[ccp4bb] Fwd: [ccp4bb] Summary: measure detergent concentration
Hi Folks, The paper Phil recommended looks very nice. Below is a summary when last time I asked people about this. Regards, Weikai Original Message Subject: [ccp4bb] Summary: measure detergent concentration Date: Wed, 28 Oct 2009 16:49:32 -0400 From: wei...@crystal.harvard.edu To: CCP4BB@JISCMAIL.AC.UK Dear All: Thanks a lot for all the responses. My question was about measuring the detergent concentration after concentrating a membrane protein sample. Here is the summary. 1. The simplest way to control the detergent concentration is to use a higher cut-off concentrator if you protein plus detergent micelle is large enough. Michael Matho: “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%. This did not happen when using a 100kDa cutoff, and DES concentration remain pretty much constant.” It is easy enough to test the “maximal cutoff you can use w/o loosing your membrane protein in the flow through”. 2. Patrick Loll, Edward A. Berry and John K. Lee suggested TLC, which seems to have the least requirement for equipments -- “silica gel TLC plates and a chromatography jar”. Patrick: “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. Edward: “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 MeOH.” 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 3. For sugar-based detergents (maltosides and glucosides), one can use some traditional chemistry to measure the sugar. Bert Van Den Berg: “do a fehling-type assay” Zhenfeng Liu: ”phenol-sulfuric acid reaction for quantification of sugars.” Biochemistry, vol 36, no. 19, 1997, p. 5887 Hari Jayaram: “sulfuric acid and phenol followed by Absorption measurement; using a standard curve against the same detergent ”. 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. 4. Christopher Law: Use surface tension properties and look at the drop shape (measure contract angle). “A small droplet of the detergent solution is deposited on a piece of Parafilm M and side views are recorded by two orthogonally arranged TV cameras.” A Novel Method for Detergent Concentration Determination. Biophys J. 2006 January 1; 90(1): 310–317. Thomas C. Kaufmann, Andreas Engel, and Hervé-W. Rémigy. 5. Ezra Peisach: by Refractive index. “Refractive index measurements were performed using an OPTILAB DSP instrument (Wyatt Technology) with a P10 cell.” Refractive index-based determination of detergent concentration and its application to the study of membrane proteins Pavel Strop and Axel T. Brunger. Protein Sci. 2005 August; 14(8): 2207–2211. 6. Michael Matho: NMR is the most accurate method “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.” 7. David Veesler and Kornelius Zeth suggested ATR-FTIR (Fourier transform infrared spectroscopy). “very accurate, fast (10min) and requires as low as 10uL of protein sample.” PVeesler, D. et al. Production and biophysical characterization of the CorA transporter from M. mazei. Analytical Biochem. (2009). 388 :115-121. Regards, Weikai
Re: [ccp4bb] How to detect the concentration of detergent?
I like the phenol-sulfuric acid colorimetric assay (Anal Biochem. 2005 Jan 1;336(1):117-24). Easy, very linear, no special ingredients/equipment required. In agreement with their paper, I also found that DDM passes through the 100 kD concentrator membranes. Ho
Re: [ccp4bb] shape complementarity
Hi Reiner I haven't seen this problem before; if you send me all the relevant files I will try to check it out. It may relate to the fact that the GRASP files were made with Chimera. sincerely Mike Lawrence > Hello everybody, > > I recently tried to feed the shape complementarity program (sc) with GRASP > surface files for visualisation. Sc is supposed to output modified surface > files, again, in GRASP format. However, the program terminated without > producing a file, or, sometimes, writing truncated ones. The GRASP files > passed the compatibility check of sc. I tested two different pdb files > with the according GRASP surface files (made with Chimera). > > The first run ended with: "At line 2825 of file > /usr/local/xtal/ccp4-6.1.13/src/sc_/sc.f > Fortran runtime error: End of file" > > In the second run, I removed all hydrogen atoms before I calculated the > surface files. Sc showed the following error: " Header information from > grasp file 1 > > format=2 > Keywords=vertices,normals,triangles > Properties= > > Decoding property list > > Updated property list is:,gproperty > Number of vertices=76375 > Number of triangles= 54697 > Size of grid= 65 > Midpoint 91.033806 91.542160 89.700684 > vertices read > accessibles read > > SC: read error in s/r read_real_line > SC: read error in s/r read_real_line > Times: User: 8.3s System:0.4s Elapsed: 0:09 > > > > > I can't make head or tail of it, maybe someone else can. > Thanks in advance for any information and advice > > > Reiner > > > __ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. __
Re: [ccp4bb] R factor & R free struck
How certain are you of the space group? I ask because in all versions of PHASER that I have used to date, it chooses the space group (when SGALTERNATIVE is set) after placing the first molecule and with additional molecules it then assumes the space group result from the first molecule is the correct space group (the PHASER team is supposed to be rectifying this). While this is often the correct space group, I have seen several cases with both PHASER and other molecular replacement programs (e.g. AMoRe) where the correct space group only becomes evident after the placement of the second molecule. In particular, I have seen a case where after the first molecule was placed, one would have chosen P 21 21 2 on the basis of statistics provided, but after the second molecule was placed, it was clearly P 21 21 21. Steven
Re: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?)
