Re: [ccp4bb] Question about merging of data from different crystals
Hi Yuan Cheng, You have received plenty of advice as to how to proceed with your problem. There is still one thing that no one mentioned, and in my opinion, is the simplest way to go about what you want to do. There are plenty of monoclinic cells with a beta that is very close to 90, but when you start with orthorhombic, you don't necessarily know which axis is the unique one. So I would go back to the beginning, and at the autoindexing step, choose the same cell setting for both crystals, whichever space group you choose. You have already identified that a and b had been switched. You don't have to live with that. You can choose a different setting when autoindexing gives you the options. Now, if you are already convinced that it is monoclinic, you still need to know which is the correct b axis, but at least the two data sets should be internally consistent, i.e. R-merge ought to be similar for the overall data set as it is for the individual one, usually an average of the two. Deciding on the correct b axis will have to be done in a different way. Usually MR (I assume you do have a starting model) is very good at selecting the correct setting. You start with your chosen setting, and if that gives you a good solution, then you have got the correct setting very likely. Just to convince yourself, you can try the other setting. It could much better or much worse. It rarely is about the same, because that would require a tetramer in the asymmetric unit, which has a 4-fold local symmetry. I doubt this is what you are dealing with. If I understand the gist of your message you have 2 copies in the a.u. Good Luck. Pierre ** Dr. Pierre Rizkallah, Senior Lecturer in Structural Biology, WHRI, School of Medicine, Academic Avenue, Heath Park, Cardiff CF14 4XN email: rizkall...@cf.ac.uk phone + 44 29 2074 2248 Yuan Cheng ych...@email.unc.edu 22/10/09 9:52 PM Hi everyone, I have some question about the merging of diffraction data from different crystals. I have two (room-temperature) datasets in my hand and each of them can be scaled in P21 space group and the screw axis is along k. However,each dataset only have below 55% completeness. To get better completeness, I am trying to merge this two datasets. The problem is one dataset has cell dimension (80.632,87.085,114.977),(90,90.026,90) and the other one has (85.497,79.857,114.003) (90,90.004,90),. It looks like that the a,b dimensions are switched between these two datasets. Unsurprisingly,the chi^2 is very high when I tried to merge these two datasets in scalepack.I am wondering whether there is any way to reindex these two datasets to make their a/b dimensions match. Any suggestion will be highly appreciated. By the way, as you might notice, the beta angle is pretty close to 90 degree. I scaled the datasets into P212121 or P2221 or P21212 at the beginning,I had no trouble to merge them in these space groups. However,I could not make the Rfree go below 45% during following refinement (2.7 angstrom cutoff). Phenix.xtriage indicated that there might exist twinning but no twin law is given. Then I reindexed the data into P2 and scaled them in P21 SG.Phenix.xtriage indicates there exists a pseudomerohedral twinning operator. When I used the twin law given in phenix.refine,the Rfree could go down to 29%. So I think P21 might be the correct space group. Thanks again for any suggestion. Yuan
Re: [ccp4bb] Changes in Cell Constants
Hi Jacob That doesn't surprise me at all, though the example you heard is probably towards the extreme end of what we've seen. We have seen individual cell parameter changes up to 10% on soaking/freezing which could easily add up to 20-30% change in cell volume. One problem with freezing is that it's almost impossible to reproduce the freezing conditions exactly (e.g. due to variations in concentrations of organic solvent, buffer, ligand, protein in the rates of freezing), even using the same ligand, so that you can easily get variations in cell parameters just induced by freezing alone. Note this puts a 'spanner in the works' of maintaining the same Rfree set across a series of ligand complexes (as discussed in a recent thread), because if the cell parameters change by even a few per-cent the structures become non-isomorphous, and then what you think are the same set of reflections in different datasets, actually define quite different points in the molecular transform. Since I don't believe this 'Rfree bias' exists beyond the first refinement anyway this doesn't bother me, but it should bother others who do believe that it's necessary to take extreme measures to 'shake out' such bias. Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: 22 October 2009 20:21 To: CCP4BB@JISCMAIL.AC.UK Subject: Changes in Cell Constants Dear Crystallographers, I have just returned from a seminar in which Robert Sauer said that they saw a change in cell volume by 25% upon soaking crystals with substrate (ATP-gammaS). He also showed that one of the cell constants changed by about -25 Ang from 200 Ang. I am assuming that the space group did not change--otherwise this phenomenon would not deserve comment. I asked whether he could see the crystals change size under the microscope, and he said a hard-to-interpret yes. Has anybody seen such large changes in cell constants in soaks, without cracking? If so, could you actually see the crystals shrink as the ligand entered the crystal? Regards, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu *** Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
