Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage
Hi Rafael, the simple answer is that the EDTA and DTT( or any other reducing agent) is not required for the TEV activity. You can have your TEV protease in 20 mM Tris pH 7.5 (RT) and 150 mM NaCl, plus either 10% Glycerol or 50% glycerol depending on storage conditions preference - 80 or -20C respectively. Normally you need to take care that you are dilating the TEV several fold (at least 5) in order to drop the Glycerol concentration below 10% not to have an inhibitory effect on the activity. Also your on-column cleavage buffer should not contain higher than 300 mM salt, otherwise the TEV activity will be decreased as well. Go ahead and give it a try. Good luck! Kind regards, Nikolay On Tue, Oct 31, 2023 at 3:21 PM Rafael Marques wrote: > > Hi everyone, > > I have been looking on this bb and other websites as well but I could not > find a veredict. We are suspecting that when I elute my sample from my Ni-NTA > column, the imidazole concentration (250 mM) is making it to precipitate. > Once my sample has a cleavable TEV site, I was planning to incubate my loaded > resin overnight with TEV and get my sample back simply using my lysis buffer. > And here lies the problem. Most of the TEVs are kept in EDTA and DTT and I > wonder if they are essential for its protease activity or if I could use > another reducing agent more compatible with my resin (or maybe do not add > both). I saw that someone did not have EDTA and used b-mercap. instead of > DTT. May I have your comments if you guys already faced a similar situation? > > Best wishes > > __ > > Rafael Marques da Silva > > PhD Student – Structural Biology > > University of Leicester > > > Mestrando em Física Biomolecular > Universidade de São Paulo > > Bacharel em Ciências Biológicas > Universidade Federal de São Carlos > > phone: +55 16 99766-0021 > >"A sorte acompanha uma mente bem treinada" > > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 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] Tagging a membrane protein after the Signal peptide
Dear Samer, that is quite an important point for construct design especially in the context of signal sequence. That is not a trial task, but based on the information you have shared with us I would assume that your protein does not get properly processed in the ER if you cannot detect it by Flow cytometry (assuming you Ab against the HA tag works properly). If you are detecting it on WB, the question is that is fit the expected MW or is it smaller sized protein? Is it possible to be an unspecific band? The tag should not be cleaved as the cleavage should happen between the SS and your HA/Myc tag. Also normally the SS will be cleaved co-transcriptionally therefore if you can detect it via WB it is already confirmation that the tag is there. It is mostly you are disturbing the post-translation folding/targeting which keeps your protein in the ER and therefore you might detect it via WB on the WCL, but not on Flow cytometry as it is not represented on the surface. I hope that helps, Best wishes Nikolay Dobrev On Thu, Jun 22, 2023 at 11:57 AM samer halabi <30c2162795b2-dmarc-requ...@jiscmail.ac.uk> wrote: > > Dear All, > > Sorry to be disturbing you all with my inquiry. > > I am tagging a membrane protein (MHC I) just after the Signal peptide with > either HA-tag or Myc-tag and expressing it in mammalian cells. I am able to > detect that protein by immunoprecipitation followed by Western blotting using > anti-Myc antibody or anti-HA antibody. However, I could not detect it by > flowcytometry, which could mean the tag was cleaved off or the protein is > incapable to be delivered to the cell surface among other possibilities . > > Does anyone has any experience with tagging membrane proteins after the > Signal peptide? Is there any restrictions against that? Or a better tag to > use? > > Thank you. > 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 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] TEV vs HRV3C
Dear all, I agree with all said so far: Here are some points from my experience and discussion on this topic. Most of the lab's due use TEV or 3C, depending on the cleavage site in their constructs and make their purification strategy work with what they have. Here are some points from my side: * 3C protease is much faster in cleaving the fusion protein in the 1:100 mass ratio (a test we use in cleavage protocols, even though we should use a molar ratio. Consider the difference between 20 kDa protein and 200 kDa protein, it is 10x in the amount of substrate difference) 3C cleaves in 30 mins more than 95% of the protein at 4-8C. TEV does need much longer, and as default, people go for overnight dialysis and cleavage