Two separate crystals, but very similar data collection strategies P On 04/10/2010, at 21.48, Jacob Keller wrote: > - Original Message - From: "Poul Nissen" > To: > Sent: Monday, October 04, 2010 2:21 PM > Subject: Re: [ccp4bb] Radiation damage with crystals containing metal centers > (TaBr people chime in?) > > > [1] the signal from Ta6Br12 is enormous and one will typically focus on low > resolution (below 7 Å) so radiation sensitivity can be handled by a fairly > low dose data collection > We collected several data sets with Ta6Br12(2+) on the Na+,K+-ATPase (Morth > JP et al. 2007) and found that although we got the strongest anom. diff. > Fourier peaks from a data set collected on the Ta peak, we got far better SAD > phases from a data set collected on the high-energy remote wavelength. This I > think is also often observed for SeMet. > > > > > > Interesting phenomenon--has it been documented, I wonder? I wonder which > datasets were collected first? If the peak was collected first, as usual I > think, and one assumes an exponential decay of the resonant signal as a > function of radiation dose, it makes sense that the resonant signal would be > more constant after the first data set, where the decay curve would have > flattened out a bit. This would also be true for two consecutive data sets > collected at high energy. Also, I think the decay function itself is steeper > at the peak wavelength, leading to a less-internally-consistent data set at > the peak. Does this argument hold water? > > Jacob Keller
Re: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?)
- Original Message - From: "Poul Nissen" To: Sent: Monday, October 04, 2010 2:21 PM Subject: Re: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?) [1] the signal from Ta6Br12 is enormous and one will typically focus on low resolution (below 7 Å) so radiation sensitivity can be handled by a fairly low dose data collection We collected several data sets with Ta6Br12(2+) on the Na+,K+-ATPase (Morth JP et al. 2007) and found that although we got the strongest anom. diff. Fourier peaks from a data set collected on the Ta peak, we got far better SAD phases from a data set collected on the high-energy remote wavelength. This I think is also often observed for SeMet. Interesting phenomenon--has it been documented, I wonder? I wonder which datasets were collected first? If the peak was collected first, as usual I think, and one assumes an exponential decay of the resonant signal as a function of radiation dose, it makes sense that the resonant signal would be more constant after the first data set, where the decay curve would have flattened out a bit. This would also be true for two consecutive data sets collected at high energy. Also, I think the decay function itself is steeper at the peak wavelength, leading to a less-internally-consistent data set at the peak. Does this argument hold water? Jacob Keller
Re: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?)
[1] the signal from Ta6Br12 is enormous and one will typically focus on low resolution (below 7 Å) so radiation sensitivity can be handled by a fairly low dose data collection We collected several data sets with Ta6Br12(2+) on the Na+,K+-ATPase (Morth JP et al. 2007) and found that although we got the strongest anom. diff. Fourier peaks from a data set collected on the Ta peak, we got far better SAD phases from a data set collected on the high-energy remote wavelength. This I think is also often observed for SeMet. [2] With a loosely bound ionic compound like Ta6Br12(2+) you will tend to loose occupancy if you backsoak. 1 mM normally works fine for initial screening to see if it will form a useful derivative - then you can always play with parameters later Poul On 04/10/2010, at 18.28, Francis E Reyes wrote: > Hi all > > I'm reading a recent review by Elspeth Garman regarding radiation damage > (Acta Cryst D) and in this she mentions two ideas regarding metal centers: > > [1] They (The metal complexes themselves) are quickly reduced > > [2] Their absorption causes localized heating in the crystal. (This causes > the temp to increase above freezing thus allowing OH radicals to diffuse). > > How is it that people can phase off of TaBr considering [1]? Presumably as > TaBr is absorbing x-ray's whatever anomalous signal that was present at the > beginning of the collection is either absent/significantly altered by the > time data collection is complete? > > Regarding [2], is TaBr usually backsoaked (into cryoprotectant not containing > TaBr) to reduce it's concentration in the crystal to highly occupied sites? > It would seem to me that having a crystal soaked with TaBr would cause more > heating than one backsoaked. > > > > Thanks > > F > > > > - > Francis E. 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
Re: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?)
