Re: [ccp4bb] How to find out Rmerge from the refinements..?
Hussain Bhukyagps wrote: Dear all, i want to know that how can we find Rmerge from the refinements done in CNS..?? Hi, I think you have the terminology wrong: Rmerge (or Rsym nowadays when most diffraction data is recorded from a single crystal) is provided by the data frame diffraction software, not by a refinement program. Internal agreement of the data so to speak. Can you tell us more what you are looking for? R-factor and R-free perhaps? Fred.
[ccp4bb] monomer-dimer
Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL
[ccp4bb] International Conference on Antivirals for Neglected and Emerging Viruses (ICAV-9) October 10 to 13, 2010
*The International Conference on Antivirals for Neglected and Emerging Viruses (ICAV-9) will take place in the historic capital of the Hanse, Lübeck, Germany, from October 10 to 13, 2010* Dear colleague, Infectious viral diseases exert a shocking toll on the developing world. Over 2.5 million people die each year from AIDS, mostly in sub-Saharan Africa. More than 3 billion people are at risk of infection with dengue fever. Rotavirus, a cause of common diarrhoea, kills an estimated 600,000 children each year. Three percent of the world's population, around 170 million people, are chronically infected with hepatitis C. In West Africa alone, there are some 500,000 cases of Lassa fever every year. Furthermore, many RNA viruses such as the new H1N1 subtype of influenza or the enteroviruses are emerging in developed countries. Despite these facts, few drugs are available for the treatment of these viral diseases; even fewer reach those in need. The International Consortium on Anti-Virals (ICAV) was founded to discover and develop antiviral therapies for neglected and emerging diseases and to ensure their accessibility to all those in need. ICAV is currently developing therapies for viral diseases such as influenza, HIV/AIDS, hepatitis, Dengue fever, Lassa fever, yellow fever, chikungunya fever, and other infectious viral diseases. ICAV has developed new funding schemes that capitalize on the discoveries of antiviral compounds made in academia. (See more on ICAV at www.icav-citav.ca http://www.icav-citav.ca). You are cordially invited to join us for ICAV's 9th International Symposium, the International Conference on Antivirals for Neglected and Emerging Viruses (ICAV-9), which will take place from October 10 -- 13, 2010, at the Media Docks in the old port of the historic Hanseatic City of Lübeck, Germany. We have assembled an interesting scientific programme with a focus on the discovery of antiviral therapies of disease caused by dengue virus, influenza virus, enteroviruses, chikungunyavirus, coronaviruses, and other emerging or neglected viruses. We will also have a microsymposium on Targeting Host Factors in HIV/AIDS Therapy. Please see the conference webpage, www.icav-9.org http://www.icav-9.org, for the scientific programme and for registration. The early-registration deadline is August 31, 2010, and the same applies for hotel booking. Also, by September 13, 2010, you are encouraged to submit an abstract describing your scientific results in the field. While the oral presentations to be selected from the submitted abstracts will focus on antivirals for neglected and emerging viruses (as well as on antivirals directed at host factors in HIV/AIDS therapy), the poster session will be open for presentations of antiviral work of any kind. Please visit the conference webpage frequently, as the scientific programme will be continuously updated. We are working on obtaining funding for bursaries for PhD students (please provide a letter of confirmation signed by your supervisor) and for participants from low-and-middle-income countries. If we are successful, partial reimbursement may become available after the conference. If you want to apply for this, please send an e-mail to loc-ica...@biochem.uni-luebeck.de. For any questions you may have, please do not hesitate to contact the ICAV-9 convenor, Professor Rolf Hilgenfeld, at hilgenf...@biochem.uni-luebeck.de. We very much hope to see you at ICAV-9 in Lübeck in October! Sincerely, Rolf Hilgenfeld Jeremy Carver, CEO, CSO Institute of BiochemistryInternational Consortium on Antivirals University of Lübeck www.icav-9.org http://www.icav-9.org