[ccp4bb] Converting map to mtz file
Dear All, I have masked a map in coot and exported it. Now I'm trying to use SFall to convert the map file to mtz file but I get the error msg: FATAL DISAGREEMENT BETWEEN INPUT INFO AND MAP HEADER. How to solve this? Any suggestion? Thanks in advance. Regards, Anita
Re: [ccp4bb] Sigma Cutoff in HKL2000 Data Processing
I guess you mean higher redundancy/completeness in the HIGHER resolution shells. Rather than decreasing that sigma cutoff, a better solution is to increase the profile fitting radius. The default value is fine for most lab-based detectors, but too small for most (larger) synchrotron detectors. Making that value larger allows reflections further away to be included in the profile fitting, rather than including weaker reflections. In my hands this has often changed the completeness of the high resolution shell from 30% to 90% as weak reflections will no longer be ignored. There is a button in one of the graphic windows to show the profile fitting radius, it will show a circle that follows the mouse pointer around. The profile fitting radius should be made large enough that everywhere (within your resolution limit) you go, 5-10 strong reflections will be inside that circle. Jose. Ed Pozharski epozh...@umaryland.edu wrote: On Thu, 2009-10-22 at 10:18 -0400, protein.chemist protein.chemist wrote: What is the Sigma Cutoff that one should use for Data Processing using HKL2000. Since you say HKL2000, I assume that you mean the Refinement Sigma Cutoff in index tab. The parameter, imu, determines which reflections will be considered strong and used in parameter refinement and (?) profile fitting. The default value is 5.0, which is just fine for good data. I do, however, routinely set it to lower value of 3.0, since it was my observation that then you get higher redundancy/completeness in lower resolution shells. There is some evidence that the mechanism here is related to rejections due to incomplete profiles. Obviously, as you reach the outer rim, strong reflections become sparse and if you are also using the relatively small default value of the profile fitting radius, large number of reflections may be rejected because denzo can't calculate average profiles in their vicinity (I expected that in the absence of the profile the integrated intensity should be used instead, but perhaps it's not the case). Is there a minimum or maximum value. I'd say it makes no sense to go below 1.0, but you can sure try and see what happens. Upper limit is obviously defined by the point where you don't have enough strong reflections for robust refinement of parameters. The absolute values will, of course, vary from dataset to dataset. -- -- *** Jose Antonio Cuesta-Seijo Biophysical Chemistry Group Department of Chemistry University of Copenhagen Tlf: +45-35320261 Universitetsparken 5 DK-2100 Copenhagen, Denmark ***
[ccp4bb] Beam time is available at the NSLS
Please visit the website at www.px.nsls.bnl.gov and select Get Access to Beam Time. The PXRR (Macromolecular Crystallography Research Resource at the NSLS) operates five beamlines for macromolecular crystallography (MX). Two of these beamlines are undulators: X29 is the most efficient MX machine east of Chicago in the US, often serving three different research groups in a day. X25 is as bright, and has counted Rod MacKinnon, Tom Steitz, and Venki Ramakrishnan among its prize-winning users. Typical beam sizes at these undulators are 50 microns. If your crystal is only 20 microns, we can give you a beam that size. There are ALS-style automounters at X29 and two of our dipole beamlines, X12-B and X12-C. We have a new station at X26-C for the coordinated measurement of optical absorption spectroscopic and x-ray diffraction data. Several publications have come from this work and many projects are underway. A Raman spectrometer is being installed now. The PXRR is funded jointly by the NIH's National Center for Research Resources and the DOE's Office of Biological and Environmental Research. = Robert M. Sweet E-Dress: sw...@bnl.gov Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Biology Dept Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) =
[ccp4bb] Alterate conformers destroyed by Refmac!
All - we're having a problem with Refmac (version 5.5.0102) in CCP4 6.1.2 that I compiled from source using ifort v11.0 on Centos 4.6. When I refine a structure with a HIS in alternate conformations (all atoms except N, C O doubled up) it completely destroys the sidechains of both copies. Same thing happens with any other residue type (e.g. SER). All single-conformer residues, including some ligands are fine. Has anyone else noticed this? Is this a known problem with this version/compiler/OS? Cheers -- Ian Ian J. Tickle, DPhil. Director of X-ray Technology Astex Therapeutics Ltd 436 Cambridge Science Park Milton Road, Cambridge CB4 0QA, UK Tel: +44(0)1223 226214 Fax: +44(0)1223 226201 www.astex-therapeutics.com Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] Alterate conformers destroyed by Refmac!
