Re: [ccp4bb] Resuspension of bacterial cell pellets
Roger Rowlett wrote: This goes straight into a bead beater for complete, gentle homogenization in 8 min. Didn't you mean complete, foam-producing, surface denaturation-inducing homogenization? I am not saying that bead beater is worse than the locally near boiling temperatures-producing more conventional sonication but surely it's a stretch to call anything related to bead beater as gentle? Also, I always wondered but never seem to find an answer to: has anyone ever measured temperature progression in a bead beater? I'd imagine that there is a lot of heat generated - how does it compare with sonication? (Which, at least, can be easily monitored with a thermometer in real time). - Dima
Re: [ccp4bb] Resuspension of bacterial cell pellets
Roger Rowlett wrote: No air in the vessel, no foam. What manufacturer/model do you use? I can't quite imagine a beater that would have no air in the chamber but maybe there is something new under the sun. Yield of soluble, active protein from broken cells is quite comparable or better than French press or sonication, but with no aerosols. The bead-beating unit is encased in ice water, and is used 15 s on and 45 sec off to minimize heat buildup. The solution still feels cold when transferred to centrifuge tubes for clarification. I suppose I could measure it next time to see how much it actually warms up during beating. Based on these observations, I conclude that the cell lysis by bead-beating is no more disruptive to proteins than a French press, but much, much faster. As a bonus, genomic DNA is sheared, so no more slimy lysates. We have used bead beaters exclusively since 1997 or so when I learned about them from a Swedish research group I was visiting. They are handy in both the teaching and research lab. Interesting. I've never used bead beaters for E.coli work and only used it once with yeasts. What I saw went well with what I read in the past from others: http://www.bio.net/bionet/mm/methods/1994-May/014416.html Quote: A comparison of sonication and beating showed that beating was much harsher FOR OUR HEAT-LABILE ENZYME IN THE PLASTIC CUP than sonication. Metal chamber is surely going to help but foaming is still a concern in my mind. - Dima
Re: [ccp4bb] Poor baculovirus stability at 4C?
We began experiencing a sudden reduction in stability of baculovirus stock stored at 4C, like the titer drops more than 5 fold in 3-5 month. The only difference compared with previous preparations is a switch from Gibco SF900-II to a media from Lonza (Insect-XPRESS). Could it be due to the media switch, or something else? What is the best way to store baculovirus? Absolutely could be. For long term storage, add 10% FBS. Helps A LOT. I've used 2 years old stocks stored in serum-containing medium and they worked fine. We don't titer (because it's kind of pointless) but the optimal virus to culture volume ratio has not changed more than twoi-fold. - Dima
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 There is really absolutely no problem co-expressing two proteins both from near-identical pET vectors as long as the two plasmids carry difference selection marker. We've used pET24 in combination with pET28 or pET31 on several complexes and it always works as long as you keep both antibiotics around. - Dima
Re: [ccp4bb] offtopic: packing gel filtration columns
Sorry for the slew of offtopic posts, but does anyone here have any experience repacking the large 120mL Superdex75/200 columns? Any advice/tips on doing it? I've got an older column that's gotten clogged while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure alarm), and not sure the bossman would be thrilled w/buying another column. There is really nothing special or magical about Superdex or Pharmacia columns. Good gel-filtration column packing can be tricky but it's been done well decades ago and can still be done today. Things to keep in mind: 1. De-fine the matrix before packing. 2. Strictly vertical column, no local heating from one side. 3. Lowering surface tension helps packing == add 0.05-0.1% Triton X-100. 4. Prevent electrostatic repulsion of beads through residual charges == use 100-200 mM NaCl in packing buffer. 5. Avoid particles sorting by size == have slurry that is about 65-70% matrix 6. Avoid discontinuities whenever possible == use packing reservoir if possible and pour definite excess of the matrix. 7. Avoid bubbles like plague == degas the slurry and packing buffer before pouring, pour in one nice careful motion, over a glass rod inserted inside (letting the slurry slide on it down rather then dropping and making bubbles). Do not allow sedimentation - connect to pump and start packing ASAP. 8. Pack at as high flow rate as possible == use absolute maximum that the matrix is spec'ed for (andthe column, of course; for Superdex prep grade it means using medium pressure columns). Pack until matrix level no longer changes. 9. Pay extra attention inserting an upper adapter without bubbles - right to the top of the matrix. Pack until matrix level no longer changes, always moving the adapter down to the matrix. 10. Compress the column: *slowly* (in several stop and go steps of compressing/running buffer through) compress by moving an upper adapter down by a total ~ 2-3% of the column height. - Dima
Re: [ccp4bb] Akta vs HPLC
What makes the Aktas different from HPLCs? Nothing. Akta Purifyer *is* HPLC. Dima
Re: [ccp4bb] Off-topic-Cleavage with TEV protease
I want to digest a tagged protein with TEV protease, it has disulfide bridges. Is there any way of doing cleavage without DTT? Yes, no problem. TEV is slowly inactivated oxidation of the active site cysteine but that's about it. If you absolutely must have no reducer during cleavage, simply up the amount of the enzyme. But it's almost certain that most proteins with S-S bridges will be perfectly happy at low reducer concentration (0.2 mM TCEP, 1 mM DTT or something along these lines; particularly if there is more than one bridge - mass action is a powerful thing and, e.g., IgG has no problem at 10 mM DTT). So I wouldn't worry about it - just add enough TEV under conditions that make your protein happy. Dima
Re: [ccp4bb] Desalting columns
I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. ~ 1 ml spin columns with Sephadex G25 or Biogel P6. Typically, when loading 200 ul of the sample onto 1 ml column, desalting efficiency is ~ 95% and protein recovery is ~ 90-95% without volume change. Lowering sample volume will increasing dealting and reduce yield but I never had the yeild worse than 75% even with 50 ul volume. Bio-Rad sells prepacked Bio-Spin P-6 or you can buy empty columns and dry P6 (by fine grade in this case) and pack to 1.2 ml per column, which will take your 250 ul volume and still result in decent desalting. Pharmacia probably sells something similar and many companies sell 0.5 ml spin columns. I like P6 (polyacrylamide) better than Sephadex (dextran) because usually (but not always) non-specific binding is lower. - Dima Thanks a lot! Best regards, Sangeetha.
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 Not really. Phusion *protocol* requires phosphorylated primers but seemingly the only reason for that is that they needed to find a way to bypass Stratagene's patent on QuikChange protocol. There is quite obviously no need for ligation at all as evidenced by any QuikChange reaction. (Even more, there is no need for two primers! We *only* ever use a single primer and the frequency of positive clones is the same). As for the Pfu, there are now much better derivatives of it than the good old Turbo. Ultra has lower mutation rate and Ultra Fusion II is a lot better in every respect (and in our hands significantly better than Phusion). - Dima
Re: [ccp4bb] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]
I can observe the amplified PCR product before and after DpnI digestion (see image in http://ompldr.org/vY2x3aw), but cannot get any colony on LB plates. I'm using very fresh super competent cells so that I've got dozens of colonies with 60 ng of the parental/non-mutated vector as positive control. Sounds like you don't have competent enough cells. 60 ng should give millions, not dozens of colonies. (Decently competent cells are 10^5/ng of plasmid). Only a tiny proportion of the observed polymerase product in QuickChange reactions is actually transforming, so you have to assume that you start with a lot less than 1 ng. So if you are not seeing at least a 1,000 of colonies after 1 ng plasmid transformation, chances that you will have positive clones in your QCh transformation are pretty low. - Dima
Re: [ccp4bb] Refmac and metal on a two-fold?
I've seen this happening to water molecules as well (in a somewhat unpredictable fashion). In the latest refmac versions, you can try harmonic restraints, although these will only slow down the atom drift, as the target position is updated every cycle. Perhaps you can use distance restraints against a dummy atom to fix the metal ion in place. Thanks, Ed! Just in case anyone else has the same issue: With Garib's help, I have forced the atom into its position by using external distance restrains of zero length against the same symmetry-related atom. The cause is unclear because the same program handles special positions in another structure just fine. Here is the exact command I used: EXTERNAL DISTANCE first chain M residue 3 atom MN - second chain M residue 3 atom MN value 0.0 sigma 0.00 symm Y Occupancy set manually to 0.5. - Dima
[ccp4bb] Refmac and metal on a two-fold?
