[ccp4bb] Clashes in Phaser
I am currently trying to solve two datasets of two different protein-protein complexes. One of the proteins is common to both complexes and there is a deposited structure in the PDB (100% identical). The other proteins currently have not been solved and share ~50% identity between them. Both complexes crystallize in different space groups. All three proteins are ~12kDa. Both datasets diffract to ~3-3.5A. A) C2221 109.6 110.1 335.2 90 90 90 with around 9 copies of the complex. B) I4122 131.6 131.6 257.1 90 90 90 with 5 copies of the complex. I am slightly suspicious of Dataset A due to similar a and b lengths. Merging in P4, results in higher Rmerges. However in each case, searching with the common protein, results in Phaser failing to find a single copy of the protein, despite TFZ scores of 8-12, due to rejection during packing; Dataset A Packing Table - Solutions accepted if total number of clashes <= 5% of trace atoms i.e. total number of clashes <= 5 AND if number of clashes <= 5% of trace atoms for each ensemble i.e. ensemble1: number of clashes <= 5 ## #Clash Packs SpaceGroup Annotation 1 198 NO C 2 2 2 RFZ=2.9 TFZ=11.2 2 154 NO C 2 2 2 RFZ=2.3 TFZ=10.8 3 229 NO C 2 2 2 RFZ=2.6 TFZ=11.5 4 161 NO C 2 2 2 RFZ=2.5 TFZ=10.0 5 118 NO C 2 2 2 RFZ=2.4 TFZ=9.8 6 166 NO C 2 2 2 RFZ=2.9 TFZ=8.1 7 199 NO C 2 2 2 RFZ=2.6 TFZ=10.0 8 165 NO C 2 2 2 RFZ=2.5 TFZ=10.0 9 167 NO C 2 2 2 RFZ=2.4 TFZ=10.1 10168 NO C 2 2 2 RFZ=2.5 TFZ=8.8 11186 NO C 2 2 2 RFZ=2.4 TFZ=9.0 1293NO C 2 2 2 RFZ=2.5 TFZ=8.3 13155 NO C 2 2 2 RFZ=3.1 TFZ=8.8 14149 NO C 2 2 2 RFZ=2.9 TFZ=8.1 15158 NO C 2 2 2 RFZ=2.6 TFZ=9.7 16151 NO C 2 2 2 RFZ=2.6 TFZ=10.5 17147 NO C 2 2 2 RFZ=3.1 TFZ=8.6 18145 NO C 2 2 2 RFZ=2.4 TFZ=8.8 19130 NO C 2 2 2 RFZ=2.4 TFZ=10.8 20166 NO C 2 2 2 RFZ=2.9 TFZ=8.4 21215 NO C 2 2 2 RFZ=2.4 TFZ=10.0 22147 NO C 2 2 2 RFZ=2.6 TFZ=10.1 23189 NO C 2 2 2 RFZ=2.6 TFZ=9.6 24164 NO C 2 2 2 RFZ=2.5 TFZ=9.2 25166 NO C 2 2 2 RFZ=2.6 TFZ=9.3 26234 NO C 2 2 2 RFZ=2.4 TFZ=8.3 27170 NO C 2 2 2 RFZ=2.4 TFZ=9.3 28126 NO C 2 2 2 RFZ=2.6 TFZ=8.1 29189 NO C 2 2 2 RFZ=2.5 TFZ=9.4 30171 NO C 2 2 2 RFZ=2.3 TFZ=9.6 31211 NO C 2 2 2 RFZ=2.5 TFZ=8.6 32203 NO C 2 2 2 RFZ=2.5 TFZ=10.2 33162 NO C 2 2 2 RFZ=2.4 TFZ=9.7 34116 NO C 2 2 2 RFZ=2.6 TFZ=8.5 35168 NO C 2 2 2 RFZ=2.5 TFZ=8.1 36159 NO C 2 2 2 RFZ=2.4 TFZ=9.6 3791NO C 2 2 2 RFZ=2.5 TFZ=8.6 38114 NO C 2 2 2 RFZ=2.5 TFZ=8.6 39212 NO C 2 2 2 RFZ=2.4 TFZ=8.3 40162 NO C 2 2 2 RFZ=2.4 TFZ=10.1 41182 NO C 2 2 2 RFZ=2.9 TFZ=8.4 42112 NO C 2 2 2 RFZ=2.5 TFZ=8.3 43131 NO C 2 2 2 RFZ=2.4 TFZ=9.3 44166 NO C 2 2 2 RFZ=2.3 TFZ=9.4 45135 NO C 2 2 2 RFZ=2.4 TFZ=9.4 46156 NO C 2 2 2 RFZ=2.5 TFZ=8.5 47189 NO C 2 2 2 RFZ=2.5 TFZ=8.6 48133 NO C 2 2 2 RFZ=2.5 TFZ=8.6 49200 NO C 2 2 