simultaneously. Sometimes that can be too long for sensitive proteins and lead to precipitation due to chance in the buffer salt concentration etc, for an extended period. Quite prominently is observed for Nucleic acid binding protein, due to lowering the salt for IEX column and precipitation surprise overnight. * Neither 3C nor TEV requires a reducing agent, which is essential to know as many people are working with S-S-containing proteins, and they take an aliquot of the protease purified in the lab without knowing there was a reducing agent in the storage buffer. Then surprises can be observed. Consider the concentration of 1 mM DTT; even 10 or 100 diluted is 10 µM at least. * Same with a chelating agent like EDTA, no need to be added to the storage buffer; the danger comes when you cleave ion-containing proteins like Zn-fingers. * 3C is much more efficient in on/column cleavage (as was already mentioned). This simple trick gives much better purity than Imidazole elution, especially when the Ni-column is not saturated and many contaminants are bound. Releasing the protein with the protease makes it much cleaner. Most of the protease has a His-tag, which allows at the same time to bind it to do beads (of course this slows down a bit the cleavage therefore mixing is required). Alternatively, GST-(only)-tag protease is better used for the cleavage on Ni beads and then captured on the GSH resin. * 3C protease is much more efficient for cleaving in the presence of detergent, there is an extensive study on that topic (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836831/) * Both proteases are easy to produce, as mentioned, and easily 100-300 mg can be obtained from 1 L culture (TB or auto-induction with OD= 8-16) * 3C protease is not happy at high protein concentrations (>4 mg/ml) in low salt buffer (150 mM). Therefore huge loss is observed if, during the Ni-elution, low salt buffer is used as the protein comes from the Ni-NTA at a concentration 15-20 mg/ml. Please use at least 500 mM NaCl for that step or better for any step from lysis to elution. * And yes both proteases have cross-activity and can cleave the opposite target site with lower activity. These are the few points coming from the top of my head. I hope they are helpful and wish everyone happy cleaving of the fusion proteins :) Kind regards, Nikolay Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents https://twitter.com/emblevents |http://facebook.com/embl.org | http://youtube.com/user/emblmedia Visit http://www.embl.org/events for a complete list of all EMBL events. > On 12/07/2022 11:51 PM Lior Almagor wrote: > > > In my experience, 3C can partially cut TEV sites as well. If using > sequentially, it is best to plan to use the TEV first. > > > > > On Dec 7, 2022, at 2:42 PM, Lau Kelvin > <5aaf8435dbef-dmarc-requ...@jiscmail.ac.uk > mailto:5aaf8435dbef-dmarc-requ...@jiscmail.ac.uk > wrote: > > I agree. I think it depends on the lab and vectors and personal > > preference. > > > > But David, have you used them sequentially? I have once tried to > > make a His-GST-ENLYFQ-3C construct, and I found that it was self cleaving > > during expression. However I have never tried to replicate the results in > > vitro. > > > > > > > > -- > > Kelvin Lau > > Protein production and structure core facility - PTPSP > > EPFL SV PTECH PTPSP > > AI 2146 (Bâtiment AI) > > Station 19 > > CH-1015 Lausanne > > Switzerland > > Email: kelvin@epfl.ch mailto:kelvin@epfl.ch > > Phone: +41 21 69 34494 > > > > > > > > > On 7 Dec 2022, at 21:38, David Briggs > > mailto:david.bri...@crick.ac.uk > wrote: > > > Hi Gloria, > > > > > > Both can be made very
[ccp4bb] Research Technician position (HH00211) @ EMBL Hamburg
Dear CCP4BB, I would like to bring to your attention a great opportunity to join our group (the lab of Prof. Matthias Wilmanns) at EMBL Hamburg as a Research technician. Please feel free to spread the word! Closing date: 1 May 2022 link to the job advertisement: https://www.embl.org/jobs/position/HH00211 Kind regards, Nikolay Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents https://twitter.com/emblevents |http://facebook.com/embl.org | http://youtube.com/user/emblmedia Visit http://www.embl.org/events for a complete list of all EMBL events. 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] Off topic - gene toxic for expression strains