Hello, I have a metal ion sitting on a two-fold. I assigned it an occupancy of 0.5 but Refmac keeps refining it away from that position so that in the end there is a symmetry-related ion 1.6A away. I thought that this problem has been rectified long ago? Certainly I had several occasions where Refmac would automatically recognize special position and automatically assigned proper occupancy (1/n). Not sure why this particular case is different... This behavior is the same in: Refmac 5.5.0088 (Windows) Refmac 5.5.0102 (Windows) Refmac 5.6.0117 (Windows) Refmac 5.5.0109 (Linux) I also tried leaving it at occupancy 1.0 and also tried renaming MN2+ to water at occupancies of 0.5 and 1.0. All to no avail - the atom is always shifted away from the density. Anything I can do to force Refmac to keep it nice and tidy? Thanks and Happy New Year! - Dima
[ccp4bb] Summary: sealing slides on VDX plates
Thanks to everyone who replied! This is a summary in case anyone is wondering about the same: 1. A clear majority of replies was along the lines of just buy pre-greased plates. 2. Next in popularity was self-greasing with Dow Corning high vacuum grease. 3. Several replies suggested mixing petrolatum (Vaseline) with mineral oil to achieve desired viscosity. Or just using petrolatum alone instead of grease while it is warm and melted. 4. Interestingly enough, not a single reply mentioned using any of the immersion oils for this purpose. This makes me wonder why Hampton has them in the catalog. So I played with mixing mineral oil and petroleum jelly today. 15% w/w jelly is very thick, almost solid at room temperature. 10% is still considerably thicker than mineral oil alone but thin enough to be spreadable using dropper bottle. In terms of the stability of the seal, it seems to be a definite improvement over pure mineral oil. The easiest way to mix is in the beaker on a 60C water bath and mixing with glass rod followed, when completely melted, by a magnetic bar on a stirrer plate. Huge amount of the air that gets trapped is easy to remove by applying some vacuum to the glass bottle containing the mixture while it is warm (fully transparent). - Dima
[ccp4bb] sealing slides on VDX plates?
Hello, I wonder what everyone is using for sealing hanging drop slides on VDX plates? For the most part, we paraffin oil but I am unhappy with it because it is too think and too frequently there is break in the oil and the drop dries too much. I find vacuum grease to be not terribly practical because it takes too much time - particularly when the well needs to be opened (seeding, modifying well content, etc). In ideal world I would like to find a much thicker oil that 1) contains as little volatiles as paraffin oil, and 2) allows no more water vapor diffusion through it than paraffin oil. Hampton suggests Cargille immersion oils for this purpose but the MSDS for these oils states that they have slight odor, so I am a bit concerned with unknown volatiles getting into crystallization drop. So, what do you like to use? Thanks much, - Dima
Re: [ccp4bb] Akta Prime / FPLC Options / Off Topic
As a personal touch, I also find Biorad software much more intuitive. This discussion, of course, is similar to comparing car brands - experiences differ. What mystifies me is a need for a service maintenance contract at all. Has hardware become so much less reliable than in the past? Granted, this Akta line is confusing but my impression that it is three completely different machines, based on peristaltic pump, FPLC glass pumps and conventional HPLC pumps. In about 20 years next to a working old-style FPLC and two HPLC setups (Waters and Gilson), I haven't seen a single service call. Just about the only maintenance FPLC requires is changing pump seals, changing lamp in UV monitor and maybe changing valve distribution plate (all trivial even for a hardware-challenged person like me). In all these years I saw one thing wrong with HPLC - pump head ceased (related to a negligent care) and needed to be replaced completely. Well, and UV lamp replacements. That's it. So, what are these magic Akta components that need constant care and repairs? BTW, agree on Bio-Rad's software. Always felt that it is much more intuitive than Pharmacia's equivalents. - Dima
Re: [ccp4bb] detect dsDNA
There exists a less toxic chemical than EtBr to stain DNA: SYBR safe DNA stain (a fluorescence dye sold by a certain vendor). SYBR Safe is about 10X less sensitive though. I suspect that not many chemicals in the lab are less toxic/mutagenic than EthBr. The classic Ames test shows that 5 ug of EthBr results in 1012 revertants. In comparison, condensate from a smoke of a a single cigarette, in comparison, gives almost 20X more revertants (18200, Table 1 in: PNAS, 1975, 72(12):5135. Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals). Assuming linear relationship, it means that one has to eat about ten mini agarose gels (20 ml at 0.5 ug/ml each) to get approximately the same mutagenic effect as smoking a single cigarette. :-) Additional reason why EthBr is probably completely harmless to humans at concentrations that we use it is the fact that it is so hydrophilic that it cannot pass plasma and instead is very quickly cleared from the bloodstream by kidneys. - Dima
Re: [ccp4bb] detect dsDNA
There exists a less toxic chemical than EtBr to stain DNA: SYBR safe DNA stain (a fluorescence dye sold by a certain vendor). SYBR Safe is about 10X less sensitive though. Can you do the toothbrush test with SYBR Safe? I wouldn't do that. As it is considerably more hydrophobic, I'd expect it to have more interactions with the body than EthBr. If anything, I expect SYBR Safe to be a lot less safe than EthBr. - Dima
Re: [ccp4bb] is codon optimization worth it?
In theory, if the rare codons are all covered by Rosetta's extra tRNAs, codon optimization should not make any difference. In practice it does because frequently it's not codon optimization per se but changing local mRNA structure. - Dima
Re: [ccp4bb] Linux vs MacOS for crystallographic software
Simon Kolstoe wrote: Meanwhile I think windows is slowly improving as a crystallography platform - and Microsoft is perhaps no longer hated in principle - however the one student in our lab who opted to go the windows route seems very limited in the software he can run. I have a feeling that the lack of Windows software continues to be mostly due to the irrational animosity toward it rather than the platform-specific issues. After all, there seemed to be many developers who were happy to code for MacOS 7-9 but refused to release anything that runs in Windows. Meanwhile, that is the only platform we never hear about installation and dependencies issues. Given the large number of Windows versions of CCP4 downloaded, I assume this is not because nobody actually installs Windows software. - Dima
Re: [ccp4bb] Silver staining Coomassie stained gels
Can anyone suggest me a protocol for silver-staining the PAGE that is already stained with Coomassie. There is absolutely nothing wrong with silver staining of a Coomassie-stained gel without destaining. It only prevents blowout of most intense bands already well-visible with Coomassie. The only thing to do is to equlibrate the gel in water before going with silver. At least that's my experinece with the silver staining protocol that is based on tungstosilicic acid (aka Bio-Rad Silver Stain Plus described here for DNA in agarose but works equally well for proteins in PAAG: http://www.ncbi.nlm.nih.gov/pubmed/2446526 ). - Dima
Re: [ccp4bb] TCA or acetone precipitation of proteins
Is it possible to precipitate proteins (TCA, acetone) from a sample that has already been stored in protein loading dye? The protein is too dilute in my current sample and I basically want to load all of the sample (100uL) in a single well in the gel. Unfortunately, I already added protein dye with SDS and all. Methanol/chloroform precipitation is both more effiecient than TCA or acetone and it can do exactly what you want it to do. Wessel, D., and Fugge, U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Analytical Biochemistry, 1984, 138:141-143 Or google it - there are many online protocols. - Dima
[ccp4bb] N-terminal sequencing facility?
Hello, Can anyone recommend a facility that does N-terminal protein sequencing very well? It has to be able to work with PVDF-blotted protein bands as a source. Relatively inexpensive would be a plus. Thanks! - Dima
[ccp4bb] Summary: N-terminal sequencing facility?