[ccp4bb] Fw:
Can anyone supply information on the use of the following in ccp4: 1. Bulk-solvent correction 2. Refinement of individual isotropic B-factors 3. Refinement of an anisotropic overall thermal parameter Rex Palmer Birkbeck College, London
Re: [ccp4bb] Fw:
Dear Rex, I am not sure what exactly you are looking for, but why don't you start with the Refmac documentation? Item 3 probably corresponds to a single TLS group in refmac. Or maybe you could specify what you mean by the use of [...] in ccp4. Cheers, Tim On Mon, Aug 09, 2010 at 02:26:12PM +0100, Rex Palmer wrote: Can anyone supply information on the use of the following in ccp4: 1. Bulk-solvent correction 2. Refinement of individual isotropic B-factors 3. Refinement of an anisotropic overall thermal parameter Rex Palmer Birkbeck College, London -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
[ccp4bb] composite maps
Hi all, I wanted to know the best way to make a composite file using ccp4. I have tried the SF-check program GUI; but it does not give me an option to construct composite maps. Also, is it true that when one does molecular replacement, if one wants to show an electron density map in a publication, it has to be composite map and not a 2Fo-Fc map? Thanks to all in advance Rakesh
Re: [ccp4bb] composite maps
Rakesh, Composite maps are used in publications some times however many folks also publish the 2Fo-Fc or even better the 1Fo-Fc. These can be obtained by selecting a button in refmac to export the maps and then visualize them in pymol. Scott On Mon, Aug 9, 2010 at 8:37 AM, Rakesh Joshi rjo...@purdue.edu wrote: Hi all, I wanted to know the best way to make a composite file using ccp4. I have tried the SF-check program GUI; but it does not give me an option to construct composite maps. Also, is it true that when one does molecular replacement, if one wants to show an electron density map in a publication, it has to be composite map and not a 2Fo-Fc map? Thanks to all in advance Rakesh -- Scott D. Pegan, Ph.D. Assistant Professor Chemistry Biochemistry University of Denver Office: 303 871 2533 Fax: 303 871 2254
Re: [ccp4bb] Fw:
On Monday 09 August 2010, Tim Gruene wrote: Dear Rex, I am not sure what exactly you are looking for, but why don't you start with the Refmac documentation? Item 3 probably corresponds to a single TLS group in refmac. That is not quite correct. An overall anisotropic B term is applied equally to all atoms in the structure. If the atoms are otherwise described isotropically, this will turn them all into identically shaped ellipsoids instead. A single TLS group, on the other hand, describes a position-dependent anisotropic model. Every atom in the structure will get a _different_ ellipsoidal shape, where the shape is a function of its x/y/z coords. Ethan Or maybe you could specify what you mean by the use of [...] in ccp4. Cheers, Tim On Mon, Aug 09, 2010 at 02:26:12PM +0100, Rex Palmer wrote: Can anyone supply information on the use of the following in ccp4: 1. Bulk-solvent correction 2. Refinement of individual isotropic B-factors 3. Refinement of an anisotropic overall thermal parameter Rex Palmer Birkbeck College, London
[ccp4bb] Does phenix have any utilities which can do B-factor sharpening?
Hi there, Does phenix have any utilities which can do B-factor sharpening (with user-specified Bsharp values) when calculating maps? Thanks! Best Regards, Hailiang
Re: [ccp4bb] Does phenix have any utilities which can do B-factor sharpening?
