Re: [ccp4bb] crystallizing fusion proteins
Hi Herman, I will add a few points to all the excellent advice already provided to you. If you decide to try this option of crystallizing a fusion protein of your target, I would consider using N-terminal but also C-terminal fusions. We have had success using MBP in N-ter and the superfolder GFP in C-terminal for a few 'vexing' proteins from *Plasmodium falciparum. *It helped us either to obtain crystals/structures (as you seek) but also provided a way to circumvent a tendency for twinning from our target in some specific cases. GFP has the added benefit that your fusion crystals should be bright yellow so that speeds up the screening process a bit. I hope this helps. Good luck Best regards, Pascal Egea, PhD UCLA School of Medicine > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > <http://mednet.ucla.edu> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Data 3 A
Hi, Have you looked at the packing of your crystal with that single molecule in the ASU. Is it enough to build a crystal as in symmetry related mates contact each other (with probably quite a bit a solvent) but still a crystal. That would not be shocking since your resolution is average (on offense intended). Best, Pascal Egea On Tue, Jan 12, 2021 at 3:22 PM rohit kumar wrote: > Dear all, > > I am trying to solve a data with 3 A resolution, however data quality is > very bad and mathews coffi. suggest two molecules per ASU but It always > gives one molecule in AU after phaser with the TFZ and RFZ score are 4.5 > and 3.5 respectively with LLG gain 121. > And when I used that model for Refmac the final R/Rfee is 23/30 and with > satisfactory Ramachandran statistics as well as electron density and model > in agreement with each other in coot. Does It mean that I have > correct solution? > Please suggest, > > -- > Regards > Dr. Rohit Kumar Singh > Postdoctoral fellow > > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > -- Pascal F. Egea, PhD Associate Project Scientist UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Faculty position in cryo-EM at Université de Montréal
An Assistant or Associate Professor position has opened in the Department of Biochemistry at Université de Montréal: https://www.umontreal.ca/public/www/documents/offres_emploi_profs/MED_11-20_8_Biochemistry.pdf Enquiries and applications to: Lorraine Bidégaré Charette mailto:lorraine.bidegare.chare...@umontreal.ca>> To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Cell disruption
Dear Bernhard , I would recommend the emulsified from Avestin. It is great . Depending on your budget you can ask for stainless steel (probe to slow tear ) or ceramic (longer lasting but a bit more expensive). We have the ceramic but I have worked with the stainless steel one and it is really good with E. coli cells. Not the best for Yeast despite pressurizing more. Yeast are better dealt with bead beaters or cryo-mill grinders. 3 passes are usually sufficient to process up to 250 ml of extract in a decent time (15-20) minutes . We operate ours at room temperature just cool the serpentine tubing coming out of the chamber in ice . There is an option to add a cooling plate but we have never considered it. Overheating comes from overpressurizing but for E Coli at 15000 psi max 3 passes will do the trick without overhea tu bf the sample. I have processEs successfully many membrane proteins And protein complexes with it. I have never seen one in a cold room to be honest but it should be fine although I don’t think it would be necessary . I would Ask avestin about that . Maintenance and cleaning is simple . Once in a while I will replace the seal, it s not very difficult to do. The whole assembly is metal so you can sterilize the whole thing After complete disassembly in extreme cases . This is a bit more involved but not overwhelming. The people at Avestin are super nice and responsive ( Canadians) so they will guide you if necessary . And we have had their engineers show up sometimes to just check the instrument for free . I have had this one for 10 years in my lab now. And before that was using one for 7 years during my post doc. I would not lyse bacteria by any other method now. I hope this helps. All the best. Pascal On Sat, Aug 15, 2020 at 9:08 PM Bernhard Lechtenberg < lechtenber...@wehi.edu.au> wrote: > Dear colleagues, > > > > We are currently looking to purchase a cell disruptor/homogeniser mainly > for routinely processing a few 100 mls of E. coli suspensions. With the > current COVID-19 restrictions it is very difficult for us to test any > equipment. I thus hope that some of you can share their experiences with > the different models. I found a similar thread on the CCP4BB from 2013 but > wondered if anybody had had some more up to date information. > > > > We are mainly looking at the Avestin Emulsiflex C3 homogeniser and the > Microfluidics LM20 Microfluidizer. In particular we are interested to know > more about ease of use, maintenance, reliability and if anybody operates > these in a cold room (4°C). > > > > Thanks in advance, > > > > Bernhard > > > > <https://www.google.com/maps/search/1G+Royal+Parade+%0D%0A+Parkville+VIC+3052+%0D%0A+Australia?entry=gmail=g> > > > > <https://www.google.com/maps/search/1G+Royal+Parade+%0D%0A+Parkville+VIC+3052+%0D%0A+Australia?entry=gmail=g> > > -- > > Bernhard C. Lechtenberg, Ph.D. > > Laboratory Head > > Ubiquitin Signalling Division > > The Walter and Eliza Hall Institute > > 1G Royal Parade > <https://www.google.com/maps/search/1G+Royal+Parade+%0D%0A+Parkville+VIC+3052+%0D%0A+Australia?entry=gmail=g> > > Parkville VIC 3052 > <https://www.google.com/maps/search/1G+Royal+Parade+%0D%0A+Parkville+VIC+3052+%0D%0A+Australia?entry=gmail=g> > > Australia > <https://www.google.com/maps/search/1G+Royal+Parade+%0D%0A+Parkville+VIC+3052+%0D%0A+Australia?entry=gmail=g> > > Phone: +61 3 9345 2217 > > Email: lechtenber...@wehi.edu.au > > > > ___ > > The information in this email is confidential and intended solely for the > addressee. > You must not disclose, forward, print or use it without the permission of > the sender. > > The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of > the Kulin > Nation as the traditional owners of the land where our campuses are > located and > the continuing connection to country and community. > ___ > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > -- Pascal F. Egea, PhD Associate Project Scientist UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Structural Importance of Methionine
Hi Samer, In addition to what has been already mentioned to you, there are also the two well illustrated cases of methionine rich domains. - in the case of the M (for Met rich) domain of the Signal Recognition Particle protein SRP54/Ffh involved in promiscuous binding of the N-terminal hydrophobic signal sequences of nascent membrane proteins as they exit the ribosome en route to the translocon and the membrane for insertion or secretion. - in the case of the ATP-ase Get3 (Guided Entry of Tail anchored proteins) 2 that also has an hydrophobic cleft enriched in Met residues also designed to promiscuously bind the hydrophobic C-terminal transmembrane helices of so-called Tailed Anchored proteins. In both cases the substrates are hydrophobic helices and the greasy and flexible side chains of Methionines of the receptor protein (SRP54/Ffh or Get3) can accommodate a variety of hydrophobic substrates in the protein binding clefts. I hope this helps you. All the best, Pascal On Fri, Aug 7, 2020 at 5:14 AM samer halabi < 30c2162795b2-dmarc-requ...@jiscmail.ac.uk> wrote: > Dear All, > I am working on structures where Methionine is important in binding of > peptides to the MHC protein complex. > Would anyone kindly like to share their knowledge about anything they find > it important about this particular amino acid structurally? Sharing a paper > or just few comments will be greatly appreciated. > > I know my question may sound very general (and kind of superficial) but > there is definitely a reason, that I don't know and might be already known, > why certain peptides (like CLIP) are rich in Methionine, and that lowers > their affinity of binding. > Thank you and sorry to disturb you all. > Best regards, > Samer > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > -- Pascal F. Egea, PhD Associate Project Scientist UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
[ccp4bb] Post Doctoral Position at UCLA
Dear All, A NIH-funded post-doctoral position is available in my group at UCLA to study the structure, mechanisms, and functions of membrane protein complexes involved in protein or phospholipid trafficking in eukaryotic pathogens (such as fungi and parasitic protozoa) as described in: AhYoung, Jiang et* al.* in *PNAS* 2015 (doi: 10.1073/pnas.1422363112). Ho, Beck et* al.* in *Nature* 2018 (doi: 10.1038/s41586-018-0469-4). Motivated and qualified candidates should hold a PhD in the field of structural biology and be proficient in cloning, protein purification, and structure solving by either crystallography or cryo-electron microscopy. Experience with expression in insect cells and/or HEK293 cells is strongly preferred. Applicants should address their CV, a cover letter stating their accomplishments, interests, and career plans together with the addresses of three potential references to *pe...@mednet.ucla.edu .* Thanks. All the best, Pascal Egea -- Pascal F. Egea, PhD UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Should I optimize these crystals?