Thanks so much to everyone who replied! Just in case anyone needs a similar info in the future, here are the responses that I got: *** Peter Cherepanov: we had good experience with Pick 'n Post (Alphalyse: http://www.alphalyse.com/) Katya Heldwein: We've been happy with ours - Tufts University Core Facility, http://tucf.org/http://tucf.org/. They charge $50 startup fee and $10/cycle. John Pascal: I have used Dana Farber Cancer Center in the past with good results: http://mbcf.dfci.harvard.edu/http://mbcf.dfci.harvard.edu/ Debasish Chattopadhyay: I used the facility at Yale University; I think the name is Keck center. It was very good. Raji Edayathumangalam: I have used the facility at UTMB in the past and was very happy with their prompt service and good results. Daniel Bonsor: http://www.alphalyse.com/n-terminal-sequencing.html $400 6 cycles included. I used this last year though it was cheaper. I found this one but not used it, though it seems too cheap to be true. http://www.protein.iastate.edu/nsequence494.html $69 + $25 for each cycle. Thomas Brett: I use this guy here in town. He takes po's and can probably do out of town. He does an awesome job. We send all our samples to him. Does first 5 a.a. at $50 a pop (i.e., $250 for minimum). His name is Dave McCourt. Link: http://www.mastl.com/ Sangwon: I have used the service from Tufts core facility. It was relatively inexpensive and I was satisfied with the results. Thanks again! Good to know that the choice is still good. The two facilities that we used in the past are no longer doing it. - Dima
Re: [ccp4bb] Off topic - transformation problems
In this case, pGEX4T3 vector also expressed inserts constitutively. http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htmhttp://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm Not quite. pGEX vectors carry a copy of lacI^q, resulting in very low leak expression from PTAC promoter. Definitely lower than the relatively leaky T7 promoter in pET20, which does not express laqI^q. Switching to a plasmid from higher pET series that do have laqI^q (e.g. pET31) should reduce the leakiness. I also thought that target protein may have tocicity on host strain. But, why pGEX4T3-target protein was not show the same phenomena. Now, we are trying to change the host BL21(DE3) to Rosetta(DE3) and BL21(DE3)pLysS. pLysS should help with repression under non-inducing conditions. If it is true toxicity issue, you might need to switch to plates with synthetic medium that does not contain any traces of lactose. - Dima
Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
The method is that by Edelhoch, mentioned a couple of times already in this discussion. You recommended determining extinction coefficients experimentally. How is plugging number of specific residues into a formula constitute experimental determination? It's also described in the paper by Pace et al., the same paper that the formula in ProtParam is from (ProtParam does not use the values determined by Gill von Hippel). Last time I looked into this, the consensus was that the Edelhoch method is the most accurate method for protein concentration determination; more accurate than dry-weighing plus N-terminal sequencing, etc. Nothing beats quantitative amino acid determination but it's one technique that requires specialized lab and good standartization. By now, it's almost a lost art. I agree with Vaheh that absolute protein concentration is not critical in crystallization but let's not forget that the protein concentration enters, one way or another, into measurement of virtually every biochemical constant. 50-100% errors there can be extremely important. - Dima
Re: [ccp4bb] methods to capture proteins from cell culture medium
I thought about HIC too, but do not know if it would work since the binding of protein to HIC need high salt conc. and I am not sure if the salt conc. in the sf900 or Hi5 medium is high enough (the formulation is secret, LOL), thus it is good to know that someone has succesful experience with HIC. Few things: 1. With regard to the salt/ionic strength, the formulation of serum-free media cannot be far off from the traditional media. Very crudely speaking, figure an equivalent of ~200 mM NaCl. So most protein won't bind to, say, phenyl sepharose under these conditions. But your protein might be in the minority - who knows? 2. What prevents you from adding extra salt to the collected medium? Say, ~1 M ammonium sulphate final? There probably will be some precipitate forming which can be filtered away before loading onto a HIC column. 3. Hydroxylapatite. Ceramic Type I version from Bio-Rad in particular. Large size beads packed into a wide column. A great way to concentrate total protein at high flow rates. Phosphate concentration in the medium is low enough that majority of proteins will sill bind. - Dima
Re: [ccp4bb] immobilized DNA resin
heparin sulfate has the highest negative charge density of any known biological molecule. Seems to me that phytic acid (IP6, C6H6-(H2P04)6) and inositol heptakisphosphate (IP7) should have higher charge per mass and per volume. - Dima
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
It is not surprising that your bradford and BCA assays don't agree if you have no aromatic amino acids in your protein. Bradford dye binds to hydrophobic residues, mainly aromatics, so I would guess your bradford is consistantly giving lower measurements than the BCA assay. I also wouldn't be surprised if the results of your Bradford vary significantly between replicates. The BCA assay reagent interacts with the backbone amides, not with any sidechains, so I would tend to believe that measurement more than anything else you have done. I work with a protein that has very few hydrophobics (only one aromatic - a Phe) and I have found that Bradfords are unreliable, but the BCA assay tends to be consistent. Bradford reagent is colloidal Coomassie G-250 and it's binding to proteins is very complex, depending on local structure, hydrophobic interaction and basic charges (mainly Arg residues). So yes, it is quite variable protein to protein but it is not a simple function of aromatics. - Dima
Re: [ccp4bb] S-200 buffer-based peak shift?
At 07:23 PM 3/22/2011, Jacob Keller wrote: Dear Crystallographers, I have run my protein-peptide complex several times on a GE S200 10/300 in buffer A (below). Today, to make a crystallization stock, I ran the sample in buffer B, and the peak shifted from a consistent 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that SEC results change as a result of buffer conditions. Could this drastic a shift be due simply to buffer conditions, or could there actually be some buffer/ion-dependent dimerization going on? Anyone have a similar experience? A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) So, it elutes earlier in essentially zero salt. I would bet that the protein is acidic and what you see is a buffer effect. Superdex (and most other gel filtration matrices) carries residual negative charge. So in zero salt there will be repulsion between protein and beads, resulting in the protein entering pore less frequently. Hence the earlier elution. I've seen this effect for a couple of monomeric acidic proteins. Chances are, switching to a salt higher than 50 mM will also retard the elution a bit. Typical recommended salt in gel filtration is in 100-200 mM range precisely to suppress ionic interactions. - Dima
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
they buy it from some small company, idea being the applicability to SLS, optimized for minimized bleeding/sheding or material from the column, which will show up only in the light scattering detector. I don't know. Every single spec of Wyatts columns looks exactly the same as Bio SEC-5 series of columns available from Agilent. Dima
Re: [ccp4bb] E. coli mutant strains
It surely is not. An N-end rule has to do with ubiquitination, and it is absent in E.coli. Not true. There is indeed and N-end rule in prokaryotes, including E. coli. Mediated by the ClpP protease-based system. See: http://www.cell.com/molecular-cell/abstract/S1097-2765%2808%2900692-8 Ooops! I dodn't know that. Live and learn. Thanks, Miguel! Dima
Re: [ccp4bb] Density sharpening with Truncate?
