[ccp4bb] Advanced Light Microscopy Facility at EMBL Heidelberg
Light microscopy and fluorescence techniques permit the analysis not only of individual proteins but also functional macromolecular complexes, which have increasingly come into the focus of modern structural biology. The Advanced Light Microscopy Facility (ALMF) at European Molecular Biology Laboratories (EMBL) in Heidelberg offers a collection of state-of-the-art light microscopy equipment. Access for up to 4 weeks to the facility is provided free of charge under the Advanced Infrastructure Initiative P-Cube, sponsored within the European FP7 program. A wide range of light microscopy techniques can be applied from the determination of protein localisation in cells or tissue, analysis of protein interactions and dynamics to the tracking of dynamic processes in live environment. We offer the possibility to perform free of charge sophisticated microscopy experiments using the adequate equipment with the support from experts. For more information, please email wolfgang.hueb...@embl.de or visit the following websites : http://www.embl.de/research/units/scb/mueller_christoph/projects/index.html http://www.p-cube.eu/
[ccp4bb] 9th NCCR Symposium on New Trends in Structural Biology - Registration now open
Dear colleagues, NCCR Structural Biology cordially invites you to its annual symposium at the University of Zürich, Switzerland. 9th NCCR Symposium on New Trends in Structural Biology 1-2 September 2011 Register online at the symposium website: http://www.structuralbiology.uzh.ch/symposium2011 Confirmed speakers: Jamie Cate, James J. Chou, Vadim Cherezov, Jennifer Doudna, Youxing Jiang, Alan E. Mark, Tom Muir This is also an opportunity to meet the State of the Art in Swiss Structural Biology. Please contact us for any additional information you may require. We look forward to welcoming you! Best wishes, Sraboni Ghose The NCCR Structural Biology is a research initiative of the Swiss Science Foundation. Its research encompasses the fields of recombinant protein technologies, macromolecular structure determination and computational biomolecular sciences with a special focus on membrane proteins and supramolecular assemblies/interactions. 14 research groups from Swiss Universities and Research Institutions participate in this network.www.structuralbiology.uzh.ch/ -- ___ Visit the NCCR on the Internet: http://www.structuralbiology.uzh.ch Sraboni Ghose, PhD Scientific Officer NCCR Structural Biology Institute of Biochemistry University of Zurich Winterthurerstrasse 190 CH-8057 Zurich Switzerland Phone +41 / (0)44 / 635 54 84 Fax+41 / (0)44 / 635 59 08 Mail s.gh...@bioc.uzh.ch
[ccp4bb] Reproducing crystals.
Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
Re: [ccp4bb] Reproducing crystals.
Maybe your old solution evaporated then you end up in your old tube with a more concentrated solution in PEG and NaCl so try to screen with new conditions with higher PEG and/or NaCl concentration Mick 2011/4/12 Jun Yong Ha j...@princeton.edu Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance. -- -- Mickael Blaise PhD Department of molecular biology Centre for structural biology Aarhus university Gustav wieds vej 10 8000 Aarhus-Denmark
Re: [ccp4bb] Reproducing crystals.
or PEG 4000 got old. ask around in the department or university for old PEG 4000 bottles. good luck! Berta On Apr 12, 2011, at 3:28 PM, mickael blaise wrote: Maybe your old solution evaporated then you end up in your old tube with a more concentrated solution in PEG and NaCl so try to screen with new conditions with higher PEG and/or NaCl concentration Mick 2011/4/12 Jun Yong Ha j...@princeton.edu Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance. -- -- Mickael Blaise PhD Department of molecular biology Centre for structural biology Aarhus university Gustav wieds vej 10 8000 Aarhus-Denmark
[ccp4bb] Postdoctoral positions in Bioinformatics and Computational Biology (University of Michigan)
Postdoctoral positions in Bioinformatics and Computational Biology (University of Michigan) Highly motivated and creative postdoctoral candidates are sought to work at the Yang Zhang Lab, the Center for Computational Medicine and Bioinformatics (CCMB), University of Michigan Medical School. The candidates are expected to work on projects including protein structure prediction, protein-ligand docking and protein design (more details can be found at http://zhanglab.ccmb.med.umich.edu/research). Candidates with background in physics, biophysics, biology, computer science and bioinformatics are welcome to apply. The ability in computer programming using languages such as C++, fortran, or python/perl and the ability in independent article writing are necessary. Previous experience in protein structure modeling, protein docking or protein design is a plus. To apply, please send your CV, a brief description of your research background and interest to Dr. Yang Zhang (Email: z...@umich.edu).