Hi Marta, 1-- There is always a possibility of forming detergent/lipid (cholesterol in your case) -containing crystals combined with other small molecule(s) (organic or not). Although you did not specify the width of the oscillation used to collect the frame displayed in your mail (was it 45 degrees or 0.5 degrees?) I see signs of a lattice at least on the vertical direction (3 rows can be distinguished it seems), can you derive a "cell-dimension" along that direction? 2- If you have access to a fluorescence-coupled light microscope (like a a PRS-1000 Korima instrument) maybe you could see if your crystals glow (provided your proteins contain tryptophans). Crystals from drop 1 seem better candidates (they look 'thicker') for a fluorescence scan, the ones from drop 2 are really thin and curvy, so it might be difficult to see in that case. This could help you characterizing and ruling things out more easily. 3- You mentioned that you analyzed the content of washed/dissolved crystals by silver-stained SDS PAGE. Forgive me for asking if you also analyzed the last wash step on this sivler-stained SDS PAGE gel to rule out that the protein signal you observed was not cross-contamination by a carry-over effect. Good luck. Good luck On Wed, Oct 24, 2018 at 1:31 PM marta borowska wrote: > Dear all, > > I have grown crystals of a membrane protein complex, that I initially > verified on SDS-PAGE. These crystals grew only if membrane protein > component is there. The condition is polypropylene glycol 400, > cryoprotected with ethylene glycol, sample buffer has 150mM NaCl on top of > detergent with cholesterol. These long thin needles are quite sturdy and > either didn't diffract or diffracted like small molecule (I cannot exclude > the possibility of contamination stuck around crystal). After the > synchrotron trip harvested crystals were washed (most did not dissolve in > Urea or NaOH!), dissolved in 10% mild detergent and still showed protein > from 4-6 crystals when analyzed on Silver Stain SDS-PAGE. > > I would appreciate your input on whether some of you encountered similar > patterns and if you think I should proceed with more optimization on these > crystals. > > Thank you, > Marta > > > -- > > Marta T. Borowska > > Graduate Student > > Adams Lab > > Department of Biochemistry and Molecular Biology > > The University of Chicago > > 929 E. 57th Street > > Chicago, IL 60637 > > Lab: GCIS W229 > > Lab phone: 773-834-0660 > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] suggestions for cryoprotectant
Hi Firdous, I noticed that conditions 2,4 and 5 are overall harsh agents (chaotropic agents such as KSCN (probably not KCN) and Nitrate ions ), in my own personal experience I have never been very lucky with crystals grown in nitrate ions. Condition 1 however looks a bit more soft with 'gentle' ions. May I suggest trying a sodium malonate screen or seeding in such a crystallization agent and maybe trying to cryprotect those acetate crystals in malonate which is a cryo-friendly so-called 'magic salt'. crystals grown in carboxylates such as citrate and malonate are usually easier to cryo-protect. Hope this helps Pascal Egea On Fri, Oct 19, 2018 at 2:57 PM Firdous Tarique wrote: > Dear members > > I have got beautiful crystal hits in SaltRx screens which are not > diffracting to a good resoultion. All of them are salt based condition and > I am not able to formulate a good cryoprotectant for these crystals. I also > think that in my case the poor resolution is due to a poor cryoprotectant > selection. > > The conditions are as follows: > > 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0 > 2>0.5M KCN 100mM Tris pH8.5 > 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0 > 4>4M Sodium Nitrate 100mM Tris pH8.5 > 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6 > > There are few more conditions but so far not able to see good diffraction > with using lower peg and glycerol based cryoprotectants. > > Can anybody suggest me good cryos conditions for salt based > crystallization conditions or anything good for SaltRx crystallization hits. > > Thanks > > Firdous > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Protein toxicity or low expression
Hi Firdous,, As you mentioned they are many things to change and try. before changing vector it is more efficient in my opinion to change expression conditions (temperature, amount of IPTG, length of culture), culture medium (regular ones against M9 or auto induction media) and expression strains BL21, BL21gold C43 and and BL21 Lemo cells. Then you can change vector (arabinose or rhamnose controlled promoters) and fusion (MBP or GST maybe). Leaky expression can have deleterious effects on expression levels and culture stability/degenerescence (especially dependent on the type of antibiotic you are using). Tightening up the expression control maybe important in the case of a toxic target). BL21 Lemo cells can be very useful in those cases. If your gene has an odd codon usage (compared to your expression host) you may want to think about recoding it or use rare tRNA plasmids to try to compensate for that. The fact that you mentioned that you see the Sumo-his but not the following target protein is worrisome. Since you mentioned the vector is fine I would think that there is a problem of translation/folding of the target sequence, maybe check the presence of rare codons that could promote pausing for example. I don t know how easy it is to check for sequences that would affect the local structure of the mRNA but codon optimization programs should take this into account. Good luck On Sat, Dec 9, 2017 at 5:27 PM, Firdous Tarique <kahkashantari...@gmail.com> wrote: > Hello everyone. > > I am struggling with the solubility or expression of my proteins in > BL21DE3 E.coli expression cells. In one of my construct I have a Sumo-His > fusion at the n-terminal of my protein. After induction what I see is the > expression of the Sumo tag only. My sequencing results are fine and there > is no mistake in cloning. I wonder why I am seeing only the expression of > the Sumo tag although my fusion protein is in frame with this tag. > My second question is related with the very low expression of one of my > gene. A brief literature search suggests so many things to improve the > expression like changing the host, vector, media etc. It is a nuclease and > cloned in vector with a Sumo-His tag on it. I am able to purify the little > amount from E coli BL21DE3 host. The problem is related with low expression > which is clearly observed in difference in the pre and post induction > lysate on SDS PAGE. Out of so many option available in the literature I am > confused what to try first. Any general idea? > > Your suggestions can help a lot. > > Kahkashan > Ph.D student > Delhi University > India > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu
Re: [ccp4bb] the purification process of protein, used for crystallization
Hi liuqing, It is more usual to finish with a sec because you control the composition of the final conditioning buffer of your sample and remove any aggregates. We personally favor the following sequence iMac - desalt - iex if necessary - sec Desalt meaning one of those small Pd10 like column that enable the quick removal of imidazole and conditioning of your protein for either cleavage with a protease of you wish to remove tags and/or lowering of salt concentration to bind on an iex column if u wish to do one. That said . In some cases Doing a real sec straight after IMac has the advantage to remove some large molecular weight contaminants ( usually DNA) and some aggregates that are annoying. These choices depend on your target of course and the level of abundance too. Best, Pascal Egea, PHD UCLA Geffen School of Medicine On Fri, Nov 17, 2017 at 5:46 AM Liuqing Chen <519198...@163.com> wrote: > Hello everyone! > I have listened someone suggested that, first use affinity chromatography > (Ni-NTA), then use SEC (superdex200 increase), and finally used ion > exchange (monoQ), to purified protein, which will be used to > crystallization. > My question is why the monoQ used in the finally step, why not the SEC > used at the finally step? > > sincerely > Liuqing Chen > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu
[ccp4bb] Job Posting - Postdoc at University of Montreal
Postdoctoral Research Position in Structural Biology at University of Montreal A postdoctoral research position is available in the laboratory of Dr. John Pascal at the University of Montreal. Current research projects focus on the structural biology, biochemistry, and cell biology of proteins involved in genome maintenance, and members of the poly(ADP-ribose) polymerase (PARP) family of proteins. Examples of our research approach can be found in recent publications (Eisemann et al. Structure 2016; Steffen et al. Nucleic Acids Research 2016; Dawicki-McKenna et al. Molecular Cell 2015; Langelier et al. Science 2012). The position requires a PhD in structural biology, biochemistry, or biophysics. Expertise in the following areas is valuable to ongoing projects: molecular biology and protein purification, biochemical and biophysical analysis of proteins and nucleic acids, and structure determination by x-ray crystallography, small-angle x-ray scattering, NMR, or cryo-EM. The laboratory is located in the Department of Biochemistry and Molecular Medicine (https://biochimie.umontreal.ca/en/). To inquire about the position, please send a brief letter of interest, a CV, and the names of three references to john.pas...@umontreal.ca<mailto:john.pas...@umontreal.ca><mailto:john.pas...@umontreal.ca>. Department webpage: https://biochimie.umontreal.ca/en/department/professors/john-m-pascal/ _____ John M. Pascal, PhD Associate Professor Université de Montréal Biochemistry and Molecular Medicine 2900 Boulevard Edouard-Montpetit Pavillon Roger-Gaudry, D-347 Montréal, Québec H3T 1J4 Canada 1.514.343.6111 ext. 4890 john.pas...@umontreal.ca<mailto:john.pas...@umontreal.ca>
[ccp4bb] MR phasing using Negative Stain EM reconstruction
Dear All, I would like to know if it is possible to use a low resolution EM reconstruction of a complex obtained in negative stain EM (not cryo EM) to help molecular replacement in a 4.5A resolution X-ray diffraction data set of the same complex I am aware of the possibility of using low resolution cryoEM maps for MR as described in the review from Jackson et al in Nature Protocols but I was wondering if there is an intrinsically impossibility for negative stain reconstructions. Any thoughts or advice will be greatly appreciated. Best, -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu
[ccp4bb] fluorescence question
Dear All, This is more a biochemical/biophysics question but since we need to complete our structural analysis with functional or biophysical data, I thought I would ask. We crystallized a protein as an MBP fusion and solved its structure. Based on previous knowledge we know that the small protein binds peptides. So we are now trying to measure binding of the protein to peptides labeled with a fluorophore using fluorescence anisotropy. Unfortunately the protein has to be used with its MBP still attached. We have a resilient problem with the MBP carrier. It has some ‘residual’ fluorescence that interferes severely with our measurements (we are using tetramethyl rhodamine as fluorescent reporter). I was curious to know if anyone else had encountered this problem and figured out a solution. Any suggestion will be greatly appreciated. Many thanks in advance. Pascal Egea Assistant Professor of Biological Chemistry UCLA David Geffen School of Medicine