At 05:39 PM 2/25/2011, Pete Meyer wrote: Or could anyone suggest a program that would be of help? CAD scaling with a scale factor of 1.0 and negative B-factor (isotropic or anisotropic) should do the trick. I haven't had much luck with density sharpening (at least at ~4-5 Angstroms), but others have apparently had some success with it. Alternatively, CCP4i task Run FFT does the job: 1. Take MTZ from Refmac output 2. Run FFT to create simple map with SigmaA-weighted phases (i.e., PHWT label). 3. In Infrequently used options, Apply B-factor scaling to F1, specify negative B-factor scaling value, usually within -10 to -50. - Dima
Re: [ccp4bb] E. coli mutant strains
Recently I am expressing one protein in BL21(DE3) and the protein undergoes N-terminal degradation. I am trying to keep this crucial N-terminal tail on the protein, which has MRS at the first 3 positions. Digging in to the literatures, I found the N-end rule, which tell that the proteins bearing N-terminal Arg or lys have much shorter half life. I am not sure whether this is my problem. It surely is not. An N-end rule has to do with ubiquitination, and it is absent in E.coli. However, I want to found one E. coli mutant strain that lacks aat and is unable to degrade N-end rule substrates that bear N-terminal Arg or Lys. Don't think such a strain exists and totally not sure this is really your problem. (Also, what's AAT has to do with it? Is this your requirement for something else?) Try inhibiting proteases better. E.g., metal-dependent proteases are a common problem with IMAC-based purification. First thing is to find whether the degradation occurs inside the cells or during purification step(s). If the former, why not fuse the thing to an N-terminal tag, thus only purifying non-degraded N-termini? (Assuming a monomer, of course). If N-term. has to be native, His-tagged SUMO fusion cleaved with SUMO protease will leave no non-native residues (assuming none was introduced during cloning, which is rather trivial to ensure these days). - Dima
Re: [ccp4bb] off-topic: tag removal
I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? C-terminal intein fusion is a self-cleavable tag that cuts cleanly. NEB sells kit for intein-chitin binding domain fusion for affinity purification but of course you can add any other tag of your liking. - Dima
Re: [ccp4bb] off-topic: tag removal
Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, et al. 2009 Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag. PLoS ONE 4(12): e8119. doi:10.1371/journal.pone.0008119 Unless the protein in question happen to be Leu as a C-terminal residue, this tag won't give wild type C-terminus after cleavage. - Dima
Re: [ccp4bb] Could someone can help me to explain why EDTA-2Na can formate salt crystals
No, there are not any other cations, so I feel very strange. Everything brought from sigma. Nothing's strange. EDTA is very poorly soluble at pH 4.2 and would become even less soluble in the presence of 47% PEG. So it crystallizes. Dima On Tue, Feb 22, 2011 at 8:58 PM, William Scott mailto:wgsc...@ucsc.eduwgsc...@ucsc.edu wrote: What other cations are present? Any divalent cations like Mg++ or Ca++? The Ksp of magnesium phosphate is about 10^-24, so even if you have a very small amount present, say as a contaminant with citrate or EDTA, it will crystallize. On Feb 21, 2011, at 1:22 PM, Yibin Lin wrote: Dear all, I got a lot of salt crystals in reservior solution (well solution), which contains 0.1 M phosphate/citrate ph 4.2, PEG200 47%, EDTA-2Na 0-22mM. Reservior solution appears crystals from 12mM EDTA. Could someone help me to explain why? Thank you very much! Yibin William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/http://chemistry.ucsc.edu/~wgscott/
Re: [ccp4bb] SDS and IMAC
In my experience, either urea or guanidinium crashes out in gels. I can't remember--which one is it? I am thinking guanidinium. (If the answer to this email saves one grad student from the aggravation of such a phenomenon, it will have been worth it...) It's GuHCl and what crashes is dodecyl sulfate salts. Urea is fine (recall that many gels are run with urea in them and soem loading buffers contain urea). High salts is bad for gels but they are easy to remove from the samples by methanol/chloroform precipitation: http://www.abrf.org/ResearchGroups/EdmanSequencing/EPosters/ABRFhand1.doc (With low protein amounts, I'd suggest modifying this protocol by increasing centrifugation times: 10 min in the interphase forming step and 5-10 min in the last step of pelleting precipitated protein). Dima
Re: [ccp4bb] Mg2+ or water
Sorry, the attachment is in here. Doesn't look like Mg2+ at all. Distances are too long, Mg is never coordinated by amides and if it were Mg you would have seen waters around it. Looks like tightly bound water to me. - Dima On Mon, Dec 20, 2010 at 4:16 PM, jlliu liu mailto:jlliu20022...@gmail.comjlliu20022...@gmail.com wrote: Hi All, I am refining a structure and encountered a problem of modeling a difference density as water or Mg2+, and would like to hear opinions from the community. It has the following coordinations (attached): the water/Mg2+ forms salt bridge/H-bonding interaction with a carboxylate group from the ligand, it also forms salt bridge/H-bonding interaction with a Glu residue from the protein, it is also within hydrogen bonding distance to the main chain N of another protein residue. In provious publication, it was modelled as a Mg2+ and the author reasoned the dual salt-bridge stabilizes the liganding binding, also the Mg2+ is present in the protein solution for crystallization. For my case, I have no Mg2+ present in the protein buffer, also modelling it with water refines perfectly with no indication of positive difference density even at 2.0 sigma cut off. Should I modelled this density as water or as Mg2+. Your opinions are appreciated. JL Content-type: image/png; name=367-mgtest.png Content-disposition: attachment; filename=367-mgtest.png X-Attachment-Id: f_ghy0k5e31
Re: [ccp4bb] hydrohyapatite column
I was wondering if any of you would be kind enough to share her/his experience with me, and would suggest vendors and models for such columns. I really like ceramic hydroxyapatite from Bio-Rad. The only type that behaves in the columns long-term, without the need for repacking. It also gives better resolution and/or allows faster faster flow rates. Their type I has higher capacity and generally more useful. The great thing about hydrohyxapatite is that essentially all proteins bind to it as long as there is no phosphate around even in high salt (like 500 mM NaCl). So it is a great way to concentrate proteins from diluted solutions or a nice chromatography step right after AEX. Some proteins (generally few, mostly alkaline) will elute at very high NaCl (1-2 M), others with require phosphate. For ceramic hydroxyapatite I find that the useful range is 0-100 mM phosphate at pH ~ 7.0. Capacity of the ceramic hydroxyapatite is lower than the normal mineral form, only around 25 mg/ml. Dima
Re: [ccp4bb] Crystal gel band
I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is 1uG? Protein to protein variation aside (which is usually within 2X), conventional Coomassie R-250 staining gives very-easy-no-doubt detection down to ~0.1 ug. 50 ng is still visible (assuming that the gel is any good). Colloidal Coomassie G-250 (Bradford) with destaining extends the range down to 30-20 ng or 10 ng with some effort and special staining solution. Proper silver staining should easily see 1 ng. I.e., a single crystal that is a 50 um cube with 50% solvent puts you in the borderline zone of easy. Load many of them to be sure or use silver. - Dima
Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
What about the possibility of double-blind review? I have actually wondered why the reviewers should be given the author info--does that determine the quality of the work? Am I missing some obvious reason why reviewers should know who the authors are? I've always felt (and advocated long time ago on Usenet) that the current review system gets everything exactly backwards. 1) To prevent hatchet jobs of a review, reviewers should not be anonymous. 2) To prevent systematic bias by things completely irrelevant to the review job, reviewers (and handling editors!) should not be given authors' names and institutions. I think that the moment #1 happens, each review will start taking much, much longer time than it is now. This means that either a lot less would ever be reviewed and published or -oh horror- postdocs and graduate students would need to be reviewers, too. IMHO, both outcomes are perfectly acceptable. #2 is difficult in practice because self-references and all kind of hints can always be planted to make sure everyone knows the names. But maybe if such advertisements are frowed upon by the community, their incidence will be low enough to be a problem? -- Dima
Re: [ccp4bb] Quality of His-Select Resin After Regeneration
The Agarose superflow material is I think the same for Talon (Clontech) and Qiagen, not sure about Machery Nagel or Sigma's product. Superflow agarose is cross-linked differently than normal sepharose/cross-linked agarose. The nature of chelates and their attachments also differ. Qiagen, Sigma and Pharmacia use NTA while Clontech (Talon) uses carboxymethyl aspartate. Qiagen uses original chemistry through lysine spacer, Sigma's synthetic route is through bis-carboxymethyl cysteine (or -cystine), and Pharmacia opted for a clean but complex chemistry that results in a very hydrophilic spacer and higher degree of derivatization. Dima
Re: [ccp4bb] Off-topic: crystallization after refolding
However, treatment with urea is known to modify proteins (N-term/Lys/Arg), which could ultimately effect crystallization. Is this something that people generally worry about? For example -would you bother cleaning up the urea by ion exchange -get ultra pure urea (ultra $$$) -change to guanidinium chloride -hope that it might benefit crystallization (i.e. similar to methylation of lysines) Have ~ 50 mM glycine or tris in your urea solution and prepare it fresh. Then you don't have to worry about carbamylation. Dima
Re: [ccp4bb] insect cell media
We have happily made a transition last year from using Invitrogen's SFM medium and cellfectin to Insect-Xpress (Lonza) and polyethyleneimine for transfection. We are moving several protein targets to large-scale cultures and would consider cost-cutting alternatives. For example, Invitrogen and Thermo Sci both offer media in powder form at an attractive price. Has anyone made a systematic comparison of these media? Any particular recommendations regarding powder media? Last I checked, no manufacturer offered serum-free powedered medium. Fetal calf serum is very expensive - once you count in its cost, the overall price of the media becomes comparable. If your protein is secreted, serum-containing media are not even a realistic choice. If it is intracellular, buying dry medium and adding FBS work well: is a lot more hassle, is about 20% cheaper, and usually gives slightly higher titers and expression levels. Of all the common media we compared, TNM-FH works best for us with Sf9 cells. Switching to dry medium also means some initial investment: bottles, filtration equipment and filters. So it all pays off only of you need large scale cultures long-term. If you are aware of a dry serum-free insect cells medium, please let me know - I'd love to try it. Dima
Re: [ccp4bb] Phosphatase, which is the best?