Hi Hailiang, yes, phenix.maps tool (command line) can compute any number of regular kFo-jFc or sigmaa weighted kmFo-jDFc maps, where k and j are any user-defined numbers. The maps can be output as in various formats (MTZ, Xplor/CNS, CCP4). B-factor sharpening can be applied. The sharpening B-factor is determined automatically or can be supplied by the user. The equivalent thing in the GUI: Maps - Create Maps. Pavel. PS There is PHENIX bulletin board for PHENIX specific questions: http://www.phenix-online.org/ On 8/9/10 10:01 AM, Hailiang Zhang wrote: Hi there, Does phenix have any utilities which can do B-factor sharpening (with user-specified Bsharp values) when calculating maps? Thanks! Best Regards, Hailiang
Re: [ccp4bb] monomer-dimer
Hello Intekhab, Your results do not seem surprising at all. It is not uncommon for molecular interactions such as dimerization to be more stable at lower temperatures, and this is exactly why you are seeing the shift to higher elution volumes at lower tempratures. At lower temperatures, both the monomer and dimer are likely to be more compact in solution due to less thermal fluctuations in the overall structures. Remember that protein structures are always in motion, and lowering the temperature restricts these motions, and therefor lowers the effective radius of the molecule in solution as it moves through the column. And of course smaller molecules elute at higher volumes, so this probably explains what you see in the cold room. As for some of the other concerns you have with your gel filtration experiments, I can offer the following suggestions. First, remember that gel filtration elution volumes are independent of conditions like flow rate and protein concentration (unless there are nonspecific interactions at high concentration), but like I described before temp is a factor. That being said, often analytical gel filtration experiments are more informative at moderate concentrations instead of high concentrations, because this will favor the formation of relevant oligomers, instead of oligomers and aggregates that form only at high concentrations and aren't really biological. When you do your incubation experiments, try using lower protein concentrations or shorter incubation times. This might prevent the formation of precipitates and will give you more biologically relevant information - after all, most proteins are not available in the cell at very high concentrations, so if your dimer is biological, the kd is likely pretty low. Also you could try another experiment like a pulldown with tagged/untagged constructs, or SPR. These experiments (SPR particularly) would also tell you if the kd is reasonable for a biologically relevant interaction. One more thought is to be sure your protein is not degrading at high temperatures, which may be the reason your 37 degree incubation results in increased elution volume. Mass spec could help you here. Finally, to determine the biological relevance of your dimer you should do an analysis of the dimer interface seen in the crystal structure. I believe that for the average biological oligomer, the oligomerization interface buries approx 1200-2000A2 of surface area, whereas the average crystal contact buries approx 400-800A2. Some older work related to these analyses has been published by Joel Janin and Janet Thornton. Also, some webservers like PISA attempt to predict the relevant oligomerization states of proteins in the PDB based on interfaces seen in the crystal structures. You might look there for a good method. Good Luck, Mike Thompson - Original Message - From: intekhab alam faisal...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, August 9, 2010 4:37:45 AM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] monomer-dimer Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] monomer-dimer
To determine the oligomeric state of a protein (monomer or dimer in your case), it's useful to use the PISA server. You upload your pdb file from the crystal structure.The server calculates the areas of interfaces (buried area) and deltaG (change in Gibbs energy) upon oligomer dissociation. (E. Krissinel and K. Henrick (2007). /Inference of macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 2005, LNBI 3695, pp. 163--174 http://dx.doi.org/10.1007/11560500_15. E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. J. Comp. Chem., in press; published on-line 6 May 2009; DOI 10.1002/jcc.21303} If the interface area (divided by 2 per one protomer) is greater than 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's a dimer. However, don't forget that most dimers can dissociate into monomers upon dilution. There is a dynamic equilibrium between dimers (oligomers) and monomers that depends on their concentration and the Kdiss. Separating them in any method will disturb this equilibrium. If the re-equilibration time is greater than the separation time, you can see both monomers and dimers. You can even roughly calculate the dissociation constant: Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, protein needs to dissociate easily for the biological function. Maia intekhab alam wrote: Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL
Re: [ccp4bb] Does phenix have any utilities which can do B-factor sharpening?