Re: [ccp4bb] Reproducing crystals.
Hi, Some anecdotes here for your reference: One paper I read says that the authors were having trouble reproducing a crystal from an initial screen. After some debugging, they realized that it was because that they used a same pipette tip when making screens. Adding a little solution from the condition prior to the hit condition solved the mystery. Another paper I read said that they failed reproducing crystals until realizing that the only success they had was that they forgot to add well solution to the hanging drop(a very small protein domain though). Of course there comes the famous example that the pioneer crystallographer used a metal container to transfer meat from the butcher's for making his protein. Then it turned out that the magic bullet for his success in crystallization was the galvanized Zinc(or nickel?). I guess there must be tons of such stories if we read through Acta D. :) PEG is known to decompose in solution, I think something like lactate might be generated. If your solution is strongly buffered by hepes (100mM at pH7.5 for example), the pH may not changed much even if you have a few mM of lactate. -- From: Jun Yong Ha j...@princeton.edu Sent: Tuesday, April 12, 2011 7:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reproducing crystals. Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
Re: [ccp4bb] Reproducing crystals.
PS, it might be a good time to start an additive screen. -- From: Jun Yong Ha j...@princeton.edu Sent: Tuesday, April 12, 2011 7:56 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reproducing crystals. Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
Re: [ccp4bb] program to calculate electron density at x,y,z (SUMMARY)
Dear Tim, On Mon, 2011-04-11 at 10:44 +0200, Tim Gruene wrote: since you pointed it out I wonder if there is any reasonable (i.e. w.r.t. data error/ resolution) difference between the interpolated values and the calculated value. I actually doubt that That should depend on the quality of interpolation primarily (i.e. grid used, resolution, etc). With a 1.9A test case the discrepancy runs somewhere around 3%, which likely is within experimental error. This is using sftools to calculate the map and mapman peek command, all with defaults. Direct calculation versus interpolation looks like this http://tinyurl.com/67f79oe For the record, getting this via mapman is not entirely trivial. Atoms should be inside the map (expanding map to the full unit cell and shifting all the atoms by symmetry inside the (0,1) range was my solution). Also, it fails for some atoms (~0.5% in this case) with Spline interpolation error, in which case the output pdb file has ** instead of B-factor (perhaps switching to interpolation can fix that). Also for the record, I believe there is a bug in sftools that messes up the expansion to P1, but only for some space groups/circumstances. For example, another test case in P3121 looks like this (~30% average discrepancy) http://tinyurl.com/69wqhvp but upon closer inspection, the maps after expansion into P1 (or those calculated with sftools FFT command) show a lot of noise. Cheers, Ed -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] program to calculate electron density at x,y,z (SUMMARY)
On Fri, 2011-04-08 at 18:06 -0700, Pavel Afonine wrote: phenix.map_value_at_point map_coeffs.mtz label=2FOFC point=1 2 3 point=4 5 6 Cool. Afaiu, this is interpolation. A useful extension would be automatic picking of (x,y,z) from a pdb-file (a la mapman), although a determined person can definitely come up with a script that converts a pdb file into a list of point statements. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] Reproducing crystals.