Re: [ccp4bb] Zinc binding protein expressed from insect cells
Hi Heng, DTT can react with metal cations such as Zinc or Iron. This is why people to tend to use little DTT or no DTT at all on metal affinity columns or replace it b-mercaptoethanol or TCEP that do not interfere. Regarding the incorporation of Zinc into the culture media. I recall that Zinc finger domain expression in E coli (for nuclear receptors dna binding domains) is classically described to be done in presence of Zinc acetate added into the culture media (around 100 microMolar I think ) this is already a lot of zinc (more might be toxic to the cells). depending on what your process of purification is. I would try to add zinc in the expression media (this has probably been descrived also for insect cells) if you use a Ni or Co chelating column I would NOT add any free zinc at this stage, you can, if you wish it, add it to eluate of your affinity column and to gel filtration buffers or ion exchange buffers. That said, I would suspect that having enough zinc around is mostly beneficial at the expression stage as the protein folding machinery is dealing with your target protein.zinc misloaded protein is likely to be unsoluble. for nuclear receptors DBDs there are protocols describing reincorporation (or even exchange) of metal ions inside the domain (maybe even starting from inclusion bodies) but the more cysteines you have the more likely it is to be difficult to get the right folded protein back. so I would rather favor a strategy trying to get as much folded protein as possible by natural means (let the cells do what we biochemists still don t know to do very well). if you add free zinc I would be careful with the concentration and the pH of your buffers. at basic pH zinc and other ions (such as Ca and Fe) form insoluble hydroxydes. if you have managed to purify some protein I would try to do some emission spectroscopy to see what ions are bound (zinc and or iron ) you may be surprised by what you will see. sorry for the lengthy response but I hope this helps. all the best, Pascal Egea On Fri, Aug 15, 2014 at 8:03 AM, Harvey Rodriguez h.rodriguez.x...@gmail.com wrote: Dear all, Sorry for the non-crystallographic question. Currently I am working on a zinc binding protein which is expressed in insect cells and may contain 4-6 zinc ions. As we know, so many zinc binding proteins can absorb the iron ions from the culture medium and the protein looks from yellow to dark red when concentrated. But when I concentrate the protein, I didn’t see the red color even in the very high concentration. I am just wondering if a zinc binding protein is expressed from insect or mammalian cells, can the zinc binding sites grab the irons instead of zinc or the zinc binding site can be empty loaded if there is not enough zinc in the culture medium? If so, do I need to include some zinc salt into the culture medium when doing expression or I can add some zinc ions when purifying? Usually, how much zinc and at which step of purification can we add the zinc into the solution when doing purification? Another question is that we know DTT can react with the heavy atoms to form the insoluble sulfide precipitates and if the zinc binding protein is purified with DTT at a final concentration of 1-5 mM, can it strip the zinc ions from the protein? I am appreciated if someone has this kind of experimental experiences and thanks in advance! Heng -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Difficult MR with MBP fusion protein
Dear Niu, It is very unlikely that MBP will be disordered. We use that protein a standard for SAXS calibration and gel filtration it is extremely well behaved. There is an excellent review about MBP enhanced crystallization (by Moon et al) and you can do a quick survey of the PDB to find the cornucopia of structures obtained using this strategy. I would suggest maybe an ensemble MR search in phaser using a sum of all conformations adopted by MBP in these different structures. You have 4 A data and it is difficult sometimes to pull out the solution at low resolution. Trimming the few disordered loops in the MBP model could help you, so you avoid throwing away the good solution because of a few clashes when it packs solutions after rotation and translation searches. We recently solved one structure MBP + target protein of about 10 kDal (2.7A) and it worked extremely well. A linker of 30 aminoacids connected the target to MBP and it ended up tightly packed against the carrier MBP protein even if we were still missing about 25 residues of linker that was actually part of the natural sequence of the protein (we had to generate 5 constructs to get it right). Another approach, may not be smart but it should get the job done, is to selenolabel your fusion and crystallize it. MBP has 5 or 6 methionines , even if you target has none, this will be enough to phase (although you are only at 4 A if I understood well). That should enable you to lock the MBP in position and see what is going on in your crystals. Good luck Pascal Egea On Fri, May 16, 2014 at 8:03 AM, Niu Tou niutou2...@gmail.com wrote: Dear All, Recently we collected some data of a MBP fusion protein, at around 4A resolution. The protein itself is about half of the MBP size. However when we tried to solve it with MR, it failed. We tried to use MBP alone, homology model of target protein alone, and MBP+model. It is very strange that MBP alone can not yield any reasonable solution at all, so does searching with MBP and model together. While searching with model alone could get some better results, but when fix it to search MBP, it failed. There are 1 molecule per ASU with solvent content 55%. The spacegroup should be right and we tried to search all possible alternatives in each run, we also tried to lower it down, but did not work either. When running Phenix.phaser, there is a warning at the beginning saying eLLG suggests placing of ensembles will be very difficult. I wonder if anybody has encountered similar situation before. Any suggestions will be greatly appreciated! Regards, Niu -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] FORTRAN still rules?
Le 09/05/2014 15:36, Adam Ralph a écrit : Can it be parallelized? That is how you reduce run-time. One of the tests matrix-matrix multiplication has been successfully speeded up by using GPUs. CUDA is the language used for this, which is a derivative of C. To be fair you only see the benefit for really large matrices, smaller ones which actually be slower on GPUs. Talking about matrix multiplication, the benchmark is biased as they used openblas. The core multiplication is done in assembly. So it's probably machine code rules! mathematica and octave probably use mkl, same as openblas. This is the reason why fortran, C, julia, mathematica and matlab perform the same. They are all linked to mkl or openblas. There is a fantastic link on the optimization on general matrix matrix mutiplication: http://wiki.cs.utexas.edu/rvdg/HowToOptimizeGemm Python uses numpy which is a wrapper around lapack aka fortran... Same for R. I think octave too. One general advice to give would be not try to optimise, use a library instead. openblas/mkl/acml for linear algebra fftw for fourier transform sleef for trigonometric functions and other math functions mixmax for random numbers And so on... Pascal
Re: [ccp4bb] off-topic: bug busting
Hi Phoebe, Another possibility is the Emulsiflex (from Avestin, in canada). Not a cheap piece of equipment, but very sturdy and efficient to up to 200 mL of extract (runs on house-air). Can deal with E coli (and even yeast if you have the models with internal compressor). It comes in 3 sizes I think we have the middle one (C-3) with a compressor. It is basically a french press but without the inconvenient of being french (I am french myself). It is quite gentle, and does not overheat samples as much as the sonicator. We make a lot of membrane protein purifications and I have been working with this since my post-doc Hope this helps. Best regards, Pascal Egea On Tue, Feb 4, 2014 at 8:49 AM, Phoebe A. Rice pr...@uchicago.edu wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
[ccp4bb] membrane protein and phase separation
Dear All, I have a question tailored for the membrane protein and detergent folks. We are purifying a membrane protein that associates into an homoligomeric pore and we have been successfully preparing it in two detergents: FC-12 or a mild lipid. The two Protein Detergent Complexes look very homogenous by SEC and can be concentrated without protein loss on membranes with MW cutoffs (100kDal) way larger that the expected their respective free detergent micelles. Everything looks good so far... until we get to the crystallization stage. While the PDC in FC12 does not tend to form too much phase separation, the PDC in the lipid does. This looks a bit odd to me since these lipid micelles are supposed to be a bit smaller than the FC-12 micelles. We are working at twice the CMC and besides lowering the detergent concentration, I am a bit perplex about what I am observing. Intuitively I would have expected to observe the reverse behavior: worst in FC-12 than the lipid. This lipid is a very mild solubilizing/reconstituting agent that has already been successfully used for structure determination. Any advice or thoughts will be greatly appreciated. Is this something that some of you have already observed? Many thanks in advance, -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
[ccp4bb] post-doctoral in membrane protein structural biology
We are seeking a post-doctoral researcher to join my research group at UCLA. The lab focuses on the structural studies of challenging targets: membrane protein complexes and channels from eukaryotes (yeast and several parasites) and seeks to characterize their architecture and molecular mechanisms of action combining X-ray crystallography, SAXS and cryo-EM when necessary and possible. We will exclusively consider applicants with proven experience in protein crystallography, cloning, protein expression and purification. Previous experience with membrane proteins is highly desirable but not required. Candidates should hold or expected to soon hold a PhD degree in a relevant area (Biophysics, Structural Biology) and have excellent social and communication skills in english (very important). Interested candidates should send a CV and personal statement together with the name and addresses of three references. I will be attending the ACA-2013 meeting in Hawaii this coming week and will present a poster presented from 5:30-07:30pm on Sunday, July 21. Motivated individuals are invited to directly contact with me. Pascal F. Egea, PhD - Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Membrane Protein Optimisation