Hi Pavel: Thanks a lot! I found it in the latest version of phenix, and it works well! Best Regards, Hailiang Hi Hailiang, yes, phenix.maps tool (command line) can compute any number of regular kFo-jFc or sigmaa weighted kmFo-jDFc maps, where k and j are any user-defined numbers. The maps can be output as in various formats (MTZ, Xplor/CNS, CCP4). B-factor sharpening can be applied. The sharpening B-factor is determined automatically or can be supplied by the user. The equivalent thing in the GUI: Maps - Create Maps. Pavel. PS There is PHENIX bulletin board for PHENIX specific questions: http://www.phenix-online.org/ On 8/9/10 10:01 AM, Hailiang Zhang wrote: Hi there, Does phenix have any utilities which can do B-factor sharpening (with user-specified Bsharp values) when calculating maps? Thanks! Best Regards, Hailiang
[ccp4bb] cphasematch
Dear colleagues, here is one cunning plan: to quickly evaluate the anomalous signal of a test data set with a non-interactive script that: 1. solves the structure using SAD 2. does some solvent flattening 3. compares the resulting phases against calculated phases from a refined, isomorphous structure. Generates some global measure of phase error (deviation from refined, calculated phases). Not very original, but still failing in my hands. 1., 2. (shelxc/d/e) - check. 3. a) take model amplitudes, phases and weights (F, PHIC, FOM(C)) from a refmac MTZ file - check. 3. b) cad PHI, FOM from the shelxe output with the selected columns from the refmac MTZ - check. 3. c) display reasonable maps using either of F-PHIC-FOM(C) or F-PHI-FOM combinations - check. 3. d) cphasematch -mtzin cad_ori.mtz -mtzout phasematch.mtz \ -colin-fo /*/*/[F,SIGF] -colin-phifom-1 /*/*/[PHIC,FOM] \ -colin-phifom-2 /*/*/[PHI_ori,FOM_ori] Now, I did expect to see a reasonable map using the F- phasematch.Phi_fom.phi-phasematch.Phi_fom.fom combination that would resemble F-PHI-FOM, but superimposed onto F-PHIC-FOM(C). But the F- phasematch.Phi_fom.phi-phasematch.Phi_fom.fom map does not resemble the protein structure at all. It is slowly coming to me that I must be doing this the wrong way. Can anyone spot the problem? By the way, the crystal is cubic insulin (a=78 A.) I would be happy to send out the data, the phasematch.mtz is just 140 kB in size. Many thanks, Wolfram Tempel
Re: [ccp4bb] monomer-dimer
Dear Intekhab Let me just add to this that gel filtration is not an accurate method for determination of molecular mass, because the migration on the column depends on the shape of the protein. The following methods can be used to determine molecular mass irrespective of shape: - MALLS (multi-angle laser light scattering or static light sxattering) - sedimentation equilibrium on analytical ultracentrifuge (AUC) - native mass spectrometry For a short recent review on issues associated with determining oligomeric state from crystal structures, with older references and relevant bioinformatic tools cited in there, please see http://www.ncbi.nlm.nih.gov/pubmed/19021571 Bostjan On 10/08/10 6:26 AM, Maia Cherney ch...@ualberta.ca wrote: To determine the oligomeric state of a protein (monomer or dimer in your case), it's useful to use the PISA server. You upload your pdb file from the crystal structure.The server calculates the areas of interfaces (buried area) and deltaG (change in Gibbs energy) upon oligomer dissociation. (E. Krissinel and K. Henrick (2007). /Inference of macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 2005, LNBI 3695, pp. 163--174 http://dx.doi.org/10.1007/11560500_15. E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. J. Comp. Chem., in press; published on-line 6 May 2009; DOI 10.1002/jcc.21303} If the interface area (divided by 2 per one protomer) is greater than 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's a dimer. However, don't forget that most dimers can dissociate into monomers upon dilution. There is a dynamic equilibrium between dimers (oligomers) and monomers that depends on their concentration and the Kdiss. Separating them in any method will disturb this equilibrium. If the re-equilibration time is greater than the separation time, you can see both monomers and dimers. You can even roughly calculate the dissociation constant: Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, protein needs to dissociate easily for the biological function. Maia intekhab alam wrote: Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL --- Bostjan Kobe ARC Federation Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience (Division of Chemistry and Structural Biology) and Centre for Infectious Disease Research Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.au URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html Office: Building 76 Room 329 Notice: If you receive this e-mail by mistake, please notify me, and do not make any use of its contents. I do not waive any privilege, confidentiality or copyright associated with it. Unless stated otherwise, this e-mail represents only the views of the Sender and not the views of The University of Queensland.