Regarding backsoaking [2], This is one of those nice exercises for the reader! Try raddose calculations with versus without 1 mM heavy-atom compound in the solvent channels. Pete -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Francis E Reyes Sent: Monday, October 04, 2010 9:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?) Hi all I'm reading a recent review by Elspeth Garman regarding radiation damage (Acta Cryst D) and in this she mentions two ideas regarding metal centers: [1] They (The metal complexes themselves) are quickly reduced [2] Their absorption causes localized heating in the crystal. (This causes the temp to increase above freezing thus allowing OH radicals to diffuse). How is it that people can phase off of TaBr considering [1]? Presumably as TaBr is absorbing x-ray's whatever anomalous signal that was present at the beginning of the collection is either absent/ significantly altered by the time data collection is complete? Regarding [2], is TaBr usually backsoaked (into cryoprotectant not containing TaBr) to reduce it's concentration in the crystal to highly occupied sites? It would seem to me that having a crystal soaked with TaBr would cause more heating than one backsoaked. Thanks F - Francis E. 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
Re: [ccp4bb] How to detect the concentration of detergent?
Hi YB, There is a nice new paper online, dealing with this subject: http://www.ncbi.nlm.nih.gov/pubmed/20850411 Cheers Phil Am 04.10.10 17:09 schrieb "Owen Pornillos" unter : >>> Could anybody tell me how to detect the concentration of detergent? > From Butler et al. (2004) J Mol Biol 340: 797-808 > > The concentration of DDM was determined by a colorimetric assay that detects > the sugar component of the detergent, which has given results identical with > the standard procedure using radioactive DDM (T. Warne, unpublished data). A > 60 μl sample containing 4–16 μg of detergent was mixed briefly with 300 μl of > 5% (w/v) phenol and then 720 μl of concentrated sulphuric acid was added > followed immediately by vortex mixing for five seconds. The samples were left > to cool for 30 minutes and then the absorbance at 490 nm was measured. A > typical standard curve contained six samples containing between 0 μg and 20 μg > of DDM, which gave a straight line (r2>0.97). > > HTH, > > Owen > > > On Oct 4, 2010, at 10:42 AM, Van Den Berg, Bert wrote: > >> Hi YB, >> >> For membrane protein crystallization it is common practice (although not >> always necessary) to dialyze the protein after the final concentration step >> (against GF buffer). The problem with DDM is that dialysis is slow due to the >> low cmc, and in general it is advisable to finish the prep and set up drops >> as quickly as possible. I would not dialyze more than 1 x O/N, although if >> your protein is really stable you could try longer. You could also try 100 >> kDa cutoff concentrators, as these may allow passage of empty DDM micelles. >> As for measuring the detergent concentration, in the case of DDM and other >> sugar-containing detergents you could do a sugar (Fehling’s) kind of assay. >> I’m not sure if anything has been published, but it should be fairly easy to >> do. You could also try TLC, but this may be less accurate. >> >> Also, 10 mg/ml is not high at all (although its a good starting point), and >> you should try much higher if most drops are clear: try 50 mg/ml and see what >> happens. >> >> Good luck, Bert >> >> >> On 10/4/10 10:28 AM, "yybbll" wrote: >> >> >>> Dear all, >>> >>> I want to crystallize a symport transporter, which contains 12 >>> transmembrane alpha-helices. We used Ni-resin column firstly, and then size >>> exclusion. After size exclusion, only one peak, it is very nice. the final >>> condition is 10 mM mes, 100 mM NaCl, 10 mM sucrose, 1 mM DTT, and 0.02% DDM. >>> The CMC of DDM is about 0.008%. However, when we concentrate protein using a >>> concentrator with 50 kDa cutoff, detergent all was concentrated. So final >>> the concentration of detergent should be very high (10 times more than CMC). >>> We don't know how to detect the concentration of detergent. We used these >>> samples to grow crystal. We found almost drops are clear, and the final >>> concentration of protein is about 10 mg/ml. For membrane protein, I think >>> this concentration is high. But for us, we can obtain so high concentration >>> easily. >>> >>> Could anybody tell me how to detect the concentration of detergent? >>> >>> And how to dilute detergent? >>> >>> Thanks all. >>> >>> Y.B. Lin >>> >>> 2010-10-04 >>> >>> >>> yybbll >>> >>> >> >> > >
Re: [ccp4bb] Radiation damage with crystals containing metal centers (TaBr people chime in?)
The radiation damage with Ta6Br12 collected on the absorption edge is very severe. There may be a case for avoiding the edge wavelength for this reason Phil On 4 Oct 2010, at 17:28, Francis E Reyes wrote: > Hi all > > I'm reading a recent review by Elspeth Garman regarding radiation damage > (Acta Cryst D) and in this she mentions two ideas regarding metal centers: > > [1] They (The metal complexes themselves) are quickly reduced > > [2] Their absorption causes localized heating in the crystal. (This causes > the temp to increase above freezing thus allowing OH radicals to diffuse). > > How is it that people can phase off of TaBr considering [1]? Presumably as > TaBr is absorbing x-ray's whatever anomalous signal that was present at the > beginning of the collection is either absent/significantly altered by the > time data collection is complete? > > Regarding [2], is TaBr usually backsoaked (into cryoprotectant not containing > TaBr) to reduce it's concentration in the crystal to highly occupied sites? > It would seem to me that having a crystal soaked with TaBr would cause more > heating than one backsoaked. > > > > Thanks > > F > > > > - > Francis E. 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] Radiation damage with crystals containing metal centers (TaBr people chime in?)