Few people had complains about this. It seems to be related with compilation. If you take the version from York's website then it should work fine. www.ysbl.york.ac.uk/refmac/latest_refmac.html I have compiled it for mac 10.5 and it does not seem to work on mac 10.4. I am trying to make it platform independent but if you want 10.4 then I can compile the latest version for this platform also. regards Garib On 23 Oct 2009, at 14:04, Ian Tickle wrote: All – we’re having a problem with Refmac (version 5.5.0102) in CCP4 6.1.2 that I compiled from source using ifort v11.0 on Centos 4.6. When I refine a structure with a HIS in alternate conformations (all atoms except N, C O doubled up) it completely destroys the sidechains of both copies. Same thing happens with any other residue type (e.g. SER). All single-conformer residues, including some ligands are fine. Has anyone else noticed this? Is this a known problem with this version/compiler/OS? Cheers -- Ian Ian J. Tickle, DPhil. Director of X-ray Technology Astex Therapeutics Ltd 436 Cambridge Science Park Milton Road, Cambridge CB4 0QA, UK Tel: +44(0)1223 226214 Fax: +44(0)1223 226201 www.astex-therapeutics.com Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing i.tic...@astex-therapeutics.com and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
Re: [ccp4bb] Converting map to mtz file
Inverting a map with sfall has different restrictions on allowable gridpoints and axis order within the electron density map (due to FFT vs slow FT). Check what the sfall documentation lists as requirements for your spacegroup, and adjust the coot map output settings as needed (or if it's easier, you can probably use a combination of mapmask and mapman to get the grid, axis and range parameters to where sfall'll be happy with them). Pete FYI - I think this is technically an inversion instead of a conversion step. Conversion usually refers to changing the same type of information into a different file format; this is changing between two different types of information. Anita paula Testa salmazo wrote: Dear All, I have masked a map in coot and exported it. Now I'm trying to use SFall to convert the map file to mtz file but I get the error msg: FATAL DISAGREEMENT BETWEEN INPUT INFO AND MAP HEADER. How to solve this? Any suggestion? Thanks in advance. Regards, Anita
[ccp4bb] polygon stand alone - updated version
Hello, An updated stand-alone version of the program POLYGON (Urzhumtseva et al., 2009, Acta Cryst, D65, 297-300) is available at http://www-ibmc.u-strasbg.fr/arn/Site_UPR9002/Polygon/Polygon.html A number of bugs were fixed; in particular this version was successfully tested at a Mac computer (in addition to linux and Windows). Best regards, Sacha
Re: [ccp4bb] polygon stand alone - updated version
Alexandre Urzhumtsev wrote: Hello, An updated stand-alone version of the program POLYGON (Urzhumtseva et al., 2009, Acta Cryst, D65, 297-300) is available at http://www-ibmc.u-strasbg.fr/arn/Site_UPR9002/Polygon/Polygon.html A number of bugs were fixed; in particular this version was successfully tested at a Mac computer (in addition to linux and Windows). Best regards, Sacha Under which license did you release the software? -- Justin Lecher Institute for Neuroscience and Biophysics ISB 3 - Institute for structural biochemistry Research Centre Juelich GmbH, 52425 Juelich,Germany phone: +49 2461 61 5385 signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] Alterate conformers destroyed by Refmac!
Not exactly the same setup, but maybe close enough. I used Intel's 11.1 compilers on RHEL4.8 to build refmac (5.5.0102 and 5.5.0105) and it will not run the refmac_tls script in the CCP4 example area. It turns out that the LAPACK libraries (3.0) that comes with RHEL4 and the Intel (11.1) compiler does not play nice (10.1 worked). Building a newer version of netlib LAPACK or using Intel's mkl_lapack makes refmac happy. This may not have anything to do with the problems you see but nevertheless, it's worth considering... Thomas On 10/23/2009 06:04 AM, Ian Tickle wrote: All – we’re having a problem with Refmac (version 5.5.0102) in CCP4 6.1.2 that I compiled from source using ifort v11.0 on Centos 4.6. When I refine a structure with a HIS in alternate conformations (all atoms except N, C O doubled up) it completely destroys the sidechains of both copies. Same thing happens with any other residue type (e.g. SER). All single-conformer residues, including some ligands are fine. Has anyone else noticed this? Is this a known problem with this version/compiler/OS? Cheers -- Ian -- Thomas Eriksson | thomas.eriks...@slac.stanford.edu SSRL/Structural Molecular Biology |Tel (650)926-4530 2575 Sand Hill Rd. MS99 |Cell (650)714-6004 Menlo Park, CA 94025 |Fax (650)926-3292
[ccp4bb] Using own orientation matrix with imosflm.