Hi Andy, just to follow up on Christian suggestion, which is exactly the way to go. In case you are using an already pET based vector, simply try BL21-AI (https://www.thermofisher.com/order/catalog/product/C607003), which has the T7 RNA polymerase under arabinose promoter should do the trick. Also, 2% Glucose is a must in this kind of situation. I have been dealing with several toxic proteins (from the family of restriction enzymes :) ) and BL21-AI was a way to go. Please also have a look if your pET backbone has the extra copy of the lacI, which makes a difference in leakage expression. As a sum up: 1) try BL21-AI (for the induction of the target protein you will need both Arabinose (T7 RNA pol induction) and IPTG for the T7 promoter) 2) or BL21 with pLysS or pLysE both simple keep 2 % Glucose and also directly from trafo goto liquid culture in parallel of the plating approach. Let me know if you need any further tips. Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents https://twitter.com/emblevents |http://facebook.com/embl.org | http://youtube.com/user/emblmedia Visit http://www.embl.org/events for a complete list of all EMBL events. > On 04/04/2022 10:32 AM Christian Roth wrote: > > > Hi Andy, > have you tried another promotor? Arabinose is much tighter, just to be > sure that it is really not leaking. > > Cheers > Christian > > > On Mon, Apr 4, 2022 at 10:20 AM Andrew Lovering mailto:a.lover...@bham.ac.uk > wrote: > > > > > > Dear Board, > > > > > > > > Perhaps off-topic, but in the wider scope it’s relevant to many on > > here. > > > > > > > > We have a gene that we are able to clone, and propagate in DH5a etc > > non-expression cells (hence nucleotide sequence is non-toxic) > > > > > > > > But, when we attempt to transfer to an expression strain we get no > > colonies > > > > > > > > We have tried pLemo, Glucose addition, 30 degrees, C41/C43 and > > still no joy > > > > > > > > We’d welcome any suggestions here – it’s a fun protein > > > > > > > > Thanks > > > > Andy > > > > > > > > - > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > > > > > - > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > 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] [External] Re: [ccp4bb] protease inhibitor and 3c protease
Dear Dhiraj, yes, on-column cleavage works as well quite efficiently with 3C protease. I am performing it in a cold room (8-10C) for 1h normally reaches similar efficiency. Depending on your tags (if the 3C has a His-tag) you can add even more protease 1:50 then for sure 1h is more than enough. The only thing you need to ensure is that upon cleavage your protein does not bind to the IMAC resin due to internal Histidines. If that is the case you can, just collect the flow-through and pass it over the beads couple of more times to ensure efficient binding of the His-3C protease then you should have a highly pure target. Please note, I am referring to Ni-NTA Agarose beads, which you use in bach mode. The efficiency will be lower most like if you are using pre-packed columns. I hope that is helpfull. Best, Nikolay > On 02/03/2022 6:56 PM Srivastava, Dhiraj > wrote: > > > Thanks Nikolay > I am doing on column cleavage, so I am > incubating it overnight at 4-degree C. Do you think even on column digestion > is also going to be done in one hour. > > Thank you > Dhiraj > > > - > From: CCP4 bulletin board on behalf of Nikolay > Dobrev > Sent: Thursday, February 3, 2022 11:53 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [External] Re: [ccp4bb] protease inhibitor and 3c protease > > Dear Dhiraj, > the 3C protease is from the Cysteine protease family. > Therefore for sure, you can add EDTA (if it is not indented for follow up > IMAC purification) and that will inhibit most of the metalloprotease. > Furthermore, you can resort to Serine type protease inhibitors like PMSF or > Aprotinin. > > One side not, the 3C cleavage does not need to be long, as it is an > extremely potent protease. If the cleavage sequence is well accessible in > less than 1 hour at 4*C you can obtain >95% cleavage when you use 1:100 ratio > protease to substrate. Increasing the temperature speeds up the cleavage > dramatically and at room temperature, you can obtain the same efficiency for > 10 mins approx. > I am assuming you are interested in using the 3C protease to cleave a tag > from recombinantly expressed protein. > > > Kind regards, > Nikolay > > > > > On 02/03/2022 6:25 PM Srivastava, Dhiraj > wrote: > > > > > > Hi > > sorry for off topic question. does anyone know which protease > > inhibitors we can include safely while cleaving with 3C protease? > > > > Thank you > > Dhiraj > > > > > > --------- > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > > > > Nikolay Dobrev > Postdoctoral Fellow @ Wilmanns group > EMBL Hamburg, c/o DESY, Building 25A, > Notkestraße 85, 22607 Hamburg, Germany > T +49 40 89902 165 | M +49 173 684 0532 > twitter.com/emblevents https://twitter.com/emblevents > |http://facebook.com/embl.org | http://youtube.com/user/emblmedia > Visit http://www.embl.org/events for a complete list of all EMBL events. > > > > - > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > > - > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents https://twitter.com/emblevents |http://facebook.com/embl.org | http://youtube.com/user/emblmedia Visit http://www.embl.org/events for a complete list of all EMBL events. 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] protease inhibitor and 3c protease
