103: 19243-19248). See http://ismagilovlab.uchicago.edu/positions.html for details. Experience in expression, purification, crystallization, and structural analysis of macromolecules is essential. Experience with membrane proteins, strong leadership and communication skills, interest in technology development are highly desirable. Experience in microfluidics is not required. We welcome outstanding postdoctoral applications from talented, motivated individuals. Respond with a CV and a brief statement describing your expertise and interests to [EMAIL PROTECTED]
Re: [ccp4bb] Highest shell standards
Isn't automatically included fabricated data for missing reflections a really bad idea for anisotropic data where most reflections are missing at high resolution? Shouldn't there be a big flashing red flag alerting the user to what's been done? Phoebe At 01:22 PM 3/26/2007, Edward A. Berry wrote: Actually I was thinking of a somewhat earlier paper: Rayment,I. Molecular relacement method at low resolution: optimum strategy and intrinsic limitations as determined by calculations on icosahedral virus models. Acta Crystallogr. A 39, 102 116 (1983). But thanks for bringing the Caliandro et al. paper to my attention. Thanks also to Fred. Vellieux for his comments, and to Pete Dunton for explaining to me that while fft doesn't do fillin by default, the 2MFo-DFc map coefficients from refmac5 do have fillin values for the missing reflection, making model bias a problem when many missing residues are included. Now I understand Petrus's question. Ed Michel Fodje wrote: You are probably referring to the following works: Caliandro et al, Acta Cryst. D61 (2005) 556-565 and Caliandro et al, Acta Cryst. D61 (2005) 1080-1087 in which they used density modification to calculate phases for unmeasured reflections, and used the phases to extend the resolution by calculating rough estimates unmeasured amplitudes. Using this technique they actually could improve the electron density. If I'm not mistaken, George Sheldrick has implemented this Free Lunch algorithm in SHELXE. /Michel On Fri, 2007-03-23 at 08:05 -0800, Edward Berry wrote: If instead you allow the missing F's to float, calculating them on each cycle from the previous map using the fillin option, someone has shown (don't have the reference handy at the moment) that the F's tend toward the true F's (in the case that they weren't really missing but omitted as part of the test). Ed --- 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/index.html http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
: misbound ligand examples?
A biochemist friend asked for examples of cases were a protein was co-crystallized with or soaked in a ligand that bound in the wrong place - say, because the ligand used wasn't quite the right one or because other important ligands were absent. I'm sure such examples are out there, especially when soaks were done at high concentrations, but I'm having trouble thinking of concrete examples. Help? thanks, Phoebe Rice --- 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/index.html http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