Hi all I'm reading a recent review by Elspeth Garman regarding radiation damage (Acta Cryst D) and in this she mentions two ideas regarding metal centers: [1] They (The metal complexes themselves) are quickly reduced [2] Their absorption causes localized heating in the crystal. (This causes the temp to increase above freezing thus allowing OH radicals to diffuse). How is it that people can phase off of TaBr considering [1]? Presumably as TaBr is absorbing x-ray's whatever anomalous signal that was present at the beginning of the collection is either absent/ significantly altered by the time data collection is complete? Regarding [2], is TaBr usually backsoaked (into cryoprotectant not containing TaBr) to reduce it's concentration in the crystal to highly occupied sites? It would seem to me that having a crystal soaked with TaBr would cause more heating than one backsoaked. Thanks F - Francis E. 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 Available
Dear All, A position is available for a postdoctoral fellow to pursue structural and functional studies of proteins involved in the innate immune response of Anopheles gambiae to malaria infection. The successful candidate will be part of a new research group combining biophysical techniques such as x-ray crystallography and small-angle x-ray scattering with biochemical characterization and in vivo validation through collaborative studies. The position offers access to in-house x-ray diffraction facilities within the Chemistry Dept., numerous local core facilities, synchrotron access at NSLS and APS, and expanding opportunities for high-throughput biology at Yale's West Campus facility. Please email a cover letter, resume and contact information for three references to richard.bax...@yale.edu, or visit http://baxterlab.sites.yale.edu for further contact information. For scientific details see the following publications: Baxter, R.H.G., et. al (2010) "A heterodimeric complex of the LRR proteins LRIM1 and APL1C regulates complement-like immunity in Anopheles gambiae" PNAS 107, 16817-16822. [doi:10.1073/pnas.1010575107, PMID:20826443] Fraiture, M. et. al (2009) "Two mosquito LRR proteins function as complement control factors in the TEP1-mediated killing of Plasmodium" Cell Host Microbe 5, 273-284. [doi:10.1016/j.chom.2009.01.005, PMID 19286136] Baxter, R.H.G. et. al (2007) "Structural basis for conserved complement factor-like function in the antimalarial protein TEP1" PNAS 104, 11615-11620. [doi:10.1073/pnas.0704967104, PMID:17606907]
Re: [ccp4bb] Coot cannot read mtz or pdb files
Hello Petr, if you put this line into .profile, it affects all your programs, which might be unwanted (otherwise one would not have changed the locale in the first place). It would be better/ cleaner to put this line (actually without the 'export', just 'LC_NUMERIC=C' would be enough there) into the coot startup script. That way it only affects the coot session and not everything else. Tim On Mon, Oct 04, 2010 at 02:35:02PM +, Leiman Petr wrote: > Again, many thanks to all who responded. The simplest fix is to include this > line in your .profile file: > > export LC_NUMERIC=C > > Cheers, > > Petr -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] How to detect the concentration of detergent?
Could anybody tell me how to detect the concentration of detergent? From Butler et al. (2004) J Mol Biol 340: 797-808 The concentration of DDM was determined by a colorimetric assay that detects the sugar component of the detergent, which has given results identical with the standard procedure using radioactive DDM (T. Warne, unpublished data). A 60 μl sample containing 4–16 μg of detergent was mixed briefly with 300 μl of 5% (w/v) phenol and then 720 μl of concentrated sulphuric acid was added followed immediately by vortex mixing for five seconds. The samples were left to cool for 30 minutes and then the absorbance at 490 nm was measured. A typical standard curve contained six samples containing between 0 μg and 20 μg of DDM, which gave a straight line (r2>0.97). HTH, Owen On Oct 4, 2010, at 10:42 AM, Van Den Berg, Bert wrote: Hi YB, For membrane protein crystallization it is common practice (although not always necessary) to dialyze the protein after the final concentration step (against GF buffer). The problem with DDM is that dialysis is slow due to the low cmc, and in general it is advisable to finish the prep and set up drops as quickly as possible. I would not dialyze more than 1 x O/N, although if your protein is really stable you could try longer. You could also try 100 kDa cutoff concentrators, as these may allow passage of empty DDM micelles. As for measuring the detergent concentration, in the case of DDM and other sugar-containing detergents you could do a sugar (Fehling’s) kind of assay. I’m not sure if anything has been published, but it should be fairly easy to do. You could also try TLC, but this may be less accurate. Also, 10 mg/ml is not high at all (although its a good starting point), and you should try much higher if most drops are clear: try 50 mg/ml and see what happens. Good luck, Bert On 10/4/10 10:28 AM, "yybbll" wrote: Dear all, I want to crystallize a symport transporter, which contains 12 transmembrane alpha-helices. We used Ni-resin column firstly, and then size exclusion. After size exclusion, only one peak, it is very nice. the final condition is 10 mM mes, 100 mM NaCl, 10 mM sucrose, 1 mM DTT, and 0.02% DDM. The CMC of DDM is about 0.008%. However, when we concentrate protein using a concentrator with 50 kDa cutoff, detergent all was concentrated. So final the concentration of detergent should be very high (10 times more than CMC). We don't know how to detect the concentration of detergent. We used these samples to grow crystal. We found almost drops are clear, and the final concentration of protein is about 10 mg/ml. For membrane protein, I think this concentration is high. But for us, we can obtain so high concentration easily. Could anybody tell me how to detect the concentration of detergent? And how to dilute detergent? Thanks all. Y.B. Lin 2010-10-04 yybbll
Re: [ccp4bb] How to detect the concentration of detergent?