Dear Dhiraj, the 3C protease is from the Cysteine protease family. Therefore for sure, you can add EDTA (if it is not indented for follow up IMAC purification) and that will inhibit most of the metalloprotease. Furthermore, you can resort to Serine type protease inhibitors like PMSF or Aprotinin. One side not, the 3C cleavage does not need to be long, as it is an extremely potent protease. If the cleavage sequence is well accessible in less than 1 hour at 4*C you can obtain >95% cleavage when you use 1:100 ratio protease to substrate. Increasing the temperature speeds up the cleavage dramatically and at room temperature, you can obtain the same efficiency for 10 mins approx. I am assuming you are interested in using the 3C protease to cleave a tag from recombinantly expressed protein. Kind regards, Nikolay > On 02/03/2022 6:25 PM Srivastava, Dhiraj > wrote: > > > Hi > sorry for off topic question. does anyone know which protease > inhibitors we can include safely while cleaving with 3C protease? > > Thank you > Dhiraj > > > - > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents https://twitter.com/emblevents |http://facebook.com/embl.org | http://youtube.com/user/emblmedia Visit http://www.embl.org/events for a complete list of all EMBL events. 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] Searching Sequence Motif
Hi there, do you want only to search for motifs in the PDB database for E. coli proteins or are you interested in the search for all E. coli proteins? This tool might work for you: https://www.genome.jp/tools/motif/MOTIF2.html you need to pick for favourite organism eco for Escherichia coli K-12 MG1655 Cannot be 100% sure if you select PDBSTR, if it will really use the PDB database, but it is worth trying. you can look up the list for all organisms here: https://www.genome.jp/kegg/catalog/org_list.html Best wishes, Nikolay Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents https://twitter.com/emblevents |http://facebook.com/embl.org | http://youtube.com/user/emblmedia Visit http://www.embl.org/events for a complete list of all EMBL events. > On 09/05/2021 5:54 PM Jon Cooper > <488a26d62010-dmarc-requ...@jiscmail.ac.uk> wrote: > > > How long and specific is the motif? If its something like GxxG, then I > can't help, sorry, but if it's something like a domain then will the search > by sequence tool at RCSB help at all or have I missed the point? Good luck, > Jon.C. > > > Sent from ProtonMail mobile > > > > Original Message > On 5 Sep 2021, 16:23, Cryo EM < cryoemfa...@gmail.com> wrote: > > > > Hi all, > > > > I want to search NCBI/PDB for all E.coli proteins with a specific > > sequence motif. > > Is there any server/software in which I can search and display all > > the proteins in E.coli with this specific sequence motif? > > Suggestions are highly appreciated. > > > > Thanks! > > > > > > > > > > - > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > > > > > > - > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 > > > > > 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] protein oligomer
It really depends from the nature of the protein and if is oligomerizing/agregating/forming polymers if any of this is reversible. On the other side if you are working with one of the fibril forming protein it will require optimizaiton on its on as they will form naturally polymers. Do you observed different specises when you analyze your protein by SEC or if you are able to perfom DLS? Additional information regarding your protein will be really helpful for more detalied suggestions how to overcome your protein. Best, Nikolay Nikolay Dobrev Scientific Officer, Protein Expression and Purification Core Facility EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany T +49 6221 387 8633 | M +49 173 684 0532 twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia Visit www.embl.org/events for a complete list of all EMBL events. On 19/07/2020 14:15, S. Mohanty wrote: Keep the protein concentration low during purification steps along with using other anti-aggregation agent/s. Make sure that the pH at which you are purifying is not close to the pI of the protein. Until completely purified, all purification steps should be performed in a cold room if it is a soluble protein. Smita On Sunday, July 19, 2020, 04:28:45 AM CDT, Sorin Draga wrote: I am not sure what you mean by polymer formation. Presuming that you have optimized your protein concentration, pH and salt concentration, you could try arginine as an anti-aggregation agent in your purification (I presume you do FPLC). Have a look at chaotropic agents used in protein purification, The answer is generally dependent on the protein/proteins you are trying to purify and is not necessarily straightforward. Kinds regards On Sun, Jul 19, 2020 at 12:08 PM 张士军 <21620150150...