Re: [ccp4bb] monomer-dimer
Dear That was a quite enlightening discussion!! I am grateful to you guys for your time!! I will definitily try some of these to get a clear answer. Regards Intekhab alam On Tue, Aug 10, 2010 at 8:38 AM, Bostjan Kobe b.k...@uq.edu.au wrote: Dear Intekhab Let me just add to this that gel filtration is not an accurate method for determination of molecular mass, because the migration on the column depends on the shape of the protein. The following methods can be used to determine molecular mass irrespective of shape: - MALLS (multi-angle laser light scattering or static light sxattering) - sedimentation equilibrium on analytical ultracentrifuge (AUC) - native mass spectrometry For a short recent review on issues associated with determining oligomeric state from crystal structures, with older references and relevant bioinformatic tools cited in there, please see http://www.ncbi.nlm.nih.gov/pubmed/19021571 Bostjan On 10/08/10 6:26 AM, Maia Cherney ch...@ualberta.ca wrote: To determine the oligomeric state of a protein (monomer or dimer in your case), it's useful to use the PISA server. You upload your pdb file from the crystal structure.The server calculates the areas of interfaces (buried area) and deltaG (change in Gibbs energy) upon oligomer dissociation. (E. Krissinel and K. Henrick (2007). /Inference of macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 2005, LNBI 3695, pp. 163--174 http://dx.doi.org/10.1007/11560500_15. E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. J. Comp. Chem., in press; published on-line 6 May 2009; DOI 10.1002/jcc.21303} If the interface area (divided by 2 per one protomer) is greater than 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's a dimer. However, don't forget that most dimers can dissociate into monomers upon dilution. There is a dynamic equilibrium between dimers (oligomers) and monomers that depends on their concentration and the Kdiss. Separating them in any method will disturb this equilibrium. If the re-equilibration time is greater than the separation time, you can see both monomers and dimers. You can even roughly calculate the dissociation constant: Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, protein needs to dissociate easily for the biological function. Maia intekhab alam wrote: Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL --- Bostjan Kobe ARC Federation Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience (Division of Chemistry and Structural Biology) and Centre for Infectious Disease Research Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.au URL: http://profiles.bacs.uq.edu.au/Bostjan.Kobe.html Office: Building 76 Room 329 Notice: If you receive this e-mail by mistake, please notify me, and do not make any use of its contents. I do not waive any privilege, confidentiality or copyright associated with it. Unless stated otherwise, this e-mail
Re: [ccp4bb] monomer-dimer
Small angle x-ray solution scattering (SAXS) can also give you molecular weight, though not quite as accurately as the best static light scattering. While SAXS is preferably done on monodisperse systems extrapolated to infinite dilution, cases in which the monomer and dimer are in equilibrium under various conditions can be treated ... especially if you already know the monomer and possible dimer structures. These are relatively easy experiments at a synchrotron source, but lab source SAXS facilities also can be found. Richard Gillilan MacCHESS On Aug 9, 2010, at 7:38 PM, Bostjan Kobe wrote: Dear Intekhab Let me just add to this that gel filtration is not an accurate method for determination of molecular mass, because the migration on the column depends on the shape of the protein. The following methods can be used to determine molecular mass irrespective of shape: - MALLS (multi-angle laser light scattering or static light sxattering) - sedimentation equilibrium on analytical ultracentrifuge (AUC) - native mass spectrometry For a short recent review on issues associated with determining oligomeric state from crystal structures, with older references and relevant bioinformatic tools cited in there, please see http://www.ncbi.nlm.nih.gov/pubmed/19021571 Bostjan On 10/08/10 6:26 AM, Maia Cherney ch...