2 RFZ=2.5 TFZ=9.1 50146 NO C 2 2 2 RFZ=2.6 TFZ=9.2 51117 NO C 2 2 2 RFZ=2.4 TFZ=8.5 52128 NO C 2 2 2 RFZ=2.5 TFZ=8.2 53144 NO C 2 2 2 RFZ=2.4 TFZ=9.0 54148 NO C 2 2 2 RFZ=2.5 TFZ=8.1 55119 NO C 2 2 2 RFZ=2.4 TFZ=8.5 56131 NO C 2 2 2 RFZ=2.4 TFZ=8.5 57131 NO C 2 2 2 RFZ=2.5 TFZ=8.4 58173 NO C 2 2 2 RFZ=2.6 TFZ=8.9 59114 NO C 2 2 2 RFZ=2.6 TFZ=8.1 60150 NO C 2 2 2 RFZ=2.4 TFZ=8.6 61150 NO C 2 2 2 RFZ=2.4 TFZ=8.4 62138 NO C 2 2 2 RFZ=2.3 TFZ=8.6 63123 NO C 2 2 2 RFZ=2.5 TFZ=8.3 64122 NO C 2 2 2 RFZ=2.5 TFZ=8.2 65116 NO C 2 2 2 RFZ=2.6 TFZ=8.4 66118 NO C 2 2 2 RFZ=2.4 TFZ=8.0 67131 NO C 2 2 2 RFZ=2.4 TFZ=8.3 6896NO C 2 2 2 RFZ=2.4 TFZ=8.4 6998NO C 2 2 2 RFZ=2.4 TFZ=8.0 70198 NO C 2 2 2 RFZ=2.5 TFZ=8.6 71137 NO C 2 2 2 RFZ=2.4 TFZ=8.9 72110 NO C 2 2 2 RFZ=2.5 TFZ=8.2 7370NO C 2 2 2 RFZ=2.4 TFZ=8.4 74128 NO
Re: [ccp4bb] Crystallization with no precipitant
Thanks for the all the comments on/off board. Especially for papers describing this method as it may be handy if the structure is solved. The crystals are reproducible. They have started to appear after 4 days. I can accelerate it to a day using just 1.5M NaCl as a reservoir solution, though I get excessive nucleation. Thanks again for the knowledge. Dan
[ccp4bb] Crystallization with no precipitant
Dear All, I have produced crystals from a robotic screen (API) in two conditions. However, I have not been able to reproduce them. They originally grew in 2.5 days (over the weekend, though it could have been sooner). I noticed that when I first set up the tray, these two wells were clear but when I reproduce the condition, I get immediate precipitation. Furthermore, in the original screen the two drops were smaller than the rest. I have tried different ratios of reservoir solution though I get less precipitation, the drops are not clear. This brings me to my question. Could it be possible that the robot never dispensed the reservoir solution and the protein just crystallized against the reservoir solution through simple concentration? I have just set up a couple of drops just against reservoir solution. If this is the case, has anyone else experienced this and had luck with this and got diffraction? Thanks in advance, Dan
[ccp4bb] Anisotropy and temperature
A PhD student asked me what causes diffraction anisotropy. Quoting from the Diffraction Anisotropy Server webpage that it is caused by whole-body anisotropic vibration of unit cells. He asked whether a colder cyrostream could improve anisotropy. My answer would be yes, as colder temperatures would lower the vibrations. My two questions are; (1) am I right? and (2) if so, has it ever been done before in practice? Thanks, Dan
[ccp4bb] Phenix
Is the phenix website down? Anyone know when it will be back up?