@stu.xmu.edu.cn <mailto:21620150150...@stu.xmu.edu.cn>> wrote: Dear all: Any ideas to decrease protein polymer formation? my protein was easy to form oligomers and precipitation when do purification,I have tried add glycerol and DTT,both didn't work. Does anyone has experience to avoid it happening. Thanks! Best Regards To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 -- Nikolay Dobrev Scientific Officer, Protein Expression and Purification Core Facility EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany T +49 6221 387 8633 | M +49 173 684 0532 twitter.com/EMBLorg | facebook.com/embl.org | youtube.com/user/emblmedia Visit www.embl.org/events for a complete list of all EMBL events. 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] Open position: Scientific Officer - Biophysical Characterization at EMBL Heidelberg
Dear CCP4 community,I would like to bring to your attention to a recently opened position for Scientific Officer (Biophysical Characterization) at Protein Expression and Purification Core Facility in EMBL Heidelberg: Closing date: 1st May 2020 Link to the job add:https://www.embl.de/jobs/searchjobs/index.php?ref=HD01727=1[]=1 (https://www.embl.de/jobs/searchjobs/index.php?ref=HD01727=1[]=1) Best Regards,Nikolay Dobrev Nikolay Dobrev Scientific Officer, Protein Expression and Purification Core Facility EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany T +49 6221 387 8633 | M +49 173 684 0532 twitter.com/EMBLorg (http://twitter.com/EMBLorg) | facebook.com/embl.org (http://facebook.com/embl.org) | youtube.com/user/emblmedia (http://youtube.com/user/emblmedia) Visit www.embl.org/events (http://www.embl.org/events) for a complete list of all EMBL events. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] CCP4BB vs COVID19
Dear all, I assume most of you are aware of the EMBL-EBI datahub which was set up in January to provide essential virus research data to all scientists, but in case someone missed it I would like to share the link: https://www.ebi.ac.uk/ena/pathogens/covid-19 (https://www.ebi.ac.uk/ena/pathogens/covid-19) You can find all relevant data, from COVID-19 genome sequencing data up to x-ray and cryo-EM structures of relevant proteins. Stay healthy, Nikolay Nikolay Dobrev Scientific Officer, Protein Expression and Purification Core Facility EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany T +49 6221 387 8633 | M +49 173 684 0532 twitter.com/EMBLorg (http://twitter.com/EMBLorg) | facebook.com/embl.org (http://facebook.com/embl.org) | youtube.com/user/emblmedia (http://youtube.com/user/emblmedia) Visit www.embl.org/events (http://www.embl.org/events) for a complete list of all EMBL events. On Sun, Mar 22, 2020 at 17:26, DUMAS Philippe (IGBMC) wrote: Relevant to the discussion: * Cell, Vol. 110, 551–561, September 6, 2002, Copyright 2002 by Cell Press An RNA Thermosensor Controls Expression of Virulence Genes in Listeria monocytogenes * Bacterial RNA thermometers: molecular zippers and switches Jens Kortmann and Franz Narberhaus NATURE REVIEWS | MICROBIOLOGY VOLUME 10 | APRIL 2012 | 255 *An RNA Thermometer Activity of the West Nile Virus Genomic 30-Terminal Stem-Loop Element Modulates Viral Replication Eciency during Host Switching Viruses 2020, 12, 104; doi:10.3390/v12010104 * Temperature triggers immune evasion by Neisseria meningitidis Edmund Loh1*, Elisabeth Kugelberg2*, Alexander Tracy1, Qian Zhang2, Bridget Gollan2, Helen Ewles2, Ronald Chalmers3, Vladimir Pelicic2 & Christoph M. Tang1,2 Nature (2013) Philippe Dumas De: "James Holton" À: "CCP4BB" Envoyé: Dimanche 22 Mars 2020 16:38:28 Objet: Re: [ccp4bb] CCP4BB vs COVID19 Thank you Patrick, RNA structure is still structural biology, so I think relevant here. It seems to me that RNA as a thermometer would be an easy hypothesis to test? Has anyone measured virulence vs temperature in cell culture? The 3D structure of the genome is no doublt important. I wouldn't want to try crystallizing the whole thing, but I wonder if this might be an excellent target for cryoEM? A challenge for that "we can classify our way out of anything" philosophy? And the result would most certainly be interesting. -James Holton MAD Scientist On 3/21/2020 8:41 AM, Patrick Shaw Stewart wrote: James, this isn't conventional structural biology, but may be of interest, and I haven't been able get any