I need to dephosphorilate blunt ends and, surfing on internet I found this two enzymes: - Shrimp Alkaline Phosphatase (SAP) - Calf Intestinal Alkaline Phosphatase (CIP) Have you any experience about these phosphatases, I mean wich is the best for blunt ends? Either will do dephosphorylation part just fine. SAP is A LOT easier to kill, so other things being equal, go with it. That said, more frequently than not, using phosphatases creates more problems than it solves (precisely because of the incomplete kill). If it is blunt ligation you are thinking of, better way of doing is to use huge excess of insert (say, 30-fold). Works fine. Also worth mentioning: In one special case blunt ligation is a piece of cake. If it is a single blunt site you are cloning into, chances are that ligation of your insert will destroy this restriction site. If you are lucky and your insert does not have the same site then do this: ligate in the presence of large excess of insert, heat kill ligase, digest the reaction with that blunt cutter, transform. Since linear plasmid is 1,000-fold less transforming in E.coli, essentially none of your clones will contain self-ligated backbone. Dima
Re: [ccp4bb] FW: pdb-l: Retraction of 12 Structures....
It could also be that the high impact factor of these journals, and their 'tabloid' character ensures that they are read by more people than other journals. So any bad data or fraud that gets published in Nature, Cell or Science is more likely to get noticed and talked about, than something that appears in smaller impact journals. And even some very big names aren't spared, which is a good thing. This one, for example http://www.nature.com/nature/journal/v452/n7183/full/nature06819.html The first author of this paper (the one who provided all figures and data that are inconsistent with the original data) has never admitted anything, continues to publish and hold tenure-track position. Ganesh On Fri, 11 Dec 2009 10:48:41 +0100, Vellieux Frederic frederic.velli...@ibs.fr wrote: Hi all, Like everyone else, I was appalled. My two cents worth: Nature and Science are not scientific journals in the strict sense of the term. They are more like magazines (I won't go all the way to say tabloids), and as such will do anything to publish what seems to be hot. And will reject very good scientific papers. So it's not a surprise that retractions affect magazines such as Science and Nature. Fred. -- What is true for E. coli is also true for an elephant - Jacques Monod.
Re: [ccp4bb] Solubilization buffer
We use 8M urea solubilization buffer for our protein in inclusion bodies and recommended temperature is 10-15C. but in 8M conc the urea does not dissolve and is in crystalline form only, will it have any effect on solubilzation efficiency. Our solubilization time is 1 Hr and after that we centrifuge and use the supernatant for refolding via dialysis. however the pellet after centrifugation of solubilzation show presence of our protein on sds page analysis. what should we do so that the process of solubilization is complete and our protein is not lost in pellet. Few things come to mind: 1. There is not much difference betweeen 15 and 20C. 8M urea is certainly soluble at 20C (in fact, it takes quite a bit of time for it to start crystallizing even on ice; nucleation problems, apparently). 2. Chances are good that you can use as high temperature as you need to dissolve. I've come across protocols using =65C in the presence of 100 mM bME to ensure complete unfolding of the polypeptide and reduction of disulfides (seems essential for refolding of some proteins and very detrimental for others). 3. You may need to increase solubilization solution/inclusion body ratio. 4. Some inclusion bodies simply won't dissolve in 8M urea, period. In those cases the typical choices are GuHCl at 6-8M or high pH (11-12.5 depending on protein). Good luck, Dima
Re: [ccp4bb] Lipid Removal from Proteins
I dug around on the net and found this method to remove lipids from proteins: More precisely, from denatured proteins. That's what methanol/chloroform phase does for most proteins. Wessel Fluegge (1984), Anal. Biochem. 138:141-143. Itґs a methanol/ chloroform precipitation and gives you a pellet that is easily redissolved. The method was especially devised for removing lipids or detergents, so it should be perfect for you. -- http://www.bio.net/bionet/mm/methods/1996-December/052513.html By far the best method of concentration/desalting/de-lipidizing proteins for SDS gels. I've used it extensively over the years. Even then, the efficiency of precipitation drops off very significantly for most small proteins at low [protein]. Is this still the preferred way? I do not want to use reagents that are *themselves* likely to denature my protein. Has anyone tried cyclodextrins? Lots of people did. They work. So if you have protein that you can easily immobilize, washing the matrix extensively with b-cyclodextrin will do the trick. But immobilized cyclodextrins are not readily available for reasonable price. So for untagged protein your next bet would be various detergent removal sorbents available from Calbiochem, Pierce, Bio-Rad and likely many others. All of these WILL bind your protein to various extent, but usually not completely because they are also work as size exclusion. I'm specifically trying to strip sarcosyl. I want to do it completely. What's the definition of completely? If you are lucky and your protein binds to cation exchangers, simply washing the column with 20 CV of low salt buffer (even better with non-denaturing concentrations of alcohols or glycols) usually will decrease sarcosyl concentration by ~ 100X. Pretty much the same if your his-tagged protein is bound to IMAC sorbent. - Dima
Re: [ccp4bb] DNA binding protein
I had a simple question about DNA binding protein. Is there an easy way to detect if your heterologously expressed protein is bound to DNA post purification. Yes. UV absorbance. DNA absorbs UV strongly, proteins do not. DNA absorbs 260 more thn 280, the opposite is true for proteins. In fact, it's always a good idea to check 260/280 ratio because quite frequently even non-DNA binding proteins purified by a single tag affinity chromatography contain enough DNA to skew estimation of protein concentration from 280 nm absorbance by several fold (9X in one case of my personal experience). Also is there an easy way to strip the protein of DNA without any damage done to the protein in doing so. No generic solution. Greatly depends on the protein and its properties. Dima
Re: [ccp4bb] Best 3D stereo combination
This chart: http://www.nvidia.com/object/IO_11761.html combined with the CCP4 wiki's advice (that the card should say stereo under display connectors) imply that we need to spend at least $760 on the video card alone. Is that really true??? EEK!! Not nesessarily. Most GeForce cards can be soft-hacked with RivaTuner into thinking they are Quadro and using the Quadro drivers - thus providing proper stereo support. http://www.techarp.com/showarticle.aspx?artno=539pgno=0 http://nvworld.ru/downloads/rivatuner.zip Dima
Re: [ccp4bb] TEV nucleotude sequence with restriction site
Does anybody have a TEV-protease-site-coding nucleotide sequence with a commonly-used restriction site in it, preferably right at the end? Alternatively, does some somebody know of a program to determine all equivalent codon permutations for a small coding region, filtered for resulting restriction site possibilities? It seems like it would be an easy enough script to write... Our main vectors (His- and His-MBP cleavable fusions) use blunt site right after TEV site. This way, you are not dependent on whether the same site is present in your gene and the cloning can be done without introducing any artefacts (besides Gly leftover). Here is the sequence: AGTGCCTGTACAAGGCCT AGGCCT is a StuI blunt cutter site (cheap and good enzyme) and GGC is a Gly So your insert is typically a PCR product that supplies the last letter in the Gly codon and you have a complete freedom of what follows. Blunt/sticky directional ligation, if done right, is extremely efficient and you only need to cut PCR product with one enzyme (no need to phosphorylate primers, the single non-ligatable joint gets repaired in E.coli). But then, we have by now almost completely switched to QuickChange cloning - clone anything anywhere anytime completely artefact-free (at least as long as the final plasmid is not larger than ~ 9 kbp). Dima
Re: [ccp4bb] issues with TLS refinement