Hi Rhys, I suspect that what you call a gel might be phase separation (correct me if this is wrong) like an oily phase enriched in protein and detergent. you may have too much detergent in your drop. may I ask what detergent you are using (low or high CMC) and at what concentration of detergent do you think you are when you setup your drops. you might have concentrated more detergent than you think along your protein. the MW cutoff of the membrane you are using to concentrate is important relative to the size of the free detergent micelle and of course the protein-detergent micelle you are trying to concentrate. too much detergent staying around is a major cause of trouble (i.e. poor diffraction and phase separation competing with productive crystal growth) besides many other parameters Hope this helps -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu On Thu, May 9, 2013 at 8:34 AM, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: Hi All, A quick question if you've ever worked on membrane proteins, I'm trying to optimize crystals for bacterial integral outer membrane protein I'm working on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of birefringent gel forming in the same condition, I get the feeling that this is Detergent/Protein complex and is robbing my crystals of material to grow bigger. These crystals diffract to 5 A so I'd quite like to make them bigger and better, Cheers, Rhys
[ccp4bb] gelification of a pure protein
Dear All, I am presently faced with a peculiar case in the lab. We are expressing a protein in E. coli and we are able to express it as a fusion protein without problems . Fusion cleavage goes well and the final product looks homogenous by size-exclusion chromatography with the expected molecular weight. There are no signs of aggregation. However when we lower the salt concentration by dialysis then the protein forms a gel. transparent , optically clear, with no fluffy material (in the cold room). Gelification seems to occur when we lower the concentration below 100 mM NaCl. This protein has a fairly high pI (~9.0). Attempts to reverse the process by gentle heating or salt addition have been so far unsuccessful. It is not a thermophilic protein. We have not been able to obtain crystals so far. Has anyone already observed this kind of behavior and/or have any suggestions? Many thanks in advance . -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] gelification of a pure protein
Thanks to All for the diligent answers to my query, The protein is not thermophilic and has only one cysteine. We are working in presence of freshly added reducing agent and glycerol to promote solubility (well kinda it seems). This is not an RNA or DNA binding protein and it has no low-complexity regions except at the N terminus, there maybe some left over from a cryptic transit peptide (somehow basic) that supposedly targets the protein to a specific organelle. We are probably going to truncate further to see if it solves our problem I appreciate all the comments and suggestions, Cheers, Pascal -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
[ccp4bb] pdb/mmcif to cif
Hi, Is anyone know how I can convert a pdb or mmcif file into the cif format ? I am trying to convert this file: http://pdb.org/pdb/explore/explore.do?structureId=3NIR Thanks, Pascal smime.p7s Description: Signature cryptographique S/MIME
Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag
Hi Raji, Thrombin is a rather good protease and behaves well in a large set of different detergents ( there is a paper by Michael Wiener that describes the relative efficiencies of several usual proteases, amongst those thrombin is inlcuded, used routinely for cleavage of membrane protein fusions in detergents. This is a rather extensive survey henceforth it is very informative. The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal James M. Vergis 1, Michael C. Wiener in Protein Expression and Purification 78 (2011) 139–142 thrombin tends to be sensitive to reducing agents so I would stay away from DTT and TCEP , b-mercapto is acceptable in reasonable amounts. We cut in salt concentrations as high as 500 mM (NaCl) at pH 7.5-8.5 with 5-10% glycerol around no chelating agents should be present and imidazole in our hands tends to be an inhibitors (probably because it has some chelating/complexing activity). we have good cleavage in DDM , OG and somehow more difficulties in foscholines but it still cuts reasonnably well given the cost of the enzyme and the targets, the most crucial parameter is Protease/target ratio and incubation time and temp. you can do trials on small scale digests in PCR tubes at different temperatures. we usually cut at 4 or room temp. I hope this helps, Best regards, -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Off-topic: heterologous co-expression in yeast
Hi Andre, There is set of plasmids allowing coexpression in yeast they are described in the following article Constructionand characterization of bidirectional expression vectors in Saccharomyces cerevisiae Aimin Li1,2, Zengshan Liu1, Qianxue Li2, Lu Yu1, Dacheng Wang2 Xuming Deng 1,2 in FEMS Yeast Res 8 (2008) 6–9 !c Those plasmids are designed for coexpression and use two different promoters a constitutive one (GPD) and a strong inducible one (GAL). You have several selection markers and they are shuttle vectors for easy manipulation and amplification in E. coli. Hope this helps. Best of luck -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] 24 well screw-cap crystallization plates
Hi Brad, I am afraid that there is no alternate source for these plates. The screw cap system , I believe, was patented by the canadian company NEXTAL that was then assimilated by Q...N and the patent is probably still holding. Pascal -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
[ccp4bb] recombinant sources of N-glycanase
Hi All, We are trying to deal with a eukaryotic membrane protein that crystallizes but, as usual, diffracts poorly so far. As it is heavily glycosylated, we are considering enzymatic deglycosylation. Does anyone know about a recombinant source of N-glycanase ; we would like to prepare it ourselves because the commercial sources are apparently too expensive. Thanks in advance. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
[ccp4bb] Subject: [ccp4bb] Soaking Kinase Crystals with ATP analogues
Hi , To add to the previous comments, crystallization of GTP or ATP (or their analogues) with their kinase/ A- or G-tpases can depend on a lot of factors that were mentioned (such as packing). A simple common problem is that ATP solutions should be carefully buffered prior to their use for soaking, people tend to forget about this. A 100 mM ATP solution is pH 3 (probably not good for your protein and it has 3 acidic groups) For some classes of ATP binding proteins, acidic pH have also been shown to lower chances of successful soaking or co-crystallization. The crystallization condition is also important. High concentrations of sulphates or phosphates tend to complicate things . Same thing for high concentrations of di or tri carboxylic acids (such as citrate, tartrate or malonate). Sulfates tend to occupy the beta phosphate binding sites and at high concentrations they can outcompete an analogue. For first hand experience, I would not assume that all analogues behave the same. Especially between AMPPNP, ATPgammaS and AMPPCP (or their Guanine counterparts). the Cp analogues in our hands tend to have lower affinities. You can always try ADP AlF4 combination or ADP BeF3, if you are not afraid of beryllium . Hope this helps Good luck -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] writing scripts-off topic
Hi, Le lundi 23 janvier 2012 23:29:39 vous avez écrit : On Jan 23, 2012, at 9:46 PM, Yuri Pompeu wrote: Hello Everyone, I want to play around with some coding/programming. Just simple calculations from an input PDB file, B factors averages, occupancies, molecular weight, so forth... What should I use python,C++, visual basic? thanks Python is the most practical. Here is a simple python program: Python is certainly a nice idea, especially with cctx. But it's not perfect as it's not a compiled language. It really depends on the task you have to acomplish. To play around, it's an excellent language. STAY AWAY from proprietary nonsense like visual basic and from languages that do not facilitate reusability, like perl or other 1980's era shell languages. You will find yourself porting or abandoning your code, which is not a good use of your time. Fortran come from the 60's and is not evil. depends on what you want do. There are also quite some code already in Fortran. But so not much in fortran 90. Fortran is also not too difficult. For example, if you stay away from pointers, there is no memory leak possible by design. And python can be VERY fast for calculations if you use free and popular libraries like numpy and scipy. These librares are wrappers around optimized fortran and C libraries that you will never have to use directly. Python is fast if you don't use python that's true :D A good start is not to use loops or at least as few as possible. loops are horribly slow. Instead you need to rely heavily on numpy, it's a good way to remember your linear algebra :) I recommend staying away from very low level languages like C or fortran, too. It is good to know these languages, but not so good to use them. Your creativity should go towards implementing cool ideas and should not be squandered on plugging memory leaks. It's better to use high level languages that leverage your time most effectively. Low level languages have their advantages. It's not a good idea to stay away from them just because there is more work or it's more difficult. You cannot have 50M reflections*atoms*s^-1 processed for structure factor calculations in python. But it's true you probably won't need it every day. Brute force has some advantages over cool ideas sometimes ;) Pascal
Re: [ccp4bb] phaser openmp
Le Tue, 8 Nov 2011 16:25:22 -0800, Nat Echols nathaniel.ech...@gmail.com a écrit : On Tue, Nov 8, 2011 at 4:22 PM, Francois Berenger beren...@riken.jp wrote: In the past I have been quite badly surprised by the no-acceleration I gained when using OpenMP with some of my programs... :( You need big parallel jobs and avoid synchronisations, barriers or this kind of things. Using data reduction is much more efficient. It's working very well for structure factors calculations for exemple. Amdahl's law is cruel: http://en.wikipedia.org/wiki/Amdahl's_law You can have much less than 5% of serial code. I have more problems with L2 misse cache events and memory bandwidth. A quad cores means 4 times the bandwidth necessary for a single process... If your code is already a bit greedy, the scale up is not good. Pascal
[ccp4bb] map file specification
Hi, I am looking at the specifications of the ccp4 map file format and I am confused with the number of columns and the number of intervals. I assume that the number of columns is the grid size but what is the number of intervals (elements 8-9 in the header)? Regards, Pascal
Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.