Frances Jurnak published a paper in 1986 on PEG impurities and purification. As I recall, it turns out that different manufacturers put different additives in PEGs as preservatives. These are generally anti-oxidants. PEGs do get oxidized. I suggest you heat up your new PEG solutions to say 80 deg C and cool them down, then use them. Let us know what happens. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jun Yong Ha Sent: Tuesday, April 12, 2011 6:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reproducing crystals. Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
[ccp4bb] Promoting oligomer dissociation
Hi, I have a protein that shows high and low MW peaks on gel filtration (which run at the same MW on SDS-PAGE). There is a slow equilibrium because rerunning the individual peaks on gel filtration a couple days later shows both peaks. The higher MW peak is ~2 orders of magnitude more dominant...the lower MW peak is not yielding much. However, as nature would have it I've only gotten the lower MW peak fractions to crystallize, and only when the affinity tag is clipped (the higher MW species is resistant to clipping). I would like to do something to shift the equilibrium towards the lower MW species. So far, I've tried (without success or clues for success) changing pH, increasing NaCl from 200 to 600mM, adding glycerol to 12%. Things that I've seen in papers but have not yet tried are temperature jumps, other salts, limited Gu/urea, Arg/glu, dioxane, limited SDS/triton. Any suggestions are greatly appreciated, thanks very much, Mike
Re: [ccp4bb] Reproducing crystals.
You might also try to control the degree of oxidation using the microwave, and setting up trials after different numbers of cycles of heating. Kendall On Apr 12, 2011, at 12:41 PM, Jim Pflugrath wrote: Frances Jurnak published a paper in 1986 on PEG impurities and purification. As I recall, it turns out that different manufacturers put different additives in PEGs as preservatives. These are generally anti-oxidants. PEGs do get oxidized. I suggest you heat up your new PEG solutions to say 80 deg C and cool them down, then use them. Let us know what happens. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jun Yong Ha Sent: Tuesday, April 12, 2011 6:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Reproducing crystals. Hi all, Recently, I produced crystals with MBClass1-64 which contains PEG4000, HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up tray with different batch of solution. I got the crystals only from 2008 solution, but not from fresh ones. I asked technical service of Qiagen, but they did not have any stock. pH between fresh and old solution is the same. I could reproduce crystals with this old solution 100% when setting up. Do you have any experience like this? Is PEG4000 degraded or oxidized? Please help me. Thanks in advance.
Re: [ccp4bb] Promoting oligomer dissociation
Do you have a reducing agent in your solutions? I.e., maybe you are seeing disulfides? JPK On Tue, Apr 12, 2011 at 1:27 PM, Michael Kenneth Fenwick m...@cornell.edu wrote: Hi, I have a protein that shows high and low MW peaks on gel filtration (which run at the same MW on SDS-PAGE). There is a slow equilibrium because rerunning the individual peaks on gel filtration a couple days later shows both peaks. The higher MW peak is ~2 orders of magnitude more dominant...the lower MW peak is not yielding much. However, as nature would have it I've only gotten the lower MW peak fractions to crystallize, and only when the affinity tag is clipped (the higher MW species is resistant to clipping). I would like to do something to shift the equilibrium towards the lower MW species. So far, I've tried (without success or clues for success) changing pH, increasing NaCl from 200 to 600mM, adding glycerol to 12%. Things that I've seen in papers but have not yet tried are temperature jumps, other salts, limited Gu/urea, Arg/glu, dioxane, limited SDS/triton. Any suggestions are greatly appreciated, thanks very much, Mike -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Promoting oligomer dissociation
Thanks for all your suggestions so far...as a quick reply to some: You say the fractions are in equilibrium - how about keeping the oligomer fraction each time and adding it to the subsequent preparation? I did this once. The equilibrium is sort of a gift that keeps on giving, but the problem is the amount it's giving. In the end, this might be the only way to go, but it's very tempting to find a chemical solution. Do you have a reducing agent in your solutions? I.e., maybe you are seeing disulfides? For native preps I used 1mM DTT throughout...for the SeMet prep I used 3mM DTT. To confirm it's not S-Ss, I'm about to run SDS-PAGE lacking reducing agent. Changing buffer from Tris/Hepes into phosphate or citrate or acetate, or if higher pH borate? When I tried lower pH I used citrate. I might give the others a try. Stay away from SDS/Triton because they will almost certainly kill your crystallization and it will be hard, very hard if not impossible to get rid of them. Thanks for the tip! Another person off the bulletin board suggested differing how cell lysis is done I used sonication.