The issue is: While running tls and restrained refinement, the program doesn't perform the given no. of tls refinements. For e.g., if you ask it to perform 10 TLS and 5 restrained refinement rounds, it'll run may be 2 or 3 rounds of TLS and then jump to restrained refinement rounds and finish the job. It is, however, finishing the given no. of restrained refinements every-time. There is no error message or any indication of abrupt ending to tls refinements in the log file either. FWIIW, I am getting the same thing. Sometimes no TLS refinement is done at all, sometimes the program does 2-3 cycles and sometimes the requested number of cycles. Seems to be PDB input-dependent but I could never find what causes the behavior. Dima
Re: [ccp4bb] CCP4BB Digest - 4 May 2009 to 5 May 2009 (#2009-124)
I incubated my pelets in 8M urea for a couple of hrs at 37C, spun and collected the supernatant, and then refolded the protein over a Ni column, as I had a his tag protein. To do this, you load and wash the protein normally, but before eluting, run b-cyclodextrin (i think 0.1%, you'll have to try a few concentrations) and then a detergent (I used Triton X-100). Then you can elute. Here's the paper I used as a guide: Biotechnol Appl Biochem 2006, 43:137-145 Except it's the other way around - the column is washed in the presense of detergent which is then slowly removed by b-cyclodextrin washes. If the reversal thing worked for you, your protein likely does not require neither b-cyclodextrin nor Triton for refolding. I've had more luck in on-column refolding by steps of decreasing urea concentrations: 2, 1, 0.5, 0 M (3-5 column volumes each with 30 min in between at room temperature). Dima
Re: [ccp4bb] IPTG induced protein expression
I was wondering if anyone has used regular IPTG (not IPTG-dioxane free, special grade) for protein expression. Are there any problems using such regular samples (5mg dioxane per Kg of IPTG) for protein expression? I have. No problem whatsoever. I belive the whole dioxane-free thing is about dioxane being carcinogen and has nothing to do with minute amounts of it affecting E.coli in any way. The amounts of dioxane in regular grade IPTG is nothing in comparison to the carcinogenicity of the heavy metals we use for phasing (or even cigarette smoking). Dima
Re: [ccp4bb] Off-topic: chemical modification on thiol groups
I am looking for a reagent (and vendor) that will irreversible put a raher bulky substituent on a free SH group and that does not react with free amines (or other potential reactive groups present on a protein surface). The connection with crystallography is that it is required for an experiment asked by a referee necessary to confirms or reject a hypothesis that results from a crystal structure. For this experiment it is essential that the reaction is not reversible (so no S-S bond formation). Any maleimide-based fluorescent dye will be bulky, irreversible and when done right will only go for thiols. Also easy to quantify extent of modification. Dima Remy Loris Vrije Universiteit Brussel
Re: [ccp4bb] Replacement for arp_waters?
Donnie Berkholz wrote: One of the major reasons our lab started using Coot instead of O is that you didn't have to memorize or look up a million obscure functions to do everything. It was in the Coot GUI. When we're training new people, this helps immensely for the 99% of them who have never used Linux or any type of command line before. If there isn't a GUI for it, it effectively doesn't exist for them. That's right. When there are 20 programs with 20 command line switches on average in each, there is no way an average user can memorize it all and use them to their full capabilities. GUI is the only way for many programs being used efficiently by many users. Command line and scripting are great for automation and programming but that's no what an average user normally does or want to do. Dima
[ccp4bb] Refmac and MSE?
Hello, I am at a loss on what's going on: I am refining SeMET containing structure and using REFMAC 5.2.0005 on Linux and, the same thing happening, using REFMAC 5.5.0070 on Windows. When MET were modelled, there were no difference peaks anywhere. When I changed them all to MSE, the large difference density peaks showed up. So either the protein does not contain SeMet or Refmac somehow uses sulfur scattering factors during refinement. I have hard time believing the former because 1) the protein was checked by mass spec to be correct size for SeMet derivative, 2) the structure was solved by SAD with all five sites correctly found (196 residues total), 3) first 50 aa of the protein are identical to a known structure. This is 2.4A resolution and at this point R/Rfree = 25/29. Refmac log shows correct scattering factors read out: SE17.0006 2.4098 5.8196 0.2726 3.9731 15.2372 4.3543 43.8163 2.8409 The PDB has this for MSE: ATOM 1140 N MSE A 143 35.708 161.163 13.715 1.00 78.82 N ATOM 1141 CA MSE A 143 35.995 162.467 13.106 1.00 77.86 C ATOM 1142 CB MSE A 143 36.307 162.307 11.617 1.00 78.79 C ATOM 1143 CG MSE A 143 37.755 162.127 11.119 1.00 80.89 C ATOM 1144 SE MSE A 143 37.503 161.104 9.363 1.00 91.22 SE ATOM 1145 CE MSE A 143 39.169 160.021 9.696 1.00 86.47 C ATOM 1146 C MSE A 143 34.709 163.226 13.030 1.00 77.70 C ATOM 1147 O MSE A 143 34.682 164.436 12.737 1.00 77.33 O Any clues greatly appreciated! Dima
[ccp4bb] Correction: Refmac and MSE?
In the previous message I forgot to mention: When MET were modelled, there were no difference peaks anywhere. When I changed them all to MSE, the large difference density peaks showed up. Large *negative* difference density.
Re: [ccp4bb] 3D modeling program
Having a generic dictionary definition is nice and dandy. However, in the present context, the term 'homology' has a much more specific meaning: it pertains to the having (or not) of a common ancestor. Thus, it is a binary concept. (*) But how do we establish phylogeny? - Based on simple similarity! (Structural/morphological in early days and largely on sequence identity today). It's clearly a circular logic: Lets not use generic definition; instead, lets use a specialized definition; and lets not notice that the specialized definition wholly depends on a system that is built using the generic definition to begin with. Plus, presumably all living things trace their ancestry to the primordial soup - so the presence or a lack of ancestry is just a matter of how deeply one is willing to look. In other words, it's nice and dandy to have theoretical binary concept but in practice it is just as fuzzy as anything else. IMHO, the phylogenetic concept of homology in biology does not buy you much of anything useful. It seems to be just a leftover from pre-Darwinian days - redefined since but still lacking solid foundation. Dima
Re: [ccp4bb] 3D modeling program
But how do we establish phylogeny? - Based on simple similarity! ah! the old rhetorical trick of changing the problem or question a posteriori! all i pointed out was that things can't be 25% homologous Well, you were right that in today's definition things can't be. But you seem to be missing my point that today's definition is essentially meaningless (relies on circular logic and has no epistemologic value) and that nothing would be lost if the term reverted to its generic usage, similar. There would still be a question to be asked similar for what reason? - same question that is presumed to be answered whenever one invokes phylogeny-based homology. i'm glad your opinion is humble here, because it has much to be humble about :-) do you really think that property (e.g., structure and function) prediction is not useful? and i can't even begin to understand how you can think that 'homology' in its present-day meaning is a pre-darwinian concept. Homology is a pre-Darwinian concept that was *redefined* post-Darwin. That's what I wrote. okay, so can we all agree now that we won't be saying and writing things like the two proteins are X% homologous anymore from now on? IMHO, it truly does not matter if we do or do not as long as we understand each other. Like I wrote in the original reply, paying too much attention to definitions of fuzzy abstract concepts is not worth it. Dima
Re: [ccp4bb] 3D modeling program