mainstream virologists to think about it. The protein sequences are obviously of interest, but so are the RNA sequences at both ends of the Covid genome, which have conserved secondary structure. A few years ago a paper came out suggesting that wild-type influenza has multiple "RNA thermometers", which may play an important role in the tropism of influenza. Similar mechanisms may exist in other respiratory viruses, including Covid. My take on this, and the relevant papers, are below. Good luck to everyone and stay well, Patrick https://oldwivesandvirologists.blog/Covid-19-and-the-trade-off-model-of-selection/ (https://oldwivesandvirologists.blog/Covid-19-and-the-trade-off-model-of-selection/) My paper in Medical Hypotheses http://douglas.co.uk/f_ftp1/ShawStewart_final_1-s2.pdf (http://douglas.co.uk/f_ftp1/ShawStewart_final_1-s2.pdf) Narberhaus, Franz, Torsten Waldminghaus, and Saheli Chowdhury. "RNA thermometers." FEMS microbiology reviews 30.1 (2006): 3-16. Chursov, Andrey, et al. "Specific temperature-induced perturbations of secondary mRNA structures are associated with the cold-adapted temperature-sensitive phenotype of influenza A virus." RNA biology 9.10 (2012): 1266-1274. Yang, Dong, and Julian L. Leibowitz. "The structure and functions of coronavirus genomic 3′ and 5′ ends." Virus research 206 (2015): 120-133. On Fri, Mar 20, 2020 at 10:59 PM James Holton wrote: You might think that as a structural biologist you won't be able to do much about COVID-19 anytime soon, but that is not true. Yes, real-world therapeutics and vaccines take time, but we have already seen just how fast we can get started. There are 21 PDBs already and some even have bound ligands. Good job Frank et al. BTW! And my personal thanks to all of you out there who are already hard at work on this. I believe this forum is an ideal place to share information and ideas on the structural biology of SARS-CoV-2 as we move forward. It's a big virus, but there are not that many proteins in it. If all of us independently do the same bioinformatics and literature searches and end up trying exactly the same thing in every lab all over the world, then that would be more than unfortunate. To that
[ccp4bb] Open position: Scientific Officer at PepCore EMBL Heidelberg
Dear CCP4 community, I would like to bring to your attention recently opened position for Scientific Officer at Protein Expression and Purification Core Facility in EMBL Heidelberg: Description: Your role: You will be responsible for the expression and purification of recombinant proteins with genetically encoded phosphorylated residues. You will also perform the functional and structural characterization of these phosphorylated proteins using various biochemical and biophysical methods and X-ray crystallography. You have: The minimum requirement for this position is a suitable degree in biochemistry, molecular biology or in a closely related scientific area. Experience with protein expression and purification methods and profound knowledge of various chromatographic techniques is essential. Teamwork, good interpersonal skills and the ability to interact with a varied multicultural scientific community are expected. Fluency in English is required. You might also have: Expertise in working on high throughput approaches would be beneficial. Experience with setting up and refining crystallization screens would be considered a plus. Please feel free to spread it out! Closing date: 7 November 2019 Link to the job add: https://www.embl.de/jobs/searchjobs/index.php?ref=HD01639=1[]=1 (https://www.embl.de/jobs/searchjobs/index.php?ref=HD01639=1[]=1) Best Regards, Nikolay Dobrev Nikolay Dobrev Scientific Officer, Protein Expression and Purification Core Facility EMBL Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany T +49 6221 387 8633 | M +49 173 684 0532 twitter.com/EMBLorg (http://twitter.com/EMBLorg) | facebook.com/embl.org (http://facebook.com/embl.org) | youtube.com/user/emblmedia (http://youtube.com/user/emblmedia) Visit www.embl.org/events (http://www.embl.org/events) for a complete list of all EMBL events. To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Anderson?Evans polyoxotungstate (TEW)
Dear all, I apologize for my off-topic question. I want to ask if anyone has so far used the Anderson-Evans polyoxotungstate (https://www.jenabioscience.com/crystallography-cryo-em/screening/xp-screen/x-tew-anderson-evans-polyoxotungstate). Did someone see an improvement in crystal quality/diffraction? Any comments will be highly appreciated. Nikolay Dobrev Nikolay Dobrev PhD Student in AG Sinning & AG Fischer Biochemie Zentrum Heidelberg University Im Neuenheimer Feld 328 69120 Heidelberg Germany Phone: +49 6221 54 4796 Email: <nikolay.dob...@bzh.uni-heidelberg.de