[ccp4bb] methods to capture proteins from cell culture medium
Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 joybeiyang
Re: [ccp4bb] possible pseudo-merohedral twin?
Without seeing your NZ or L-test plots, but looking at the logs, your data does not appear to be twinned. It will not be R32 because the R-merge is too high. The probable space group is C2. The listed twin fractions will approach 0.5 if your data has perfect twinning or you processed in too low symmetry so the twin operator is actually crystallographic (not a twin operator). The latter is most likely your case. You may have pseudo-symmetry but without the plots, cannot say for sure. You probably cannot solve it by MR because your search model may not be accurate... Jon -- Jonathan P. Schuermann, Ph. D. Beamline Scientist NE-CAT, Building 436E Advanced Photon Source (APS) Argonne National Laboratory 9700 South Cass Avenue Argonne, IL 60439 email: schue...@anl.gov Tel: (630) 252-0682 Fax: (630) 252-0687 On 04/12/2011 04:27 PM, Teresa De la Mora wrote: Hello all I have data that I'm trying to see if it is a pseudo-merohedral twin. The data process well in C2 or P1 but it does flag R32. I tried to process in R32 but it didn't work so I processed in both C2 and P1. C2 cell: 104.4 61.6 96.9 90.0 112.9 90.0; P1 cell: 60.7 60.6 95.5 107.1 91.9 118.9. I tried MR in both unit cells looking for 2 mol/ASU in C2 or 4 mol/ASU in P1 and got nothing. I tried Phaser, molrep, epmr and no solutions was given for any of these programs using both C2 or P1. I use a complete model as well as a trimmed model (obtained from SMM) and didn't work either. I used a monomer as well as a combination of dimers as seen in other published models for this protein, again no solution. I also ran an SDS-gel of these crystals and it showed the same MW as the protein I placed for crystallization. So I looked at back posts from the mail list and I ran phenix.xtriage with C2.mtz and P1.mtz. Both runs resulted in no pseudo-translation nor -symmetry found. However, in P1 there is a pseudo-merohedral twin operator: k, h, -h-k-l and the Britton analysis gives 0.441, H-test gives 0.450 and ML gives 0.458. This is not shown on C2 analysis. In the P1 analysis it recommends to process data in C2 so I'm guessing that's why in the C2 analysis there was no similar twin operator. But it makes me think that perhaps I don't have a twin crystal since I'm assuming that if it was twin, it should have been shown on C2 analysis, am I making the right assumptions? I tried to reprocess the data using the unit cell recommended in the P1 analysis which is the same as the C2 cell except that beta=126.76 but it doesn't index well. This is my first encounter with a possible twin data, could you please look at the attached logs files and please tell me if I read them correctly? And if it is not twin, not pseudo-translated nor -symmetry, what other things could be hindering the structure solving? Thank you for your kind suggestions/answers. Teresa Teresa De la Mora-Rey Ph.D. Dept. Medicinal Chemistry University of Minnesota 8-101 Weaver-Densford Hall 308 Harvard St. SE, Minneapolis, MN 55455 Lab phone (612) 626-5226 If you never did you should. These things are fun and fun is good Dr. Seuss
Re: [ccp4bb] methods to capture proteins from cell culture medium
Bei, I had a former labmate who had the same situation and would load somewhere between 6-8L of media directly onto a column. I don't remember what type of column it was, ion exchange may not be ideal if the ionic strength of your medium is high. I think it may have been a phenyl sepharose column. Good luck, Mike - Original Message - From: joybeiyang joybeiy...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, April 12, 2011 2:13:49 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] methods to capture proteins from cell culture medium Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 joybeiyang -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] program to calculate electron density at x,y,z (SUMMARY)