To estimate the total concentration of detergent, you can use TLC beside standard amounts of the detergent- see: Analytical Biochemistry 323 (2003) 234�C241 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* Interestingly this paper also discusses concentration of detergent by ultrafiltration. You need to be clear about total concentration of detergent vs concentration of free detergent. The protein will bind some detergent, up to maybe 1 g/g protein as you approach CMC, so if protein is 10 g/l you might have 10 g/l total detergent (100 x cmc) and the concentration of the monomer still be below CMC. I take it your 0.008% is concentration in the column buffer, so assuming the protein is equilibrated with that, it is the free detergent concentration. If the pass-through from ultrafiltration contains the same concentration, it means the detergent is not really being concentrated, only the protein-bound detergent is being retained by the filter. Even if all the free detergent is retained, it might be an insignificant increase in the total concentration, and the free concentration could be "buffered" to below CMC by protein binding. eab yybbll wrote: > Dear all, > I want to crystallize a symport transporter, which contains 12 > transmembrane alpha-helices. We used Ni-resin column firstly, and then > size exclusion. After size exclusion, only one peak, it is very nice. > the final condition is 10 mM mes, 100 mM NaCl, 10 mM sucrose, 1 mM DTT, > and 0.02% DDM. The CMC of DDM is about 0.008%. However, when we > concentrate protein using a concentrator with 50 kDa cutoff, detergent > all was concentrated. So final the concentration of detergent should be > very high (10 times more than CMC). We don't know how to detect the > concentration of detergent. We used these samples to grow crystal. We > found almost drops are clear, and the final concentration of protein is > about 10 mg/ml. For membrane protein, I think this concentration is > high. But for us, we can obtain so high concentration easily. > Could anybody tell me how to detect the concentration of detergent? > And how to dilute detergent? > Thanks all. > Y.B. Lin > 2010-10-04 > > yybbll
Re: [ccp4bb] How to detect the concentration of detergent?
Dear Y B Lin, Measuring detergent has been discussed before on this list - one way is to do thin-layer-chromatography against a set of standards. http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg12915.html >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 To reduce detegent concentration you can reload your protein onto a small Ni-resin or ion exchange column, wash, then elute in a small volume. James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of yybbll [yyb...@gmail.com] Sent: Monday, October 04, 2010 3:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to detect the concentration of detergent? Dear all, I want to crystallize a symport transporter, which contains 12 transmembrane alpha-helices. We used Ni-resin column firstly, and then size exclusion. After size exclusion, only one peak, it is very nice. the final condition is 10 mM mes, 100 mM NaCl, 10 mM sucrose, 1 mM DTT, and 0.02% DDM. The CMC of DDM is about 0.008%. However, when we concentrate protein using a concentrator with 50 kDa cutoff, detergent all was concentrated. So final the concentration of detergent should be very high (10 times more than CMC). We don't know how to detect the concentration of detergent. We used these samples to grow crystal. We found almost drops are clear, and the final concentration of protein is about 10 mg/ml. For membrane protein, I think this concentration is high. But for us, we can obtain so high concentration easily. Could anybody tell me how to detect the concentration of detergent? And how to dilute detergent? Thanks all. Y.B. Lin 2010-10-04 yybbll
Re: [ccp4bb] How to detect the concentration of detergent?