I would like to add something about the NanoDrop versus NanoPearl, I don't think that the path length is fixed on this instrument (the NanoDrop) since if I recall well, the instruments sets the path length as it scans through the droplet, hence the characteristic clicky noise that you hear as the handle moves. This instrument requires recalibration every year (or so according to the vendors, and this is of course not cheap) since there is a moving part that can get out of alignment. On the other hand, the NanoPear from Implen as a fixed geometry for the same tiny amount of sample required. Thus you do not have to deal with moving parts and recalibration. We just bought a NanoPearl and I should also mention that this instrument does both things: nano-drop size measurement like the NanoVue AND cuvette size measurements (for OD600 or old school Bradford). All the best, On Thu, Jun 16, 2011 at 4:57 PM, Shaun Lott s.l...@auckland.ac.nz wrote: Just to add my 2c worth... The department here has a couple of nanodrops as a shared facility, one for DNA/RNA and one for protein. It has been noticeable that over time people has been getting decreased reliability of measurements on the latter machine cf cuvette measurements, presumably due to the build-up of protein deposits over time - so I would say that although it's easier to clean than a cuvette, the nanodrop is not immune to the problem. The biggest issue I see with the nanodrop is evaporation of sample. Even here in moist Auckland, where RH is very often 80%+, taking a series of measurements with the nanodrop over a period of just a minute or two shows increasing concentration in the sample. So, for consistent results, one has to be careful to measure quickly. It's probably fine for comparative measurements, but as has been observed above, not great for super-accurate values for biophysics, and I think rather operator dependent. But all our students are super-careful, right? ;) Worth to note also that ProtParam calculates extinction coefficients based on Gill von Hippel, (Gill, S.C. and von Hippel, P.H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182:319-326) who claim accuracy of ~5% for 'normal globular' proteins without extra chromophores. Whilst on this subject, I would put in a plug for the good old BCA (aka Pierce) assay for protein concentration. It's a little slow, but gets away from sequence dependency somewhat as it is primarily dependent on the peptide backbone rather than sidechains and works well in micro-titre plates etc. It is certainly very superior to Bradford. (Smith, P.K., et al. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1): 76–85. doi:10.1016/0003-2697(85)90442-7). cheers Shaun -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] GTP Agarose Resin
Hi Matthew, Most GTPases required Magnesium to hydrolyze so you maybe able to reduce this by working in presence of EDTA and absence of magnesium. This may promote removal of traces of GTP or GDP ( from previous experience with SRP GTPases I would be more worried about residual GDP). Having EDTA and no magnesium during purification helps. If your GTPase has a very low basal GTPase activity ( and some do as they require a cognate GAP to really get in the mood to hydrolyze) this might be enough to minimize hydrolysis on this resin. Good luck, -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu On Mon, Jun 13, 2011 at 12:21 PM, Matthew Bratkowski mab...@cornell.edu wrote: Hi. I was considering using GTP Agarose Resin for the final clean up step of the purification of a GTPase and was wondering if anyone has had experience using this resin. My main concerns are whether it actually has a decent binding capacity for GTP binding proteins, considering that endogenous GTP/GDP may remain tightly bound during purification, and if an active GTPase would bind without cleaving the GTP off of the resin itself. I found only a few companies that still carry the resin, but the price for each is very different. The resin from Sigma is fairly cheap and is linked to the resin via ribose hydroxyls, while the resin from Innova Biosciences is more than four times as much but is linked to the resin via the gamma phosphate, which supposively prevents cleavage by contaminating phophatases. Considering that my protein should be relatively pure during this purification step, I was wondering whether or not GTP cleavage of the resin by the GTPase and loss of binding to the column would be a problem if using the Sigma resin. If anyone has any other information about purification using this resin, such as resin binding capacity, an effective protocol with relevant buffers, and the lifetime of the resin after regeneration, I would be happy to hear it. Thanks, Matt
[ccp4bb] expression of membrane proteins as GST fusions
Dear All, This is not strictly a crystallography related question. We are trying to express several membrane proteins and were considering the use of GST as a fusion partner instead of the HIS or FLAG purification tags. I would like to know if anyone had any experience (positive or negative) to share with us. Many thanks in advance. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 364 Boyer Hall Molecular Biology Institute office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 364 Boyer Hall Molecular Biology Institute office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Hydrophobic protein surface and SDS page
Hi Debajyoti, Migration of proteins in SDS containing gels is dependent on hydrophobicity, the amount of SDS that your protein binds (despite the fact that in theory all proteins should behave the same way under these conditions) and charge. Highly basic or acidic proteins will migrate anomalously, thermophilic proteins (which are usually more highly charged and hydrophobic) also tend to migrate anomalously..membrane proteins also because they usually tend to interact differently with the detergent; it is not unusual to observe stable non-covalent homo-oligomers of membrane proteins even in an SDS-PAGE gel. Phosphorylation and glycosylation will also affect the apparent MW as estimated from the SDS-PAGE experiment. What you observe is not unusual at all. If you want to be sure of your MW, Mass Spec will tell you what you want to know. Hope this helps, good luck -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] crystallization of a weird protein
Hi Wei, this milk is precipitated/microcrystals of SDS probably with some phosphate since you are in PBS buffer. I would have two suggestions. 1-Can't you find a better detergent than SDS for your membrane protein? Have you run a detergent screen for this protein to find a milder and more crystallization friendly detergent for the reconstiution/purification and handling of your sample. SDS is very very rarely used for membrane protein crystallization. 2- I would try to avoid preparing a protein for crystallization in a phosphate containing buffer (unless you have no choice). Phosphates tend to yield more salt crystals at the screening stage. Hope this helps. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 356 Boyer Hall office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] highly glycosylated protein
Hi Wei, Glycosylation usually stabilize proteins although it is a source of structural heterogeneity for us crystallographers.Since you are expressing in HEK293 cells, there is a strain of cells that is deficient for glycosylation (it was designed by Gobind Khorana at the MIT I believe). You may want to try this. This is particularly useful when you express membrane proteins, it avoids hyperglycosylation. You may want to try a lightly glycosylated version of your protein and see if it behaves correctly, The other extreme solution is to identify all occupied sequons in your protein and eventually inactivate them by mutagenesis to have a completely deglycosylated protein. This solution is probably not the best since glycosylation usually stabilize proteins and may be essential to their biological function and activity. So it is to be considered with a lot of caution. Hope this helps. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 356 Boyer Hall office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] reproducibility of protein crystals
Anita, Proteolysis and oxydation are the most common alteration affecting proteins during the course of crystallization. If you have drops of the trays that yielded crystals I would run a gel on those drops and look at the aspect of protein still around in the drop. That would give you some clues. If there was no reducing agent in the drops I would run a gel with two samples (with and without a reducing agent such as DTT or beta-mercaptoethanol for example). you could also, if you had crystals to spare (although from what you say it does not seem the case), run a gel on a crystal (it takes a little bit of practice) to characterize what is in your crystal or if you have a mass spec at hand look at the content of a crystal. How long did those crystals take to grow? Is there a skin covering your drops? Hope this helps -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
[ccp4bb] Protease inhibitor cocktails and protein crystallization.
Dear All, I apologize if the questions has already been asked on this forum. We are purifying a membrane that seems prone to proteolysis. Although we use Protease Inhibitor cocktails during lysis and the first step of purification we get rid of them after and only keep PMSF and EDTA as general anti-protease control agents. I am considering reincluding the cocktail of inhibitors at the last purification step (a size exclusion in our case) and was wondering if having this infamous mixture of peptidic inhibitors (for the most part) around during crystallization would be a problem: specifically getting crystals of these inhibitors. Does anyone have extended experience in this matter. Many thanks in advance. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] off topic: GPCR membane insertion/orientation
Hi Justin, Since GPCRs are polytopic a-helical transmembrane proteins, it is very likely that (1) insertion into the membrane is primarily performed by the Sec61 complex AKA translocon and (2) targeting to the membrane would be controlled by the signal recongition particle and its receptor. the latter implies that a N-terminal signal sequence (that may very well be the first TM of a GPCR) would control the insertion process. Does your favorite GPCR have a predicetd signal sequence? Sec61 in theory contributes to signal sequence orientation according to the positive-inside end rulebut as for any rule they are exceptions. there is a set of excellent papers dissecting this mechanism by Skach WR NSMB (2009) 16:6 606-12 (review) Pitonzo Skach Mol Biol Cell (2009) 20(2) 685-698 (article) Sadlish H Skach NSMB (2005) 12(10) 870-878 (article) Sadlish and Skach J Membrane Biol 202 115-126 (2004) (review) You may also want to look in the work of the group of Art Johnson (paper by Woolhead et al) describing the insertion process of aquaporin by the sec61 complex. they are polytopic a-helical membrane proteins and you may want to look into these articles since they dissect the process of TM insertion, orientation and protein maturation quite well. Hope this helps, Best regards -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Lysis of Pichia pastoris