Hi All I have a predetermined matrix from labelit, how can I use it with imosflm? I add the matrix file under 'Images', but I cannot integrate. Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
[ccp4bb] Postdoctoral position, MacCHESS, Ithaca NY
MacCHESS (Macromolecular diffraction at Cornell High Energy Synchrotron Source) has an opening for a post-doctoral associate to work on developing novel methods of 3D visualization of protein crystals mounted on a synchrotron beamline, for the purpose of crystal centering and motion planning during data collection. Confocal microscopy will be the initial method investigated. The position is suitable for persons with a Ph.D. in biophysics, physics, optical engineering, image processing, spectroscopy, or similar fields and a strong interest in hands-on development of equipment and techniques. Experience in Python or Java GUI programming is a plus. Located on an ivy-league university campus in picturesque upstate New York, the Cornell High-Energy Synchrotron Source (CHESS) serves a world-wide user base of structural biologists, chemists, physicists, and engineers. MacCHESS is an NIH-supported National Resource providing support for structural biology at CHESS. MacCHESS is a heavily team- oriented environment. Good clear communication skills are a must, including fluency in the English language. This is an entry-level post-doctoral position for one year, renewable for an additional year subject to mutual agreement and continued availability of funds. Please provide an application and arrange to have at least three letters of reference sent to: Dr. Marian Szebenyi, Chair Postdoctoral Associate Search Committee Newman Lab Cornell University Ithaca, NY 14853 USA Applications should include a cover letter, curriculum vita, a publication list, and a detailed summary of research experience and interests. Electronic submissions and inquiries may be addressed to search-l...@cornell.edu. Deadline for application is November 15, 2009. Starting date is negotiable. Cornell is an equal opportunity employer.
[ccp4bb] pdb submission with SS bond + alternate and author defined ss
Hello, I have two short questions about submitting a structure to the PDB: 1- How should I submit a structure that has a SS-bond with alternate free cysteines (knowing that the SSBOND record does not support alternate conformation)? 2- How can I keep my author-defined secondary structures during the conversion of the pdb to mmCIF with pdb_extract ? Thanks in advance for your comments, Lionel
Re: [ccp4bb] measure detergent concentration
Only easy if you happen to have silica gel TLC plates and a chromatography jar lying around, perhaps from some phospholipid analysis: A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins Laura R. Eriks, June A. Mayor, and Ronald S. Kaplan Analytical Biochemistry 323 (2003) 234–241 This paper recommends spotting on a TLC plate and running beside standard amounts of the same detergent. From intensity/size of the detergent spot after developing you can bracket the detergent concentration. (And by the way they found that detergents are concentrated by ultrafiltration). To increase sensitivity, speedvac a volume too large to spot on the plate, dissolve the residue in Me0H. Ed wei...@crystal.harvard.edu wrote: Hi Folks: After concentrating a membrane protein, is there a (easy) way of measuring the detergent concentration in the sample? Regards, Weikai
Re: [ccp4bb] measure detergent concentration
Weikai, We did it using NMR but you asked for a simple way so I guess I'm out of topic. Anyway, since I believe it is the most accurate method, here it is: using a high detergent concentration stock solution you can assign resonance peaks to your detergent molecule bonds. Then you can set up a standard curve using different known detergent concentrations (for example from 10% down to 0.1%) by calculating the surface of your peak(s) which is directly related to your detergent concentration. Each time you need to know the concentration of a new sample, you just need to record the peaks, and use the three-click rule to deduct the unknown value. As a colleague answered you earlier, we noticed that a 50kDa cutoff withheld a lot of detergent during concentration process and consequently your final concentration might increase significantly. For example we started with 0.25% DES and noticed increases of above 1%. Of course this will depend on the concentration factor. This did not happen when using a 100kDa cutoff, and DES concentration remain pretty much constant. Now, it will depend on your system: what detergent you are using, since micel size and CMC are obviously the critical parameters here -- but also what maximal cutoff you can use w/o loosing your membrane protein in the flow through... Good luck, Michael - Original Message - From: Patrick Loll To: CCP4BB@jiscmail.ac.uk Sent: Friday, October 23, 2009 1:12 PM Subject: Re: [ccp4bb] measure detergent concentration I'll second this. We've done this as an