Hi YB, For membrane protein crystallization it is common practice (although not always necessary) to dialyze the protein after the final concentration step (against GF buffer). The problem with DDM is that dialysis is slow due to the low cmc, and in general it is advisable to finish the prep and set up drops as quickly as possible. I would not dialyze more than 1 x O/N, although if your protein is really stable you could try longer. You could also try 100 kDa cutoff concentrators, as these may allow passage of empty DDM micelles. As for measuring the detergent concentration, in the case of DDM and other sugar-containing detergents you could do a sugar (Fehling's) kind of assay. I'm not sure if anything has been published, but it should be fairly easy to do. You could also try TLC, but this may be less accurate. Also, 10 mg/ml is not high at all (although its a good starting point), and you should try much higher if most drops are clear: try 50 mg/ml and see what happens. Good luck, Bert On 10/4/10 10:28 AM, "yybbll" wrote: Dear all, I want to crystallize a symport transporter, which contains 12 transmembrane alpha-helices. We used Ni-resin column firstly, and then size exclusion. After size exclusion, only one peak, it is very nice. the final condition is 10 mM mes, 100 mM NaCl, 10 mM sucrose, 1 mM DTT, and 0.02% DDM. The CMC of DDM is about 0.008%. However, when we concentrate protein using a concentrator with 50 kDa cutoff, detergent all was concentrated. So final the concentration of detergent should be very high (10 times more than CMC). We don't know how to detect the concentration of detergent. We used these samples to grow crystal. We found almost drops are clear, and the final concentration of protein is about 10 mg/ml. For membrane protein, I think this concentration is high. But for us, we can obtain so high concentration easily. Could anybody tell me how to detect the concentration of detergent? And how to dilute detergent? Thanks all. Y.B. Lin 2010-10-04 yybbll
Re: [ccp4bb] Coot cannot read mtz or pdb files
Again, many thanks to all who responded. The simplest fix is to include this line in your .profile file: export LC_NUMERIC=C Cheers, Petr
[ccp4bb] How to detect the concentration of detergent?
Dear all, I want to crystallize a symport transporter, which contains 12 transmembrane alpha-helices. We used Ni-resin column firstly, and then size exclusion. After size exclusion, only one peak, it is very nice. the final condition is 10 mM mes, 100 mM NaCl, 10 mM sucrose, 1 mM DTT, and 0.02% DDM. The CMC of DDM is about 0.008%. However, when we concentrate protein using a concentrator with 50 kDa cutoff, detergent all was concentrated. So final the concentration of detergent should be very high (10 times more than CMC). We don't know how to detect the concentration of detergent. We used these samples to grow crystal. We found almost drops are clear, and the final concentration of protein is about 10 mg/ml. For membrane protein, I think this concentration is high. But for us, we can obtain so high concentration easily. Could anybody tell me how to detect the concentration of detergent? And how to dilute detergent? Thanks all. Y.B. Lin 2010-10-04 yybbll
[ccp4bb] shape complementarity
Hello everybody, I recently tried to feed the shape complementarity program (sc) with GRASP surface files for visualisation. Sc is supposed to output modified surface files, again, in GRASP format. However, the program terminated without producing a file, or, sometimes, writing truncated ones. The GRASP files passed the compatibility check of sc. I tested two different pdb files with the according GRASP surface files (made with Chimera). The first run ended with: "At line 2825 of file /usr/local/xtal/ccp4-6.1.13/src/sc_/sc.f Fortran runtime error: End of file" In the second run, I removed all hydrogen atoms before I calculated the surface files. Sc showed the following error: " Header information from grasp file 1 format=2 Keywords=vertices,normals,triangles Properties= Decoding property list Updated property list is:,gproperty Number of vertices=76375 Number of triangles= 54697 Size of grid= 65 Midpoint 91.033806 91.542160 89.700684 vertices read accessibles read SC: read error in s/r read_real_line SC: read error in s/r read_real_line Times: User: 8.3s System:0.4s Elapsed: 0:09 I can't make head or tail of it, maybe someone else can. Thanks in advance for any information and advice Reiner
Re: [ccp4bb] Coot cannot read mtz or pdb files
On 4 Oct 2010, at 11:15, Leiman Petr wrote: > It cannot read in MTZ files (quote: "This is not an mtz file"). PDB files > are garbled up on reading as well. Most (but not all) connections are > broken. A screenshot is attached. Is the Mac set up with an unusual language, locale or set of formats? I experienced this with an older version of Coot when one of our group fiddled with "Language & Text" in System Preferences. They were trying to make it easier for themselves to create text files in Greek and/or Cyrillic script. The changes had the unintended consequence of breaking coot. My theory at the time was that the number separators (, and .) had reversed which was confusing an input library that was used in reading PDB and MTZ files. Setting the region back to UK or US fixed the problem, and I didn't investigate much further. Regards, Chris The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] mol rep help needed