[ccp4bb] Inclusion Bodies
We have been expressing a family of human proteins as inclusion bodies in E. coli, which we can be refolded and crystallized. However, three members show no expression either as inclusion bodies or as soluble proteins. The genes were ligated into the pE21d vector with either a His-tag or no His-tag. The genes were codon optimized for E coli. They are expressed in BL21(DE3), though I have tried the different strains for the non-expressing protein (e.g. Walker cells, Rosetta). What are the causes for no expression. I am guessing something to do with the mRNA. Should I try unoptimized/semi-optimized codons? Or is there something I could add to the media to aid in expression of the protein into inclusion bodies? Any suggestions?
[ccp4bb] Reindexing lattice
Hi, I am trying to reindex a P212121 lattice to change the axis order from 32.62, 64.89, 103.16 to 64.89, 103.16, 32.62. Looking at the REINDEXING manual I can't find how to reindex the lattice. Does anyone know how I should do this and an example of the execute script that I should use? Thanks Dan
Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!
They have, it is call PDB_REDO. You can download the maps and refined structure there. http://www.cmbi.ru.nl/pdb_redo/ Dan
Re: [ccp4bb] Cleaved peptide density!
First of all, you don't say how long it took to first set up crystals, for them to grow, harvest, freeze and collect data on. Secondly how long did leave the peptide/substrate for your SDS PAGE experiment? If they are of a different time scale e.g. 6 hours v.s. 30 days, it may be that your enzyme is not totally dead. Also how did you purify the alanine mutant? If you purified it on the same columns/beads as the WT protein you may have a residual amount of active protein which could cleave your peptide over the course of crystallization. You may want to use fresh beads, or treat columns with pepsin or sodium hydroxide. Not real answers I am afraid, more like suggestions.
Re: [ccp4bb] RMSD of dimers
Hi all, Just writing an update, as I have had two people contact me asking if I had found a solution and if I could share. I would like to apologize first for not phrasing my question to describe the situation simply. I had several suggestions that Coot, and PISA could do it but actually can not. Eugene Krissinel rephrased the question brilliantly. What is required, however, is optimal superposition on one pair of chains (one from each dimer) but measuring RMSD on the other pair of chains from same dimers. The solution was given by Thomas Holder and can be done in Pymol: Use rms_cur based on a sequence alignment obtained with align, try: # make sequence alignment object (optional: cycles=0) align A1B1 chain B, A2B2 chain B, object=aln, transform=0 # get current rms for this alignment rms_cur A1B1 aln, A2B2 aln, matchmaker=-1 This will give the RMSD for other pair of chains. Thanks Dan
[ccp4bb] RMSD of dimers
I have a family of homodimers (denoted A1B1, A2B2, A3B3...) which I have superimposed using Chain A. Several programs will produce the RMSD of Chain A2, A3, A4... to Chain A1. However, I would like to know the RMSDs of Chain B2, B3, B4... to Chain B1 when I have superimposed the structures relative to Chain A. I have tried using Pymol though there are gaps/insertions so rms/rms_cur will not work. Does anyone else have any other suggestions? Thanks in advance, Dan
[ccp4bb] Wrong Space Group?