exercise in NSLS Membrane Protein Crystallization workshop for a few years, and it works like a charm. You can stain in a warm iodine chamber and visualize by scanning the TLC plate on a garden variety scanner (we use an inexpensive Canon LIDE that probably cost less than USD 60 five years ago). We quantify the spot intensity with NIH Image or equivalent, and get lovely linearity down to the CMC, spotting only 1 uL of sample--so we haven't seen any need to concentrate. On 23 Oct 2009, at 3:41 PM, Edward A. Berry wrote: Only easy if you happen to have silica gel TLC plates and a chromatography jar lying around, perhaps from some phospholipid analysis: A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins Laura R. Eriks, June A. Mayor, and Ronald S. Kaplan Analytical Biochemistry 323 (2003) 234–241 This paper recommends spotting on a TLC plate and running beside standard amounts of the same detergent. From intensity/size of the detergent spot after developing you can bracket the detergent concentration. (And by the way they found that detergents are concentrated by ultrafiltration). To increase sensitivity, speedvac a volume too large to spot on the plate, dissolve the residue in Me0H. Ed wei...@crystal.harvard.edu wrote: Hi Folks: After concentrating a membrane protein, is there a (easy) way of measuring the detergent concentration in the sample? Regards, Weikai --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] measure detergent concentration
Also depending on the detergent , If you have a detergent like decyl-maltopyranoside ( DM) or any other glycoside based detergent you can use the reaction with sulfuric acid and phenol followed by Absorption measurement to quantitate as detailed in Anal Biochem. 2005 Jan 1;336(1):117-24. A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research. Urbani A, Warne T. Using a standard curve against the same detergent you can quantitate your detergent concentration in your final protein sample Hari On Fri, Oct 23, 2009 at 4:35 PM, Michael Matho mma...@scripps.edu wrote: Weikai, We did it using NMR but you asked for a simple way so I guess I'm out of topic. Anyway, since I believe it is the most accurate method, here it is: using a high detergent concentration stock solution you can assign resonance peaks to your detergent molecule bonds. Then you can set up a standard curve using different known detergent concentrations (for example from 10% down to 0.1%) by calculating the surface of your peak(s) which is directly related to your detergent concentration. Each time you need to know the concentration of a new sample, you just need to record the peaks, and use the three-click rule to deduct the unknown value. As a colleague answered you earlier, we noticed that a 50kDa cutoff withheld a lot of detergent during concentration process and consequently your final concentration might increase significantly. For example we started with 0.25% DES and noticed increases of above 1%. Of course this will depend on the concentration factor. This did not happen when using a 100kDa cutoff, and DES concentration remain pretty much constant. Now, it will depend on your system: what detergent you are using, since micel size and CMC are obviously the critical parameters here -- but also what maximal cutoff you can use w/o loosing your membrane protein in the flow through... Good luck, Michael - Original Message - From: Patrick Loll To: CCP4BB@jiscmail.ac.uk Sent: Friday, October 23, 2009 1:12 PM Subject: Re: [ccp4bb] measure detergent concentration I'll second this. We've done this as an exercise in NSLS Membrane Protein Crystallization workshop for a few years, and it works like a charm. You can stain in a warm iodine chamber and visualize by scanning the TLC plate on a garden variety scanner (we use an inexpensive Canon LIDE that probably cost less than USD 60 five years ago). We quantify the spot intensity with NIH Image or equivalent, and get lovely linearity down to the CMC, spotting only 1 uL of sample--so we haven't seen any need to concentrate. On 23 Oct 2009, at 3:41 PM, Edward A. Berry wrote: Only easy if you happen to have silica gel TLC plates and a chromatography jar lying around, perhaps from some phospholipid analysis: A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins Laura R. Eriks, June A. Mayor, and Ronald S. Kaplan Analytical Biochemistry 323 (2003) 234–241 This paper recommends spotting on a TLC plate and running beside standard amounts of the same detergent. From intensity/size of the detergent spot after developing you can bracket the detergent concentration. (And by the way they found that detergents are concentrated by ultrafiltration). To increase sensitivity, speedvac a volume too large to spot on the plate, dissolve the residue in Me0H. Ed wei...@crystal.harvard.edu wrote: Hi Folks: After concentrating a membrane protein, is there a (easy) way of measuring the detergent concentration in the sample? Regards, Weikai --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