Hi Cory, I am afraid to say that Pichia and Saccharomyces are tough cells to break compared to bacteria. We also purifiy membrane proteins in yeast in my lab so we have gone through this process. With regular yeast we can break in an emulsiflex but it takes some hard work and this is not really viable option on a large scale once you process several liters of cell culture. So we use a bead-beater ( from BIOSPEC) and glass beads (0.5 mm for yeast, the diameter depends on the kind of organism you want to disrupt). it works well and is quite fast , however you have to deal with the bead cleaning part and the losses due to the mass of liquid trapped in the beads (rinsing of beads with buffer is fine but it results in an increase burden at the membrane centrifugation step) It takes some optimization (mass of beads/mass of cells processed), but once this is is set this is probably the way to go, Bead beaters come in various sizes depending on the volumes you want to process. It is just a blender after all. Hope this helps. Best regards -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Confirming expression of a GPCR in HEK293
Hi Qing, I would recommend either the use of GFP as mentioned by Jacob or a his-tag or a flag-tag. C-terminal tagging is preferred to prevent interference with signal sequences at the N-terminus of the protein. Flag tag is really good for detection , the commercial antibodies for detection are really great, however it is not that great, in my hands, when it comes to purification of a membrane protein (in presence of detergent) I prefer his-tags to flag-tags in this case. You can use a his-flag to combine both advantages. Hope this helps -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] how to optimize crystallization of a membrane proteinf
Hi Qiangmin, All the comments and references that were already mentioned to you are excellent, I would stress 3 points. 1- The detergent. A clear distinction should be made between the detergent used for extraction/solubilization and the detergent (or cocktail of detergents/lipids) used for crystallization. These are two very different things. If you are lucky you may not need to change, but once you have extracted your rmembrane protein in one detergent, you should try to characterize its homogeneity by size exclusion chromatography in different detergents ( with shorter or longer chains and/or belonging to a different class (change from a choline or a phospho-glycero-lipid to an alkyloside or from a charged to an uncharged detergent etc). This scouting is tedious but is extremely informative and it can be done on analytical scales (so it does not require too much protein). If you like statistics about detergent use you can look there. *http://www.mpdb.tcd.ie/* depending on the class of membrane protein beta-barrel versus all-alpha helical etc etc you can initially concentrate your efforts on a subset of detergents. 2- The diffraction. As mentioned, starting with very poorly diffracting crystals is not uncommon (as it is for RNA crystals). My own personal experience is that you can get from 40 A resolution to the dreadful 6-4.0 A resolution barrier by tweaking the purification/extraction conditions (1/ changing detergent (shorter chain) and 2/ carefully controlling the amount of detergent present in the sample used for crystallization (to avoid or at least minimize the phase separation problem)). 3- The cryo conditions. Crystallization drops in presence of detergent are actually not as homogenous at it seems. Within the same drop you may have crystals of identical size and morphology and freeze them in the same condition and still get very different diffraction limits. When you freeze your crystals matching the detergent concentration in your cryo-condition with the 'expected' concentration in the drop can be extremely important especially with alkylosides (personal experience). Good luck, -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] problem in annealing
Hi Hussain, I think you need to edit your input file and increase the max number of chain and the max number of tree . I had this problem several times especially if the structure is big or the chain(s) is(are) fragmented. The default values in CNS usually work but you can increase them and it should go through. HTH -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
[ccp4bb] metal-chelating affinity chromatography and FosCholine detergents
Dear All, I apologize for the not strictly crystallography-related query. I am currently purifying several membrane proteins solubilized in fos-cholines detergents and I consistently observe a significant loss of protein at the binding step (done in absence of imidazole). Has anyone else experience the same quite systematic (so far in my hands) problem with this class of detergents. I would appreciate any comments or advices from biochemists that face(d) the same situation. Thanks in advance -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] question - GFP fusion - cleavage sites
Hi Celina, I cannot answer to your question concerning the GFP-related problem. Fot the thrombin vs TEV protease related question I can tell you that in my hands thrombin works really very well in most detergents (TEV is somehow more sensitive). I am working on membrane proteins purified in very different detergents such as OG, DM, DDM, FosCholines and mixed lipids/detergents mixture and I see good (very good) cleavage with thrombin. We buy it from Sigma ( bovine thrombin) and it can be stored for years at -20C in a suitable buffer without loss of efficiency. Hope this helps, Cheers Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu n Mon, May 24, 2010 at 5:05 AM, Celina R. r.g.cel...@gmail.com wrote: Dear CCP4er's, Sorry for the non-crystallography related question and was hoping someone on the bulletin board might have some suggestions to overcome my peculiar protein purification problem. I am working on several membrane proteins (for crystallization trials) that have a C-Terminal eGFP fusion partner followed by a His-tag. The membrane protein and the GFP-His tag are separated by a TEV protease site. After purifying the fusion protein by IMAC, I add TEV protease to cleave the linkage between the membrane protein and the GFP-His tag.The cleavage reaction is also dialyzed to get rid of the imidazole. This cleavage seems to go to completion as judged by SDS-PAGE. However, when I try to separate the membrane protein from the GFP-His tag by passing through a IMAC column twice (excess nickel resin), a significant amount (about 1 mg) of the GFP-His tag doesn't bind the IMAC column and flows through along with my protein. In addition, other methods such as centricons (30, 50 or 100 kDa M.W.C.O.), Gel Filtration and Ion-Exchange are also not able to separate them. All my buffers have 5 mM reducing agent and 500 mM NaCl to try and prevent any non-specific interaction between my protein and the GFP-His tag. It appears that the GFP-His tag is somehow stuck to my protein and co-elutes on any chromatographic column that i use. Has anyone encountered such a problem and managed to overcome it? Any suggestions/tricks would be helpful. I also have a question: Which is better to use for the cleavage of His-tags, in case I want to clone the membrane protein without GFP: TEV protease or thrombin? Thanks in advance. C. --
[ccp4bb] continuous flow centrifuge
Dear All, Sorry for the not strictly crystallography-related question. We are currently setting up a fermentation core and are considering purchasing a T1 Sharples continuous flow centrifuge. It is a mid-range capacity instrument. We will be processing bacteria and yeast. I was wondering if anyone had experience with this type of instrument and would be willing to share his/her thoughts about it. Thank you very much in advance. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] UV microscope for screening
Dear Scott, We had a UV inspection microscope from KORIMA to look at crystals by UV fluorescence. It works relatively well but it is expensive ( way too much in my opinion), you pay for the microscope, the UV source and the software (named Wasabi) that comes with it An alternative to that is described in the paper from Alan D'Arcy and coll. Acta Cryst D63 (2007) p550-554. They use a DUVI 204 LIght source (from PLS design GmbH , in germany) adapted to their Crystal Score system. If I understood well, this is a just a UV source light adapted to your microscope of choice. It is probably much cheaper and as efficient. Hope this helps. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Expression of large proteins in E. coli
Hi Nick, Some success has been reported for large soluble proteins using the C41(DE3) and C43(DE3) *E. coli* strains (see the paper by Miroux Walker). Also you can try another promoter/expression system , the pBAD expression system based on arabinose induction. Hope this helps. Best -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
[ccp4bb] Post Doctoral Position at UCLA. Membrane Protein Complexes.
*Post-Doctoral Researcher. Structure and Function of Membrane Protein Complexes*. We seek a motivated individual to join our laboratory in the Department of Biological Chemistry at the University of California in Los Angeles. We are interested in studying the mechanisms of protein folding/translocation and organelle biogenesis in diverse systems. Research projects involve the study of various multi-subunits membrane protein complexes using X-ray crystallography and cryoEM. State-of-the-art equipment is available in a modern research setting including robotic crystallization, cryoEM and fermentation facilities. Synchrotron radiation time is available at the Advance Light Source in Berkeley and also at the Advanced Photon Source in Chicago. *Qualifications. *Applicants should have received a Ph.D degree in a field relevant to structural biology (X-ray crystallography or cryoEM) and/or molecular biophysics and have a good background in protein expression and purification. Crystallographers applying should have some experience in experimental phasing. *Contact.* To apply please email the following to pe...@mednet.ucla.edu: a CV, the contact information for 2 or 3 references (e-mail, address, phone), a list of experimental expertise and a brief research statement describing your past research and future goals. Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
[ccp4bb] Bad geometry for alt. conformation refined in Refmac5