Hi Ed, yes, this is the eight-point interpolation, but since you can select to choose very small grid step for the map calculation (grid_step parameter), I hope this should be ok. If necessary, I can add an option so it will give you the map value at the closest grid point instead of interpolation or even both (although I guess the latter would be too much). In the next build (dev-728 and up) it will be possible to use a PDB file as a source of points. Pavel. On Tue, Apr 12, 2011 at 9:37 AM, Ed Pozharski epozh...@umaryland.eduwrote: On Fri, 2011-04-08 at 18:06 -0700, Pavel Afonine wrote: phenix.map_value_at_point map_coeffs.mtz label=2FOFC point=1 2 3 point=4 5 6 Cool. Afaiu, this is interpolation. A useful extension would be automatic picking of (x,y,z) from a pdb-file (a la mapman), although a determined person can definitely come up with a script that converts a pdb file into a list of point statements. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] Promoting oligomer dissociation
So what happened with the non-reducing gel? (If the DTT was fresh, there should be no problem, but if not...) JPK On Tue, Apr 12, 2011 at 3:21 PM, Michael Kenneth Fenwick m...@cornell.edu wrote: Thanks for all your suggestions so far...as a quick reply to some: You say the fractions are in equilibrium - how about keeping the oligomer fraction each time and adding it to the subsequent preparation? I did this once. The equilibrium is sort of a gift that keeps on giving, but the problem is the amount it's giving. In the end, this might be the only way to go, but it's very tempting to find a chemical solution. Do you have a reducing agent in your solutions? I.e., maybe you are seeing disulfides? For native preps I used 1mM DTT throughout...for the SeMet prep I used 3mM DTT. To confirm it's not S-Ss, I'm about to run SDS-PAGE lacking reducing agent. Changing buffer from Tris/Hepes into phosphate or citrate or acetate, or if higher pH borate? When I tried lower pH I used citrate. I might give the others a try. Stay away from SDS/Triton because they will almost certainly kill your crystallization and it will be hard, very hard if not impossible to get rid of them. Thanks for the tip! Another person off the bulletin board suggested differing how cell lysis is done I used sonication. -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] program to calculate electron density at x,y,z (SUMMARY)
Pavel Afonine wrote: Hi Ed, yes, this is the eight-point interpolation, but since you can select to choose very small grid step for the map calculation (grid_step parameter), I hope this should be ok. If necessary, I can add an option so it will give you the map value at the closest grid point instead of interpolation or even both (although I guess the latter would be too much). What about doing the Fourier summation at the precise location requested, in order to not calculate the map or interpolate at all? Input would be the mtz file rather than map file. eab In the next build (dev-728 and up) it will be possible to use a PDB file as a source of points. Pavel. On Tue, Apr 12, 2011 at 9:37 AM, Ed Pozharski epozh...@umaryland.edu mailto:epozh...@umaryland.edu wrote: On Fri, 2011-04-08 at 18:06 -0700, Pavel Afonine wrote: phenix.map_value_at_point map_coeffs.mtz label=2FOFC point=1 2 3 point=4 5 6 Cool. Afaiu, this is interpolation. A useful extension would be automatic picking of (x,y,z) from a pdb-file (a la mapman), although a determined person can definitely come up with a script that converts a pdb file into a list of point statements. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] methods to capture proteins from cell culture medium
Dear all, Thanks a lot for sharing, seems that either a HIC column or AS would work, and that's great, I should give both of them a try. I thought about HIC too, but do not know if it would work since the binding of protein to HIC need high salt conc. and I am not sure if the salt conc. in the sf900 or Hi5 medium is high enough (the formulation is secret, LOL), thus it is good to know that someone has succesful experience with HIC. Thank you very much again! Bei 2011-04-12 joybeiyang 发件人： mi...@chem.ucla.edu 发送时间： 2011-04-12 18:34:27 收件人： joybeiyang 抄送： CCP4BB@JISCMAIL.AC.UK 主题： Re: [ccp4bb] methods to capture proteins from cell culture medium Bei, I had a former labmate who had the same situation and would load somewhere between 6-8L of media directly onto a column. I don't remember what type of column it was, ion exchange may not be ideal if the ionic strength of your medium is high. I think it may have been a phenyl sepharose column. Good luck, Mike - Original Message - From: joybeiyang joybeiy...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, April 12, 2011 2:13:49 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] methods to capture proteins from cell culture medium Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 joybeiyang -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] program to calculate electron density at x,y,z (SUMMARY)