Dear all, I have collected ~160 degrees of data on a new crystal form of a protein which has already been solved. Data was processed with XDS and reindex, scaled and truncated with Aimless. Both XDS and Pointless suggested a Laue group of P6/mmm with a possible space group of P6122 or P6522. Stats showed an overall Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of 19.1), a completeness of 99.1% and resolution of 2.8Ang. With cell dimensions of 63.1 63.1 243, only one protein chain can be found in the asymmetric unit (two copies would leave a solvent content of 8%). I ran phaser with all alternative space groups and a single solution in P6522 with a TFZ of 10.0. I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I open the structure and map in Coot and could see that there was a large conformational change of helix-turn-helix actually becoming just a long helix (https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and then dimerizing through the long helix with one of the symmetry mates. This section was rebuilt (https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran through Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through the rest of the structure I see nothing else really to be modeled. Nothing that could bring the Rfactors down to a reasonable range. I have therefore tried several things. I ran the structure through Zanuda server to look at other space group possibilities. The server suggested I was in the correct space group. However I did reprocess the data to P6, P3, P312, P321, C2221, P2 and C2, and reran phaser in search all alternative space groups using the original search model but found no solutions. I did reprocess the data in P1, though I did not collect enough data. Twinning tests show no twinning. Although that does not mean there is no twinning, I can see that P6522 has no twin laws. Does that mean no twinning can occur in P6522 or that it can occur but there is no law to be able to separate the amplitudes? I also collected data on a single point mutation of the protein. Although this diffracted to a slightly weaker resolution (3.2Ang), I also observe the same problem of good maps in P6522 but no solution in the groups described above, a clear indication that this helix has elongated but terrible Rfactors. Based upon that the maps look good in P6522 do you believe that I have solved the structure in the correct space group but my data collection is at fault or in fact that I have some form of pseudosymmetry or something else going on and that the space group has lower symmetry but not in the space groups I have checked. Or is it something else. Any suggestions, criticisms or you need further information please contact me and enjoy your weekend.
Re: [ccp4bb] A photograph of the Arndt-Wonacott rotation camera?
Bob Sweet has a powerpoint presentation but it took a long time to download the file (http://www.google.com/url?sa=trct=jq=esrc=ssource=webcd=27ved=0CEoQFjAGOBQurl=http%3A%2F%2Fcima.chem.usyd.edu.au%3A8080%2FMMSN%2Fevents%2FRAAW_Mar_2005%2FBob_Sweet_v2.pptei=_jRxUqvjGeqrsASg8YDwBAusg=AFQjCNFXctrZtshdnzaF-zyHmg6ELyu_twbvm=bv.55617003,d.cWccad=rja) I have copied the camera and placed it here (https://www.dropbox.com/s/oceo44w5bngmdgh/Picture1.jpg) for easier access. I hope it is the camera.
Re: [ccp4bb] Philosophical question
You may want to read: Evolutionary conservation of codon optimality reveals hidden signatures of cotranslational folding Nature structural molecular biology VOLUME 20 NUMBER 2 FEBRUARY 2013 237 Here they suggest that the codon bias is such it allows translation to pause and folding of the polypeptide. Most proteins probably do not have a problem folding so it does not matter which codon is used. Dan It is so intolerant to change because reassigning a codon to a different amino acid type or stop codon affects thousands of proteins that use that codon simultaneously. The probably that none of those mutations are deleterious is extremely small. Genetic code changes are more common in the mitochondrial code. First of all the mitochondrial genome is much smaller, ~16kb for vertebrates. Moreover, in cases I have looked at the change in codon use seems to happen when first there is a case of extreme bias against using a codon. When a codon is (almost) not used at all it can be re-purposed without affecting any proteins. Bart On Tue, Mar 19, 2013 at 2:05 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: I don't understand this argument, as it would apply equally to all features of the theoretical LUCA No it won't. Different features would have different tolerance levels to modifications. Yes, this tolerance is the second (hidden or implicit) principle I referred to. So you'd have to explain why the codon convention is so intolerant/invariant relative to the other features--it seems to me that either it is at an optimum or there is some big barrier holding it in place. And you'd have to explain this without invoking interchange of DNA, viruses, etc, as we're talking about a LUCA here, right? And you'll have to make sure that whatever reason you invoke cannot be applied to other features of this LUCA which are indeed seen to be variable. JPK
Re: [ccp4bb] Off Topic GST-tag Protein Purification
Protein A must be co-expressed as it does not express well alone. Have you tried separating A+B on the Nickel column by flowing 2M urea to help tickle off protein B. The amount will vary depending on the affinity. In my case (~50nM) requires about 1000-1700ml of 20mM Tris, 2M urea pH 7.5. Then you can add unlabelled purified protein B to labelled protein A. Dan
Re: [ccp4bb] protein cleavage
You do not mention what buffer you are trying to do your cleavage in. You need a reducing agent for TEV to work (e.g. reduced gluthionine, DTT, mercaptoethanol). EDTA (0.5-1mM) is recommended as TEV is a cysteine protease and the presence of divalent metal ions will/eventually inhibit TEV. TEV becomes less active as salt concentration increases (50% active at 0.5M NaCl) If your protein contains disulphide bridges and you want to keep them intact you can use a ratio of 3 mM glutathione/0.3 mM oxidized glutathione which provides enough reducing power for TEV to work but should not disrupt disulphides. If none of these reasons are why your protein fails to cleave, there is a strong possibility that the TEV site is inaccessible. You could try 1M urea in this case. TEV again will be less active, but you could at least test this theory.