Hello All, We are trying to refine ARG residues with two conformations in Refmac5, and the refined atom positions in the output PDB file are all over the place, as if the geometry restraints are not well defined. We've tried several different formats for the input file, based on previous postings to the bulletin board and the PDB standard (two examples below), but the result is always the same. We are using Refmac5 in CCP4 Suite 6.1.2, GUI 2.0.5 on Mac OSX. We'd appreciate any suggestions. Thanks. -John Examples of ARG format: 1) ATOM 1472 N ARG A 1 -5.737 26.887 38.372 1.00 29.53 CN ATOM 1473 CA ARG A 1 -5.445 25.560 37.882 1.00 30.24 CC ATOM 1474 CB ARG A 1 -5.314 24.548 39.036 1.00 30.63 CC ATOM 1475 CG ARG A 1 -5.426 23.052 38.627 1.00 34.81 CC ATOM 1476 CD AARG A 1 -4.827 22.075 39.644 0.50 37.09 CC ATOM 1477 CD BARG A 1 -4.301 22.419 39.279 0.50 37.09 CC ATOM 1478 NE AARG A 1 -3.430 21.777 39.304 0.50 42.71 CN ATOM 1479 NE BARG A 1 -4.482 21.902 40.627 0.50 42.71 CN ATOM 1480 CZ AARG A 1 -2.998 20.868 38.402 0.50 44.91 CC ATOM 1481 CZ BARG A 1 -3.648 22.142 41.638 0.50 44.91 CC ATOM 1482 NH1AARG A 1 -3.841 20.117 37.678 0.50 45.20 CN ATOM 1483 NH1BARG A 1 -2.584 22.912 41.464 0.50 45.20 CN ATOM 1484 NH2AARG A 1 -1.688 20.715 38.210 0.50 44.99 CN ATOM 1485 NH2BARG A 1 -3.878 21.619 42.831 0.50 44.99 CN ATOM 1486 C ARG A 1 -6.518 25.176 36.830 1.00 29.97 CC ATOM 1487 O ARG A 1 -7.675 25.501 36.971 1.00 31.02 CO 2) ATOM 44 N AARG A 1 26.671 62.112 46.990 0.50 30.13 AN ATOM 45 CA AARG A 1 26.970 63.346 47.667 0.50 30.65 AC ATOM 46 CB AARG A 1 27.172 64.495 46.676 0.50 31.07 AC ATOM 47 CG AARG A 1 27.152 65.897 47.322 0.50 34.20 AC ATOM 48 CD AARG A 1 27.993 66.976 46.599 0.50 37.16 AC ATOM 49 NE AARG A 1 27.726 67.425 45.342 0.50 42.06 AN ATOM 50 CZ AARG A 1 28.315 67.639 44.168 0.50 44.78 AC ATOM 51 NH1AARG A 1 29.525 67.160 43.918 0.50 45.37 AN ATOM 52 NH2AARG A 1 27.690 68.340 43.240 0.50 45.67 AN ATOM 53 C AARG A 1 25.839 63.640 48.622 0.50 30.37 AC ATOM 54 O AARG A 1 24.690 63.377 48.340 0.50 31.24 AO ATOM 55 N BARG A 1 26.667 62.080 47.010 0.50 30.13 AN ATOM 56 CA BARG A 1 26.921 63.329 47.640 0.50 30.65 AC ATOM 57 CB BARG A 1 27.108 64.390 46.581 0.50 31.07 AC ATOM 58 CG BARG A 1 27.138 65.756 47.103 0.50 34.20 AC ATOM 59 CD BARG A 1 28.447 66.452 46.933 0.50 37.16 AC ATOM 60 NE BARG A 1 28.377 67.657 47.707 0.50 42.06 AN ATOM 61 CZ BARG A 1 29.349 68.269 48.373 0.50 44.78 AC ATOM 62 NH1BARG A 1 30.572 67.836 48.373 0.50 45.37 AN ATOM 63 NH2BARG A 1 29.066 69.368 49.034 0.50 45.67 AN ATOM 64 C BARG A 1 25.822 63.636 48.638 0.50 30.37 AC ATOM 65 O BARG A 1 24.698 63.372 48.403 0.50 31.24 AO John Pascal, PhD Thomas Jefferson University Department of Biochemistry Molecular Biology 233 South 10th Street, BLSB 804 Philadelphia, Pennsylvania 19107 ph 215.503.4596 fx 215.923.2117
Re: [ccp4bb] off topic, design of a self-cleaving tag
Hi Daniel, look at this the Profinity eXact Fusion-tag system from BioRad* * the protease i fused to your protein and self activated by halides (F or I I think). Cleavage in on column. The principles is clever, now the cleavage conditions may not suit to your protein, but it seems to work. This is for expression and purification purposes only. Hope this helps -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] small molecule library
I can suggest the following reading Discovering novel ligands for macromolecules uisng X-ray crystallographic screening Nienaber VL et al, Nature Biotechnology vol 18 october 2000 pp1105 -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] lysozyme
Hi Camille, I don't know if you have any protein labs around you but if someone is using rosetta or codon-plus type expression Ecoli strains those cells usually contain a plysS plasmid derivative that is chloramphenicol resistant and carries the gene encoding lysozyme among other things (plus the rare tRNA genes). If they don't have the plasmid at hand you can still grow the cells and make a miniprep of the plasmid (low copy) to have a lysozyme gene in your hands. Hope this helps. Let me know if you can't find it. -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry 314 Biomedical Sciences Research Building office (310)-825-1013 lab (310)-825-8722 email pe...@mednet.ucla.edu
Re: [ccp4bb] Adding a transmembrane segment
Hi,if you add the TM segment, I would expect that the protein will then get targeted to the membrane and hopefully inserted correctly. My guess is that you will have to treat your protein as a membrane protein or a membrane-anchored protein...which means prepare a membrane fraction, use detergent and look for your protein in there. Is your ectodomain N or C-terminal (I assume it is N from your message)? Have you looked at the targeting sequences present in the protein (signal sequence, reverse anchoring motifs)? Hope this helps Pascal F. Egea, PhD
Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate
Hi Brenda, You can try sugars like glucose, trehalose and sucrose for high AS contents. It has been succesfully used in really hard cases such at protein RNA crystals grown in AS. see Acta Cryst (2002) D58 1664-1669 Garber et al. HTH Pascal Egea
Re: [ccp4bb] DNA binding protein
Hi Neeraj, An absorption spectra between 220 and 400 nm (for example) should show you if there is DNA coming along with your protein. In theory A280 is about 1.7 times A260 for a pure protein sample. This is a rough estimate. If your peak is shifted towards 260 instead of 280 then you can suspect the presence of contaminating DNA or RNA. As to get rid of the DNA, I can suggest several ways to do it. 1-An heparin affinity column could out-compete your contaminating DNA while binding your protein. They work really well. 2-Ion exchange is worth trying. It has worked for me with an extremely basic archeal RNA binding protein purified in E.coli and bringing along some endogenous RNA/DNA. An ion exchange ( Mono-S type) got rid of the contaminating nucleic acid, however with some loss of protein. 3-DNAse treatment may be worth trying but the presence of traces of DNase may be a problem with subsequent crystallization trials in presence of the bona-fide target DNA sequence. 4-Precipitation of the DNA with streptomycine sulfate. You could also loss some protein. 1 and 2 are in my opinion the less invasive soutions. Hope this helps. Best regards Pascal Egea
Re: [ccp4bb] detergent crystals
Hi Jose, It is quite difficult to crystallize DDM. The main problem is to estimate detergent concentration in the final sample. One method requires the use of chromatographic workstation coupled to a light scattering detector and refraction index measurement unit. In this case you can know if there is an excess detergent (particularly likely to be the case if you are using a low CMC detergent like DDM and if you have been concentrating your sample on a membrane with a cutoff smaller than the empty micelles). This is more involved and requires a specialized equipment. But you can detect both your protein and your excess detergent (if there is). Also to test you crystals, you may want to see if you can access a fluorescence microscope. If your protein contains tryptophan (and you have the correct wavelength) then you protein crystals should glow. Your detergent crystals should not. Those microscopes are getting more and more popular so maybe you have one next by. It is very convenient. Hope this helps Pascal Egea, PhD University of California Los Angeles Department of Biological Chemistry
Re: [ccp4bb] citrate blocks the active site
Dear Samy, You mentioned that your enzyme crystallizes in 0.1M citric acid pH3.5 plus 25% PEG 3,350. I wonder if you have tried to systematically scan for other carboxylic acids as buffer/co-precipitating salts. I would suggest you to try the serie of carboxylic acids, citrate, acetate, formate and also the magic carboxylate malonate; a little bit like the Hofmeister series. You maybe able to find a surrogate to citrate that will able you to either soak your crystals or co-crystallize successfully with your substrate(s). Hope this helps Cheers, Pascal Egea University of California San Francisco Department of Biochemistry and Biophysics
Re: [ccp4bb] Tips on fitting poorly defined loop regions
Hi Drew, Have you tried arp/wARP (the LOOPY option) or the AutoBuild option in Phenix ? If you haven't tried this you can try a complete rebuilding using you model as it stands and providing the complete sequence of your protein. At this resolution (2.1A) , they may be able to rescue your loops at least partially. There is another program called XPLEO (and a derivation of it called LoopTK) and is available from the Stanford site at the Synchrotron Linear Accelerator that has been helpful in our lab in the case of a loop region in a membrane protein. Hope this helps. Good luck Pascal F. Egea, PhD University of California San Francisco Department of Biophysics and Biochemistry
Re: [ccp4bb] Acrylamide in RNA crystallization
Hi Vanessa It is better to get rid of traces of residual acrylamide that may contaminate your final purified and refolded RNA. It is usual to have contaminations with monomeric acrylamide. NMR spectroscopists studying RNA can usually detect its presence on their spectra. If you can dialyze your purified product to try to get rid of it it would be the best. Traces (sometimes it is not a negligible amount) are not good because you may not be able to reproduce your results and optimize eventual crystals. And for RNA this can be an excruciating pain. When we transcribe RNA, we usually run the preparative acrylamide-urea gels and elute the RNA out of the gel (most of the time by electroelution). The RNA usually contains urea and acrylamide so I either precipitate using the salt/ethanol technique and then resuspend the pellet and dialyze/refold or I further purifiy on an ion exchange (Q type column) to try to clean it up. If you have an NMR spectroscopist friend around, try to look at the presence of acrylamide before and after these steps and see what works the best for you. Hope this helps Pascal Egea, PhD University of California San Francisco Department of Biochemistry and Biophysics
Re: [ccp4bb] OT: Crystallisation compatible detergents
Hi Darren I believe that the most frequently used detergent for protein crystallization (not including membrane proteins) is octyl-glucoside. The most important parameter is the CMC of the detergent and the size of the micelles of free detergent if you have micelles around. These considerations do not apply if you are well under the CMC (only free molecules of detergent are present in solution). I believe tween-20 CMC is roughly 0.006% so you are at the border. The problem is you have micelles of detergent is that depending on their size and the MW cutoff of the device you use for protein concentration you may end up concentrating both components protein and detergent and then this is when trouble starts. You may have an unknown amount of concentrated detergent above the CMC, this can be very problematic for crystallization because you may have a lot of phase separation in your drops; phase separation are usually (but not always) not desirable. For membrane protein crystallization we are always facing this problem because we usually work in this regime of critical detergent concentrations. However for soluble proteins that need a little bit of detergent to remain stable this is not as usual. OG (octyl glucoside) is a rather mild detergent with a CMC of about 20 mM (check ANATRACE catalog). To simplify, maybe a little bit too much, the most important is at first to find the minimum amount of detergent you need to keep you sample stable and possibly stay as low under the CMC. And then may be go up in detergent concentration if you don't get the results expected. You can check that your complex stays functional using Biacore (in your specific case) Other detergents very popular for non membrane proteins are DDM (dodecyl maltoside) or CHAPS (zwitterionic cholesterol derivative). So in order of decreasing preference I would suggest OG, CHAPS (which is a totally different type of detergent) and DDM (this one has a very low CMC ~0.15mM I believe, so it is difficult to get rid of it, but it is fairly genlte). An alternative to detergent are non-detergent sulfo-betaines they can sometime have the same protective effect without the trouble of detergents. I hope this helps, Cheers Pascal F. Egea, PhD University of California San Francisco Department of Biochemistry and Biophysics