That is exactly what HYDENS is doing. A good interpolation with small grid steps should be equally good but with current computers and just a few hundred or even thousand points to evaluate, a classical Fourier summation is pretty fast and, for me, easier to program than a proper cubic-spline interpolation. Bart On 11-04-12 08:54 PM, Edward A. Berry wrote: Pavel Afonine wrote: Hi Ed, yes, this is the eight-point interpolation, but since you can select to choose very small grid step for the map calculation (grid_step parameter), I hope this should be ok. If necessary, I can add an option so it will give you the map value at the closest grid point instead of interpolation or even both (although I guess the latter would be too much). What about doing the Fourier summation at the precise location requested, in order to not calculate the map or interpolate at all? Input would be the mtz file rather than map file. eab In the next build (dev-728 and up) it will be possible to use a PDB file as a source of points. Pavel. On Tue, Apr 12, 2011 at 9:37 AM, Ed Pozharski epozh...@umaryland.edu mailto:epozh...@umaryland.edu wrote: On Fri, 2011-04-08 at 18:06 -0700, Pavel Afonine wrote: phenix.map_value_at_point map_coeffs.mtz label=2FOFC point=1 2 3 point=4 5 6 Cool. Afaiu, this is interpolation. A useful extension would be automatic picking of (x,y,z) from a pdb-file (a la mapman), although a determined person can definitely come up with a script that converts a pdb file into a list of point statements. -- Hurry up before we all come back to our senses! Julian, King of Lemurs -- Bart Hazes (Associate Professor) Dept. of Medical Microbiology Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521
Re: [ccp4bb] methods to capture proteins from cell culture medium
I thought about HIC too, but do not know if it would work since the binding of protein to HIC need high salt conc. and I am not sure if the salt conc. in the sf900 or Hi5 medium is high enough (the formulation is secret, LOL), thus it is good to know that someone has succesful experience with HIC. Few things: 1. With regard to the salt/ionic strength, the formulation of serum-free media cannot be far off from the traditional media. Very crudely speaking, figure an equivalent of ~200 mM NaCl. So most protein won't bind to, say, phenyl sepharose under these conditions. But your protein might be in the minority - who knows? 2. What prevents you from adding extra salt to the collected medium? Say, ~1 M ammonium sulphate final? There probably will be some precipitate forming which can be filtered away before loading onto a HIC column. 3. Hydroxylapatite. Ceramic Type I version from Bio-Rad in particular. Large size beads packed into a wide column. A great way to concentrate total protein at high flow rates. Phosphate concentration in the medium is low enough that majority of proteins will sill bind. - Dima
Re: [ccp4bb] program to calculate electron density at x,y,z (SUMMARY)
Hi Ed, yes, this is one of possible ways of doing this. Although I doubt it will make any (significant) difference in practice compared to other options. All mentioned methods should normally result in similar values. Pavel. On Tue, Apr 12, 2011 at 7:54 PM, Edward A. Berry ber...@upstate.edu wrote: Pavel Afonine wrote: Hi Ed, yes, this is the eight-point interpolation, but since you can select to choose very small grid step for the map calculation (grid_step parameter), I hope this should be ok. If necessary, I can add an option so it will give you the map value at the closest grid point instead of interpolation or even both (although I guess the latter would be too much). What about doing the Fourier summation at the precise location requested, in order to not calculate the map or interpolate at all? Input would be the mtz file rather than map file. eab In the next build (dev-728 and up) it will be possible to use a PDB file as a source of points. Pavel. On Tue, Apr 12, 2011 at 9:37 AM, Ed Pozharski epozh...@umaryland.edu mailto:epozh...@umaryland.edu wrote: On Fri, 2011-04-08 at 18:06 -0700, Pavel Afonine wrote: phenix.map_value_at_point map_coeffs.mtz label=2FOFC point=1 2 3 point=4 5 6 Cool. Afaiu, this is interpolation. A useful extension would be automatic picking of (x,y,z) from a pdb-file (a la mapman), although a determined person can definitely come up with a script that converts a pdb file into a list of point statements. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