Hi David, On Fri, Oct 01, 2010 at 11:40:13AM -0400, David Roberts wrote: > My question - finally - how can I run automolrep with one dimer fixed, > looking for the location of the other 2 monomers (so basically I want to > fix a dimer as part of my solution, and then search for the other 2 > molecules in the asu). I found the latest MOLREP binary from http://www.ysbl.york.ac.uk/~alexei/molrep.html#installation to work very well (thanks Alexei!). Something like: molrep_linux -f your.mtz \ -m monomer.pdb \ -mx fixed_dimer.pdb It doesn't get simpler than that I guess ;-) Cheers Clemens -- *** * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com * * Global Phasing Ltd. * Sheraton House, Castle Park * Cambridge CB3 0AX, UK *-- * BUSTER Development Group (http://www.globalphasing.com) ***
Re: [ccp4bb] Coot cannot read mtz or pdb files
On 4 Oct 2010, at 11:15, Leiman Petr wrote: > Dear all, > > Coot behaves in a very strange way on my student's MacBook (32bit) running > MacOS X 10.6.4. Both versions of coot are affected - the precompiled Prof. > Scott's one and the compiled from source. > > It cannot read in MTZ files (quote: "This is not an mtz file"). PDB files > are garbled up on reading as well. Most (but not all) connections are > broken. A screenshot is attached. I think this is a locale issue; try running 'LANG=C coot'. I suspect the parser is assuming that the decimal-point character is 'comma' not 'full-stop', which is why the atoms have been moved to exact-integer locations. Tom
Re: [ccp4bb] Twinned data
Hi Boaz thanks for the advice - Yes your suggestions are very useful thank you! I actually have not tried Phaser with the P4 data so I will give that a shot. Curiously Pointless selects P21 from P4 and P421/3 from P21... I tried MolRep in P1 but Phaser failed to find any solution though perhaps I did not pursue it enough... I think it is entirely possible that the space group I have , as in your example, may be something other than P21 etc Never tried Zanadu sounds interesting I'll take a look... We have new crystals... I was wanting to solve the twinned data to get some initial info etc and as a crystallography problem/exercise not having come across it yet... thanks again Best Gina From: Boaz Shaanan [mailto:bshaa...@bgu.ac.il] Sent: Friday, October 01, 2010 5:18 PM To: Clayton, Gina Martyn Subject: Re: [ccp4bb] Twinned data Hi Gina, I've had a similar situation and here are some suggestions/ ideas: 1) You could try to run Phaser on the the data processed as P4 BEFORE detwinning and ask it to try all the possible space groups including the P422 and related ones. 2) Before step 1 or in parallel or following it, I would run the P4 processed data (again NOT detwinned) through pointless and/or xtriage to see whether they can give some indications for better choice of space group. 3) In my case, only running Phaser in P1 gave me a solution (the data were initially processed as P4 but I reprocessed them in P1 for that purpose). It's worth you trying too. 4) Eventually, I ran the P1 model (8 mols.) and data in Zanuda (on the York site) and that gave me a new space group choice (P21212). When I ran Phaser in this space group (after reprocessing the data again) it also gave a good solution which I'm now refining (with twinning) either in Phenix or refmac but without detwinning, since the program do this for you internally (I'm sure about refmac and assume it's the same in Phenix). I hope you'll find some of this helpful. Cheers, Boaz - Original Message - From: "Clayton, Gina Martyn" Date: Friday, October 1, 2010 21:28 Subject: [ccp4bb] Twinned data To: CCP4BB@JISCMAIL.AC.UK > Hi there > > Just wondering if anyone has any suggestions how I can deal, if > possible, with the following situation -. My first > encounter with > twinned data... > > which initially indexed as p43/p41 cell dimensions 58.4 58.4 61 > 90 90 > 90. > Having seen the Matthews Coefficient for the solvent content of > the unit > cell as 16% I discovered the data are merohedrally twinned with twin > fraction given as 0.1. > > I reprocessed the data as p1, p2 p21, c2221 (Rsymm etc indicates wrong > space group, Mats Coeff 16%) p4 etc. In p2 the (pseudo) twin > fraction is > given as 0.43 (Mats Coeff 60% solvent 2.7 mol/ASU). > > I ran Detwin (ccp4) on the p2 p21 data with alternate operators as > indicated by Xtriage in Phenix. I have had no > molecular replacement > solution i.e. Molrep rotational searches are not giving peaks > and Phaser > has not found a solution (nor with alternates of the search "model"). > > Does anyone have any suggestions/best paper to consult etc based on > their experience of twinned data (aside from sort the crystals out...) > or should I "throw in the towell?" > > Thanks in advance for any information and advice > > Gina > > > > > Notice: This e-mail message, together with any > attachments, contains > information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, > New Jersey, USA 08889), and/or its affiliates Direct contact > informationfor affiliates is available at > http://www.merck.com/contact/contacts.html) that may be confidential, > proprietary copyrighted and/or legally privileged. It is > intended solely > for the use of the individual or entity named on this message. > If you are > not the intended recipient, and have received this message in error, > please notify us immediately by reply e-mail and then delete it > from > your system. > Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] R factor & R free struck
On Sun, Oct 3, 2010 at 6:34 PM, J. Preben Morth wrote: > hi > remember to reindex your data to P21212 in case you used Phaser to search > all alternative orthorhombic SG's and it found P22121 > Preben > > Hi, re-indexing the data without also remembering to transform the co-ordinates (using the matrix transpose of the inverse of the re-indexing operator) would almost certainly cause the R factors to increase! But even assuming you did that, why should re-indexing/transformation have any effect at all on the R factors? Cheers -- Ian