But how do we establish phylogeny? - Based on simple similarity! (Structural/morphological in early days and largely on sequence identity today). It's clearly a circular logic: Hardly. Two sequences can be similar and non-homologous at all levels. Also, two similar proteins can be homologous at one level but not at another. It's also possible for two proteins that have no detectable similarity above random sequences to be homologous. Hence there is no circularity. Of course there is. Just how do you establish that the two are not homologous? - By finding that they don't belong to the same branch. And how do you decide what constitutes the same branch? - By looking at how similar things are! Plus, presumably all living things trace their ancestry to the primordial soup - so the presence or a lack of ancestry is just a matter of how deeply one is willing to look. This is also wrong. Even if all organisms trace back to one common ancestor, that does not mean all proteins are homologous. New protein coding genes can and do arise independently, and hence they are not homologous to any other existing proteins. Just how do they arise independently? Would that be independent of DNA sequence? And if not, then why can't shared ancestry of the DNA sequence fully qualify for homology? You also ignore the levels of homology concept -- just because two proteins are homologous at one level does not mean they are homologous at others. For example, consider these three TIM barrel proteins: human IMPDH, hamster IMPDH, and chicken triose phosphate isomerase. They are all three homologous as TIM barrels. However, they are not all homologous as dehydrogenases -- only the human and hamster proteins are homologous as dehydrogenases. ... And all that is concluded based on sequence similarities [of other proteins/DNAs] to construct phylogenetic tree. So, ultimately, homology ~ similarity. The generic concept of homology used to be used as a proof of evolution. Today, things seem to be reversed and evolution is being used to infer homology. A useful concept turned into a statement with little or no utility. Dima
Re: [ccp4bb] Quikchange cloning: Insert length
I am curious to hear what is the longest insert anyone has cloned using a modification of the Quikchange cloning strategy. Basically, ligation-independent cloning by strapping on homologous regions of the vector onto the primers which also generate the initial PCR product. I plan to proceed with my insert which is ~ 2kb and am curious to get some feedback if you have successfully cloned inserts 1.5kb using the above strategy. Here, the maximum we've tried was 3 kbp and it worked just as well as with the the smaller fragments. Most of the Quickchange cloning we did involved 1500-300 bp and no obvious differences in success rate vs size come to mind. Dima
Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags
Another theory is that trace amounts of Ni may leech off the column during purification and coordinate with multiple His-tags on the pure protein, causing them to aggregate. For sure. We had a case where a protein would precipitate after post-Ni-NTA dialysis. The precipitate would go back into solution if resuspended in the presence of imidazole or EDTA/EGTA. Inclusion of a bit of EDTA into fractions and dialysis solution completely eliminated precipitation. Dima
Re: [ccp4bb] off-topic: selective reduction of surface cysteine
We have a recombinant secreted glycoprotein produced in a mammalian culture system; the native protein has 12 cysteines which form 6 intramolecular disulfide bonds. We have introduced a new cysteine residue at a surface position, with the intention of targeting this residue for an in vitro site-directed chemical modification. The mutant protein is well-expressed and soluble, but while we do see some monomer, non-reducing SDS-PAGE shows that a substantial proportion of it is probably in a homodimeric form (we suspect dimerization through intermolecular disulfide formation), Did you inhibit S-S bond formation after addition of SDS? Lots of proteins form dimers when unfolded without presence of reducing agent. Adding 20 mM NEM into SDS-PAGE loading buffer is easiest way to prevent this. Assuming that you did this and you actually see intermolecular bond, you can play with reducing agent(s) concentrations. Intramolecular bonds require much higher concentration of reducing agents (e.g., IgG is perfectly happy and not reduced at 1 mM DTT at room temperature). Dima
Re: [ccp4bb] AW: [ccp4bb] hi / cloning stuff
I found out that my glycerol stocks were not alright any more and I had no plasmid backups (do it!!!). So I had to clone, again. Hint for the future: Dead glycerol stocks, even dead (say, a year old) clone on a plate, still contain your plasmid. Do a regular miniprep with it, transform - 99% of clones will contain your plasmid. Dima But in this case I used a cloning kit from Qiagen (I have no commercial interests here). I got the insert into the cloning vector from Qiagen very easily. Then I was able to amplify it as much as I wanted and the ligation procedure was not so dependent from the yield of the PCR reaction (high molar access of insert compared to the cleaved vector) . Also in this case, cloning into the expresson vector was not completely trivial (maybe 1-1.5 months) but much easier than without this kit. So, it might be an idea to use a kit that you can amplify your insert as much as you want. I know that these kits a pricy but your time is also expensive and you also use/waste a lot of chemicals for unsuccessful trials. After I finished my thesis, another PhD student started to work with these vectors and tried it for more than one year and then gave it up! So this vector really seems to be a tough one. Thats why if nothing works, you might take another vector into consideration. But before that you should check buffers, ligase, restriction enzymes etc. and do in silico cloning. Another idea would be do to de/phosphorylation (see textbooks). Good luck! Jan --- vijay srivastava [EMAIL PROTECTED] schrieb am Sa, 30.8.2008: Von: vijay srivastava [EMAIL PROTECTED] Betreff: [ccp4bb] hi An: CCP4BB@JISCMAIL.AC.UK Datum: Samstag, 30. August 2008, 13:04 hi i am facing problem in cloning,getting my insert and vector at the correct position after digestion but after ligation colony is not coming and if how it is coming than i am not getting my insert Unlimited freedom, unlimited storage. http://in.rd.yahoo.com/tagline_mail_2/*http://help.yahoo.com/l/in/yahoo/mail/yahoomail/tools/tools-08.html/Get it now __ Do You Yahoo!? Sie sind Spam leid? Yahoo! Mail verfГјgt Гјber einen herausragenden Schutz gegen Massenmails. http://mail.yahoo.com
Re: [ccp4bb] agarose-acrylamide composite gels
Sorry for the off-topic question. I am trying to separate by SDS-PAGE really big proteins (500 kDa), and lower percentage (3-8%) acrylamide gels do not do the trick. Based on literature searches, acrylamide-agarose composite gels seem the way to go. Is anyone willing to share a protocol? I cannot get hold of any of the old journals where this was described at our rather limited library... There is no need for a protocol. You just make conventional mix but add low melting agarose stock instead of water and keep everything at 35-37C before adding APS/TEMED. Stacker can be 0% AA/0.5% normal agarose (60C at pouring). Unless you must resolve high and low MW on the same gel, I wouldn't bother with a gradient, just find AA% that works (3% AA with acrylamide:bisacrylamide at 20:1 would be my first guess in your case). Run on the cold room with a tank filled to the top or use circulated water cooling. Dima Dima
Re: [ccp4bb] Expression vector with NdeI-ClaI sites
...Or do the slightly-less-new-but-not-quite-old school technique: QuickChange. Find a vector with roughly the properties you want, and just quickchange mutagenize in NdeI and ClaI sites at the desired positions. Works like a charm for me, assuming the vector is 10Kb or so, and I typically buy the various components from the cheapest suppliers rather than the er... *cough* $$$tratagene kit.* Or, if you go the QuickChange route, might as well do the entire cloning using it! We now routinely clone in the whole genes using it: PCR your gene with primers that anneal to the plasmid, use PCR product in place of QuickChange primers. That's all. Works like a charm. I was very excited when I invented it couple years ago - only to discover that I invented a wheel - the technique (with few variations) has been published three times already (and no one paper cited another; can find refs. and give my protocol if interested). This way, just like with SLIC, you can put anything into any position on the plasmid but, IMHO, with much less fuss - no fiddling with vector besides miniprep and the only enzymes used throughout are polymerase and DpnI. Dima
Re: [ccp4bb] Concentrating protein
Nathan Cowieson: I have found that ion exchange is a good way to concentrate protein. If there is no reason that you can't pass the whole 450 mls through the column then you should do that. Another way is to load dilute protein onto small hydroxylapatite column. Provided that the buffer does not have phosphates, almost all proteins bind to hydroxylapatite regardless of the salt (at least up to 0.5 M). The obvious advantages is that one does not need to dilute and that for most proteins elution can be done with only 50-100 mM Pi. The disadvantage in comparison to ion exchangers is about 4-fold lower - usually, around 20-30 mg/ml of the sorbent. Dima
Re: [ccp4bb] Structural importance of ordered water?