Re: [ccp4bb] precipitation of deglycosylated protein
Hi Simon, Although they are a source of heterogeneity for crystallization, glycosylations usually stabilize proteins. There are a couple of things that may be important to consider before you deglycosylate your protein. Do you know how many natural sequons/glycosylation sites your protein has? And in what system/organism are you expressing your protein? I am saying that because I have the case of a bovine enzyme which has three sites. Inactivation of all sequons from Asn to Asp ( they are standards N-glycosylation sites) and expression in Pichia pastoris results in a fully-deglycosylated protein which is unstable and precipitates. However if one specific sequon is kept intact, the obtained protein is glycosylated by Pichia at this functional site, behaves very well and yields good quality crystals. The electron density maps show the two first N-acetyl glucosamine units N-linked to the Asn residue. Interestingly the protein is pretty homogenously glycosylated by Pichia. I know this may sound a little bizarre but you may get around this problems by keeping some sequons or the sequon active (if there is only one) and try to deal with a protein, glycosylated-light as you express it. This depends on what your expression system is. You can try to add stabilizing agents like glycerol, ethylene glycol or some di-sugars like trehalose. I hope this helps, Cheers, Pascal F. Egea, PhD University of California San Francisco Department of Biochemistry and Biophysics On Mon, Mar 16, 2009 at 9:29 AM, Yue Li simon.yu...@yahoo.com wrote: Hi everyone, Recently, I obtained a soluble glyco-protein. Unfortunately, after I added PNGase or Endo Hf to remove the glycans, the deglycosylated protein is precipitated. Is there any method to avoid this kind of precipitation? Thanks, Simon
Re: [ccp4bb] precipitation of deglycosylated protein
My apologies Simon, I should have been more thorough answering your question. Yes the protein was shown to be quite homogenously glycosylated using mass-spectrometry. The ED maps showed the first two ordered fully occupied NAG units and residual density for a third sugar unit although it is very poor defined. Pascal Egea
Re: [ccp4bb] Purification with ligand
Hi Mariah, You may need to specify what type of ligand (is it a nucleotide, a small synthetic molecule, a peptide etc ?) and also what is the affinity between your ligand and your protein. I have purified several protein-ligand complexes, you can go several routes. If you have a high affinity binder and fairly 'cheap' or abundant ligand it is possible for example to add it in your crude extract (or even in your bacterial culture in some extreme cases). It may end-up improving your 'expression yield' in terms of soluble protein readily available in your initial extract. I have used this approach successfully when purifying the ligand-binding domain of a nuclear receptor with its very hydrophobic natural ligand (retinoic acid) or a synthetic drug. In this case it was giving a more homogenous population of protein at the start of the purification. I included the hormone in the crude extract after sonication and centrifugation (so at an early stage of the purification). After that I kept ligand around in the buffer (Cobalt affinity chromatography and gel filtration (at low concentration) and kept adding ligand to the concentrated protein. If your ligand has some kind of specific UV absorption, it can be very easy to monitor its presence and the 'saturation level' of your protein. If you have a high-affinity binder, it can be a very efficient way to start with homogenous population of protein-ligand complex. This approach is really useful when your ligand happens to be only soluble in protein-unfriendly solvents like ethanol or acetone (this was the case for retinoic acid); in the crude extract despite the addition of alcohol, your protein won't suffer too much from the presence of added alcohol and if affinity is high and you add enough of it, you will efficiently saturate it. In another case, a complex between two GTPases, I had to use a non-hydrolyzable GTP analog. The compound was far too expensive to be used in the crude extract. In this case , we purified the two apo-proteins separately, formed the complex in presence of ligand and included ligand in the ion-exchange chromatography buffers and in the gel filtration buffer (at a low concentration though but it helped us to stabilize the complex). Again it all depends on the affinity.If you decide to include ligand in your gel-filtration buffer, keep also in mind that you will contaminate your columns and it can be hard to get rid of some ligands sometimes. Sorry, if all this was a little bit too long. Hope this helps, Pascal Egea, PhD University of California San Francisco Department of Biochemistry and Biophysics Laboratory of Robert Stroud On Thu, Feb 26, 2009 at 2:58 PM, protein.chemist protein.chemist pp73...@gmail.com wrote: Hello, I wanted to know if there is a standard procedure for purification of protein with ligand. I have never done this before so it will be nice to get some help. Thanks, Mariah -- Mariah Jones Department of Biochemistry University of Florida
Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
Hi Mo, Gene synthesis is definitely something you should try if you can afford it. However, I would suggest also trying to change expression plasmid and in particular induction system and promoter system. For toxic proteins we had some success using the pBAD (invitrogen) expression system using arabinose induction and the arabinose operon promoter different from the classical T7 promoter. It is tightly regulated and has a very fast kinetic of induction. It is worth trying even combined with gene synthesis. I also remember that some kinases are efficiently expressed in Ecoli in presence of another expression plasmid encoding two chaperones , the trigger factor and GroES/EL. This is worth trying too. Hope this helps, cheers. Pascal Egea, PhD University of California San Francisco Department of Biochemistry and Biophysics Stroud Lab
Re: [ccp4bb] Se oxidation
Hi,I believe it is not important as long as you run a proper scan of the crystal. Both forms will allow proper phasing. This is very well described in a paper from Thomazeau et al. here is the reference MAD on threonine synthase: the phasing power of oxidized selenomethionine. Acta Crystallogr D Biol Crystallogr.javascript:AL_get(this,%20'jour',%20'Acta%20Crystallogr%20D%20Biol%20Crystallogr.'); 2001 Sep;57(Pt 9):1337-40. Epub 2001 Aug 23. Cheers, Pascal Egea, PhD Post Doctoral Researcher UCSF Department of Biochemistry and Biophysics Stroud laboratory On Mon, Feb 9, 2009 at 10:27 AM, aka akaka druida...@hotmail.com wrote: Dear All I would like to know whether oxidation of Se entails any problem for SAD or MAD experiments and/ or how to resolve it. Cannot use DTT or reducing agents in my protein (extracellular and disulphide bonds are important). Thanks Dr. R.Depetris Weill Cornell Medical College -- Get 5 GB of storage with Windows Live Hotmail. Sign up today.http://windowslive.com/Explore/Hotmail?ocid=TXT_TAGLM_WL_hotmail_acq_5gb_112008
[ccp4bb] Job Announcement - FACULTY POSITION IN X-RAY CRYSTALLOGRAPHY
FACULTY POSITION IN X-RAY CRYSTALLOGRAPHY Department of Biochemistry and Molecular Biology Thomas Jefferson University in Philadelphia USA Job Description: The Department of Biochemistry and Molecular Biology at Thomas Jefferson University in Philadelphia invites applications for tenure-track or tenured faculty positions in the areas of X-ray crystallography of proteins or nucleic acids. We seek established investigators who are currently supported by extramural funding and who have demonstrated research excellence and productivity. The Department offers a highly collaborative culture and provides state-of-the-art facilities for advanced structural biology and biophysical work. The successful candidate is expected to establish a dynamic and independently funded research program and participate in graduate training at the interface between biology, biochemistry, and biophysics. How to respond: Applicants should submit a curriculum vitae, a brief statement of research interests and future plans, and names of at least three references to: Professor Ya-Ming Hou Thomas Jefferson University Department of Biochemistry and Molecular Biology 233 South 10th Street, BLSB 220, Philadelphia, PA 19107 Email: ya-ming@jefferson.edu Thomas Jefferson University is located in center city Philadelphia, adjacent to a variety of cultural, entertainment and historical attractions. Affirmative Action/Equal Opportunity Employer.
[ccp4bb] FACULTY POSITION IN X-RAY CRYSTALLOGRAPHY
FACULTY POSITION IN X-RAY CRYSTALLOGRAPHY Where: Department of Biochemistry and Molecular Biology Thomas Jefferson University in Philadelphia USA Job Description: The Department of Biochemistry and Molecular Biology at Thomas Jefferson University in Philadelphia invites applications for tenure-track or tenured faculty positions in the areas of X-ray crystallography of proteins or nucleic acids. We seek outstanding established investigators with demonstrated research excellence and a solid track record of extramural funding. The Department offers a highly collaborative culture and provides state-of-the-art facilities for advanced structural biology and biophysical work. The successful candidate is expected to establish a dynamic and independently funded research program and participate in graduate training at the interface between biology, biochemistry, and biophysics. How to respond: Applicants should submit a curriculum vitae, a brief statement of research interests and future plans, and names of at least three references to: Professor Ya-Ming Hou, Thomas Jefferson University, Department of Biochemistry and Molecular Biology, 233 South 10th Street, BLSB 220, Philadelphia, PA 19107. Email: [EMAIL PROTECTED] Thomas Jefferson University is located in center city Philadelphia, adjacent to a variety of cultural, entertainment and historical attractions. Affirmative Action/Equal Opportunity Employer. John Pascal, PhD Thomas Jefferson University Department of Biochemistry Molecular Biology 233 South 10th Street, BLSB 804 Philadelphia, Pennsylvania 19107 ph 215.503.4596 fx 215.923.2117