Re: [ccp4bb] R factor & R free struck
the reindexing have no effect on the R-factor, I did indeed mean to remember to transform the co-ordiantes according to the indexing :-) On 04/10/2010, at 11.27, Ian Tickle wrote: > > On Sun, Oct 3, 2010 at 6:34 PM, J. Preben Morth wrote: > hi > remember to reindex your data to P21212 in case you used Phaser to search all > alternative orthorhombic SG's and it found P22121 > Preben > > > Hi, re-indexing the data without also remembering to transform the > co-ordinates (using the matrix transpose of the inverse of the re-indexing > operator) would almost certainly cause the R factors to increase! But even > assuming you did that, why should re-indexing/transformation have any effect > at all on the R factors? > > Cheers > > -- Ian Jens Preben Morth, Ph.D Aarhus University Department of Molecular Biology Gustav Wieds Vej 10 C DK - 8000 Aarhus C Tel. +45 8942 5257, Fax. +45 8612 3178 j...@mb.au.dk website: http://person.au.dk/en/j...@mb.au.dk
Re: [ccp4bb] R factor & R free struck
Is that necessary? Eleanor J. Preben Morth wrote: hi remember to reindex your data to P21212 in case you used Phaser to search all alternative orthorhombic SG's and it found P22121 Preben On 03/10/2010, at 04.56, Jack Russel wrote: Hi all, I have collected a data at 2.9 Å and the solved the structure using phaser . the space group comes to be P2 21 21. There are 4 molecules in Assymetric unit and an octamer is generated according to the symmetry. But after repeated rounds of rigid body refinement with REFMAC5 and model building with coot the R factor had been struck at 40% and R free at 50%. So my first question is whether my solution after phaser is correct. And if it is how can i lower the R factor and Rfree. The second question is it possible to have such a large difference between R factor and R free. Thanks in advance
Re: [ccp4bb] Coot cannot read mtz or pdb files
Many thanks to everybody who quickly replied to my cry for help. It is a locale definition problem as the laptop in question is set up with the French Swiss locale. Cheers, Petr
Re: [ccp4bb] Twinned data
Hi there Thank you for the advice regarding my twin data (mostly sent off the board). Seems I didn't put enough info in the email sorry for that! The resolution is 2.6Ang. My first thought was that the crystal was a fragment of the full length protein. The crystals are undergoing mass spec (the proteins itself appears stable using DLS etc)... I did try molecular replacement in the different space groups prior to detwin with variants of my search model. Truncate for the centric moments of E show a value close to 1.5 (perfect twin is 1.5) H test for twinning close -lkh is close to 0.5 Pseudo twin fraction is given as 0.43 The unmerged data processed in p1 are selected as p21 by Pointless and P421/3 if given as unmerged in p21. Thankfully I have new crystals now... Gina -Original Message- From: George M. Sheldrick [mailto:gshe...@shelx.uni-ac.gwdg.de] Sent: Friday, October 01, 2010 6:35 PM To: Clayton, Gina Martyn Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Twinned data If you really have the systematic absences for P41 or P43 then you must have (at least) four molecules in the cell, twinning cannot reduce this. Since a solvent content of 16% is too low, the most likely explanation is that it is not twinned but that the protein is smaller than the one you are expecting. With an unknown sequence and no experimental phase information such as SAD or MAD, your only hope would be Arcimboldo, but that requires data to about 2A or better (you don't mention the resolution). As an even longer shot, you could search the PDB for matching cells and space groups. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Fri, 1 Oct 2010, Clayton, Gina Martyn wrote: > Hi there > > Just wondering if anyone has any suggestions how I can deal, if > possible, with the following situation -. My first encounter with > twinned data... > > which initially indexed as p43/p41 cell dimensions 58.4 58.4 61 90 90 > 90. > Having seen the Matthews Coefficient for the solvent content of the unit > cell as 16% I discovered the data are merohedrally twinned with twin > fraction given as 0.1. > > I reprocessed the data as p1, p2 p21, c2221 (Rsymm etc indicates wrong > space group, Mats Coeff 16%) p4 etc. In p2 the (pseudo) twin fraction is > given as 0.43 (Mats Coeff 60% solvent 2.7 mol/ASU). > > I ran Detwin (ccp4) on the p2 p21 data with alternate operators as > indicated by Xtriage in Phenix. I have had no molecular replacement > solution i.e. Molrep rotational searches are not giving peaks and Phaser > has not found a solution (nor with alternates of the search "model"). > > Does anyone have any suggestions/best paper to consult etc based on > their experience of twinned data (aside from sort the crystals out...) > or should I "throw in the towell?" > > Thanks in advance for any information and advice > > Gina > > > > > Notice: This e-mail message, together with any attachments, contains > information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, > New Jersey, USA 08889), and/or its affiliates Direct contact information > for affiliates is available at > http://www.merck.com/contact/contacts.html) that may be confidential, > proprietary copyrighted and/or legally privileged. It is intended solely > for the use of the individual or entity named on this message. If you are > not the intended recipient, and have received this message in error, > please notify us immediately by reply e-mail and then delete it from > your system. > > Notice: This e-mail message, together with any attachments, contains information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