Direct hydrogen bonds between sidechains are obviously important to structural stability in proteins. From time to time I see cases of water-mediated bonds in which a single water molecule seems to play an important role (sometimes taking the place of a missing ligand atom in an apo structure, for example). But what about larger chains and networks of water? Assuming a structure is high enough in resolution and well-ordered enough to observe such things, has anyone systematically studied the structural importance of multiple water interactions (I do know of a paper by Faerman and Karplus back in 94, but perhaps there is more recent work). Actin gives an example of such. It has two large domains with a metal-nucleotide binding cleft between them. Without the metal-nucleotide the protein is unstable and slowly and irreversibly denatures. When a water network coming out from the Me-nucleotide is analysed (I did it manually), the reason becomes very clear - secondary and tertiary hydration shells provide a web of interactions that ultimately join the two large domains, thus, presumably, providing the stability. Dima
Re: [ccp4bb] 3D model building without CRT monitor
Vangelis Christodoulou wrote: Philips is offering an interesting solution: http://www.business-sites.philips.com/3dsolutions/products/3dscreens/index.htmlhttp://www.business-sites.philips.com/3dsolutions/products/3dscreens/index.html Interesting! Their PDF says it works with OpenGL: http://www.business-sites.philips.com/assets/Downloadablefile//2006-05-30-Technology-backgrounder-13560.pdf Does it mean it will work in Coot as is? Dima
Re: [ccp4bb] insect expression system
I searched my mail box for possible answers you have given but couldn't find any. The question is about selecting insect expression systems for mammalian or viral glycosylated proteins. There is confirmed expression of the target proteins in mammalian cells. They are secreted into the media. 1) Baculovirus system 2) Drosophila system Which system would you recommend for high expression and convenience? For most proteins, the level of expression is far greater with bacuovirus. Dima
Re: [ccp4bb] Copper Staining
There's an even faster reagent for staining - takes abut 1 minute overall and the results look just like dear old Coomassie. This reagent works wonderfully for us. Yes, the recipe is proprietary - but if you add up the cost of reagents for normal Coomassie and compare - this one isn't much more expensive. http://www.novexin.com/products/InstantBlue.html Sounds like a good old Bradford reagent AKA colloidal Coomassie G-250. 60 mg G-250 dissolved in 50 ml ethanol and diluted into 1 L of 1/10 dilution of concentrated phosphoric acid (~8.5% final). Solubilizing agents is a smoke screen, for G-250 is fully soluble in ethanol and for the colloid particles to not penetrate the gel pores inclusion of any solubilizing agents will only be detrimental. Betcha it's a browish solution that forms blue pellet when centrifuged hard. If so, the claimed 5-10 ng sensitivity can only be achieved after 1) at least 15 min staining (30 min better; no one abolished laws of physics that requre diffusion into the gel for the interaction to take place), 2) some destaining to get rid of slight brown-blue background. Dima
Re: [ccp4bb] extra line of stain in our gel
When we develop our gel we recently keep getting a horizontal line of stain at 5 mol weight in all lanes of our gel. (This is not a feature of interest except that the protein that we are interested happens to be 5 mol weight). I would appreciate any ideas on how we can get rid of this line. Are these somewhat fuzzy bands? If yes then it's [human] keratins. Usually it is either SDS or bME stocks that get contaminated with keratins. Usually, just changing stocks and working cleanly solves the issue but occasionally bME as bought already contains them. Ochs, D. (1983) Protein contaminants of sodium dodecyl sulfatepolyacrylamide gels. Anal. Biochem. 135, 470–474. Dima
Re: [ccp4bb] Is phophorylation possible in E. coli expression system?
However, if your protein is not a protein tyrosine kinase, you may check your western condition. Yes, this is an essential control. Use lots of Lambda protein phosphatase to dephosporylate your protein and us the resulting material as a negative control in Western. My experience is that phosphorylations of over-expressed proteins in E. coli are always auto-phosphorylation. Should be the case when there is tons of overexpression. But to be fair, bacteria (and E.coli) do possess tyrosine kinases. It's still possible that one of them does work on some sites found in your protein. Dima
Re: [ccp4bb] The importance of USING our validation tools
I like to emphasize that the infamous table 1 alone should have immediately tipped off any competent reviewer. The last shell I/Isig is 1.3 and rmerge 0.11 (!). And keep in mind that this statistics comes from merging data from FOUR different crystals! (That's clearly and unambigously stated in Methods section). Dima
Re: [ccp4bb] DNase inhibitors
I have small crystals of protein DNA complex. I am worried about the possibility of presence of some DNases in the drop (I use DNase in the lysis buffer). I have Tris and MgCl2 in the crystallization condition, which makes me hesitant to use DEPC or EDTA/EGTA respectively. Are there any other DNase inhibitors that I can use to protect the DNA? Any other precautions one should take while handling DNA There is something called aurintricarboxylic acid that I've seen mentioned several times. it is supposed to be a general inhibitor of nucleases. I have no experience with it. Also, this may sound crazy but if you are worried about DNAse I that you used in previous steps, one option is to add a little bit of actin. Actin binds to DNAse I very specifically and with very high affinity, inhibiting its activity. At the concentrations that you are likely to use it at, it should be below critical concentration that makes it polymerize. Dima
Re: [ccp4bb] CCP4 GUI
The level of detail in the GUI is a matter of constant debate. The underlying programs are far far richer, so the question is how much to expose in the GUI. How about the simple and elegant way it is done in Refmac? Tacked under Developers Options is specify an external keyword script file for Refmac. I frequently use this and always wished that *all* CCP4i GUIs had this. Sounds like something that lets user enjoy the simplicity GUI yet allowing flexibility of using complete functionality of the programs. This should be very easy to implement. Dima
Re: [ccp4bb] Expression hosts (was:Inclusion body)
As has been suggested, you can try other hosts - for instance Gram positive bacteria (Bacillus megaterium is an interesting host as are other bacilli, especially if your protein is secreted), other Gram negative bacteria (Pseudomonas for instance), Can anyone comment on availability of Archaea expression systems? Based on available structures, it would appear that their chaperons are much more similar to eukaryotic one than anything present in eubacteria. For a long time, I've been puzzling over why there is still no popular/workable Archaea expression system. Reason? yeasts (Pichia, Hansenula, Yarrowia, Klyveromyces), insect cells (Trichoplusia or Spodoptera - they're different!) I've compared Sf9/Sf21 and High5 cells for many, many baculoviruses and have yet to find a difference that would ultimately matter. One clear difference is that Trichoplusia/High5 seem to require less virus to get to the same level of expression - but that level is exactly the same as what Sf9 give at higher pfu/cell infections. So the benefit is only felt by people who failed to amplify their virus properly. Same with regard to solubility. Some of the proteins I've dealt with misfold (partially or completely) even in insect cells but back to back comparison of High5 and Sf9 showed no difference. Trichoplusia are a lot more difficult to get growing in suspensions without horrible clumping. mammalian cells (dozens of options there too) All of them are essentially equivalent = pathetic, as far as overexpression is concerned. One exception though: transient expression in Cos-1 cells. It's not a system one can do on a large scale but the amount of protein/cell is 10-20X higher than constitutive expression and thus it can be appropriate to to get enough material for limited proteolysis or biochemical assays that don't require lots of protein. , or cell-free expression. If you're truly desperate you can try expression in more esoteric hosts such as plant roots, algi, parasites (Leishmania), whole insect larvae, animals (milk), and so forth. Dictyostelium discoideum (slime mold) deserves honorary mention here. Its cultures are nearly as cheap as E.coli, it grows almost as fast as yeasts, it doies not have funky post-translational processing yeasts frequently do, and it gives at least as much protein as common mammalian expression systems. The drawback is its weird codon usage but with the gene synthesis prices coming down fast it's fast becoming a viable option. I got enclusion body in many cases when I tried to express human proteins in E coli. I would like some suggestions on how to go about it. I would also like to try co-expression of GroEL/GroES or DnaJ/DnaK, and would like to know where to get the plasmids. IMHO, if you don't get *any* soluble expression at all in minimal medium expressing at 16C, the chances that anything you do in E.coli will magically make soluble protein are slim to none. If, however, there is *some* folding but it inefficient, then, As Artem mentioned, there is a long list of options you can consider... Good luck, Dima
Re: [ccp4bb] Bio-rad DuoFlow
(FPLC is a trademark. The marketing hype has been so effective that many people think that FPLC is a method.) Well, it is, in a way. The FPLC thingie was a pure marketing genius that bundled the remarkable technology of Monobeads (AFAIK, there is still no other company that offers comparable size dispersion) with an inefficent but lower cost alternative to HPLC. It worked. When the market was taken, the technology reverted back to the standard HPLC under a new marketing name. Dima