Re: [ccp4bb] off-topic: detergents for the stabilisation of water-soluble proteins
The following paper (which can be found at www.wolfson.huji.ac.il/purification/PDF/Literature/Bondos2003.pdf Detection and prevention of protein aggregation before, during, and after purification. Sarah E. Bondos and Alicia Bicknell (2003) Analytical Biochemistry, 316, 223-231 contains a table of agents that may promote protein solubility on the 2nd page. Most are non-detergents which may be worth following up as well as Tween 80, Tween 20 and Nonidet P-40 at the recommended concentrations and their references. Dan
Re: [ccp4bb] Overlapping transparent surface representations in Pymol
I am assuming you are after something like a difference map of the two surfaces. http://www.pymolwiki.org/index.php/Map_set Map_set command can average, copy, difference, maximum, minimum, sum and unique Hope this is what you are after. Dan
Re: [ccp4bb] co-express two proteins in E.coli
I have been using the Duet system from Novagen (or whatever it is called these days), specifically the pETDuet-1 and pRSFDuet-1. Co-expression of my proteins did not work in either vector. Either, one protein expressed or the other. I played around with the promotors (they are both T7) by changing one to the tac promotor. This increased the expression of this gene but shut off expression of the other. The only way I could get my proteins to co-express was to use pGEX vector with one protein, and pRSFDuet with the other protein (leaving the second MCS empty). There is a paper which sums up co-expression in E coli. http://www.nature.com/nmeth/journal/v3/n1/full/nmeth0106-55.html Dan
Re: [ccp4bb] Off-topic His-Antibody
Dear All, Thanks for all the replies on and off-board. I received around twenty replies and the majority have spoken in favor of the QIAgen BSA-free anti-5His mAb from QIAGEN. Not to be bias, a couple of people recommended the one from Abcam as well. Thanks again, Dan
[ccp4bb] Off-topic His-Antibody
Are there any good antibodies for His-tags on the market. I have never used one and I heard several stories that they were poor and not worth the money, over the past few years. The only post I could find on the BB was from 2008. Have His-Antibodies improved or are they still rubbish.
Re: [ccp4bb] about point mutation
Phusion requires that the primers are phosphorylated for mutagensis to work, unlike Pfu. If you cannot phosphorylate them use Pfu as recommended by Charlotte Dan
Re: [ccp4bb] Merging data collected at two different wavelength
Isn't it true that we cannot even agree on what MAD stands for? Is the following right? M = Multiple-wavelength. I think everyone agrees to this, although I believe I've seen the occasional (and sometime non-sensical) variant A = Anomalous (I think everyone agrees, although this term should really be changed to resonant, as there is no anomaly to it anymore...) D = Diffraction, Dispersion, Destruction, Dissolution...? JPK D =Discussion?
Re: [ccp4bb] raw data deposition
Why should we store images? From most of the posts it seems to aid in software development. If that is the case, there should be a Failed Protein Databank (FPDB) where people could upload datasets which they cannot solve. This would aid software development and allow someone else to have ago at solving the structure. If it is for historical reasons, how can someone decide whether their structure is historical? I would propose that images should be uploaded for a protein or protein-complex that has never be solved before. That way the images are there if that structure does become historical. The question is not whether or not images should be uploaded but who would use the images that were uploaded. For example, people who use crystallography as a tool to aid in characterization of their protein, would probably not look at images for 99.5% of other protein datasets, and they probably would not look at images for a protein that is related to their own protein. They are more interested in the final structure. I too would probably not be interested in reprocessing and solving a structure again when I can easily access the final product already.
Re: [ccp4bb] should the final model be refined against full datset
I may be missing something or someone could point out that I am wrong and why as I am curious, but with a highly redundant dataset the difference between refining the final model against the full dataset would be small based upon the random selection of reflections for Rfree?
Re: [ccp4bb] Off topic_protein degradation.
If you are unsure about whether the disulfides have formed treat a small amount of protein with N-ethylmaleimide. If the disulfides have not formed, when you perform mass spec on the protein you should see an increase of mass of 125Da for each exposed cysteine.
Re: [ccp4bb] Off topic - Streptactin Column Summary
A couple of people asked why the GST/His steps. The only way I can expressed this protein is by co-expression. Co-protein is GST tagged and my protein of interest is N-term His and C-term StrepTag. Even with the coexpression, there is a major degradation product, the reason why for the strep tag, but now it is not being removed by the column. I have tried size exclusion (140kDa vs 100kDa does not work) and MonoQ (pI are near identical). I have been using HABA for regeneration. I will try some of the suggestions to try and rescue it. Summaries are presented below. A summary of answers: 1) We use a Streptactin resin from IBA and they recommend regenerating with HABA, which is non-destructive and I believe can be used many times (we've done greater than 7 times I believe?). They say their linker is more affected than GE's version so NaOH is quite destructive and cannot be run more than ~ 3 times with their resin. Perhaps GE resin is also affected more by NaOH? HABA will displace any biotin. 2) I use 5 ml StrepTrap columns from GE and these columns work almost indefinitely with reproducible results if the following trick is used .I pre-clear biotin from my lysate with a pinch of avidin (Sigma #A9275-25MG) and find that this works really well. You could try adding a small amount of avidin to your protein sample after your Nickel column. After each run, I wash the column with 3 CV 0.5 N NaOH, 10 CV water, 10 CV 1 mM HABA (Sigma H5126-25G) containing buffer (pH 8) and finally equilibrate with 10 CV binding buffer. In the past, I used to see a gradient of pale orange at the top of the column and dark orange at the bottom as the column got older, presumably because of sites inactivated by biotin. Surprisingly, this phenomenon went away after avidin treatment suggesting that it might be possible to rescue old columns inactivated by biotin although I haven't rigorously tested this. 3) We have been using Strep-Tactin SuperFlow (high capacity) columns more than 100 times, even when run under preparative conditions with protein extracts from E. coli fermentations. Biotin binding to the engineered streptavidin is not irreversible but can be cured by extended washing. To check the column status I recommend our HABA binding procedure described in: Schmidt, T. G. M. Skerra, A. (2007) The Strep-tag system for one-step purification and high affinity detection or capturing of proteins. Nat. Protoc. 2, 1528-1535. 4) i am not the last authority on this, and really, IBA have in the past been very helpful (http://www.iba-go.com) with information. But here my 2 cents on this: i would give it a 20 times possibly. you CAN even apply lysate directly and iba describes on their pages how you avoid biotin contamination with addition of streptavidin to your sample. streptavidin catches all the free biotin, but does not bind the strep tag. done it and works real well, too ... 5) I used the Strep-Tactin resin from IBA Bio TAGnologies, 2-1201-010, and would regenerate it after each use with 2 mM HABA (Sigma H5126-25G). I did not observe a decrease in affinity over multiple uses. Also, I only used the one column and obtained 99% pure protein.
[ccp4bb] Could Biological Negative Results be published?
There are least two types of negative results 1) Contradiction of previously published results. Negative results of this kind is either they are wrong, you are wrong or it depends on the differences within the experimental methods used. An example of the latter case would be SPR vs ITC. SPR for a number of protein interactions just does not work but ITC does. 2) You have developed a hypothesis, tested it and it does not work. Either that you hypothesis is wrong, which may be useful if it were published as it may stop other research groups wasting time/effort/money on an experiment that would not work in the first place. Or your experimental method is wrong. Again this maybe worth publishing as it may give someone else an idea. Though of course publishing these types of negative results may not really help you but may help your rivals/competitors. So you have to be careful.
[ccp4bb] Off topic - Streptactin Column
Does anyone know how many times roughly you can re-use a Streptactin column? I know that contamination with biotin will destroy the column but the protein that I am using has gone through both a GST and Nickel purification steps before seeing the Streptactin column and I think lately that I have been seeing decreased binding. Thanks in advanced.