@ualberta.ca wrote: To determine the oligomeric state of a protein (monomer or dimer in your case), it's useful to use the PISA server. You upload your pdb file from the crystal structure.The server calculates the areas of interfaces (buried area) and deltaG (change in Gibbs energy) upon oligomer dissociation. (E. Krissinel and K. Henrick (2007). /Inference of macromolecular assemblies from crystalline state/. J. Mol. Biol. *372*, 774--797 . E. Krissinel and K. Henrick (2005). /Detection of Protein Assemblies in Crystals/. In: M.R. Berthold /et.al./ (Eds.): CompLife 2005, LNBI 3695, pp. 163--174 http://dx.doi.org/10.1007/11560500_15. E. Krissinel (2009). /Crystal contacts as nature's docking solutions/. J. Comp. Chem., in press; published on-line 6 May 2009; DOI 10.1002/jcc.21303} If the interface area (divided by 2 per one protomer) is greater than 1000 A2 and delta G is more than 5kcal/mol (the higher the better), it's a dimer. However, don't forget that most dimers can dissociate into monomers upon dilution. There is a dynamic equilibrium between dimers (oligomers) and monomers that depends on their concentration and the Kdiss. Separating them in any method will disturb this equilibrium. If the re-equilibration time is greater than the separation time, you can see both monomers and dimers. You can even roughly calculate the dissociation constant: Kdiss=[monomer]2/[dimer] where brackets mean concentrations. To give you an estimate, at Kdiss=10(-3)M, you have roughly equal concentration of dimers and monomers at 10-3 M and only 10% dimers at 10-4 M. Sometimes, protein needs to dissociate easily for the biological function. Maia intekhab alam wrote: Hi everyone Sorry for some non specific query! i am working with a protein that shows a dimer in the crystal structure but when i tried to figure out that with standard molecular markers in gel filteration (superdex-200, 24ml column) it turned out to be a monnomer. Native gel analysis after incubating the protein at 20 degree, 37 degree showed more dimer at 20 degree celcius as compared to 37. I tried similar strategy in gel filteration by incubating my protein at various temperature,where a lot of precipitation was observed at 37 degree celcius and after removing the precipitates i run the gel filteration that has 0.5 ml higher elution volume as compared to samples incubated at 20 degree celcius and 4 degree celcius.( Is this significant) Furthermore i have done some experiments in cold room (4 degree) where the elution volume is stuck at a point irrespective of the conditions (as Flow rate, concentration of protein etc) and that is higher than that of the room temperature by 1 ml. Standard moleculr weight markers also show higher elution volume in cold room in comparison to the room temperature by 1 ml. I will be highly obliged if someone suggest some literature or any otherway to do gel filtrtaion so that i can clearly resolve this issue. Also let me know if there is some literature available on effect of temperature on the elution volume of proteins. Thanks in advance -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL --- Bostjan Kobe ARC Federation Fellow Professor of Structural Biology School of Chemistry and Molecular Biosciences and Institute for Molecular Bioscience (Division of Chemistry and Structural Biology) and Centre for Infectious Disease Research Cooper Road University of Queensland Brisbane, Queensland 4072 Australia Phone: +61 7 3365 2132 Fax: +61 7 3365 4699 E-mail: b.k...@uq.edu.au
Re: [ccp4bb] cphasematch
There are two different but equally good ways of indexing the reflections in space group I213, so there is a 50% chance that your new solution and the refined structure will be indexed differently, in which case the phases will not agree. Also you can easily improve the experimental phases from shelxe by using the beta-test version that does iterative main-chain tracing (available by sending me an email while I am still developing it). 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 Mon, 9 Aug 2010, wtempel wrote: Dear colleagues, here is one cunning plan: to quickly evaluate the anomalous signal of a test data set with a non-interactive script that: 1. solves the structure using SAD 2. does some solvent flattening 3. compares the resulting phases against calculated phases from a refined, isomorphous structure. Generates some global measure of phase error (deviation from refined, calculated phases). Not very original, but still failing in my hands. 1., 2. (shelxc/d/e) - check. 3. a) take model amplitudes, phases and weights (F, PHIC, FOM(C)) from a refmac MTZ file - check. 3. b) cad PHI, FOM from the shelxe output with the selected columns from the refmac MTZ - check. 3. c) display reasonable maps using either of F-PHIC-FOM(C) or F-PHI-FOM combinations - check. 3. d) cphasematch -mtzin cad_ori.mtz -mtzout phasematch.mtz \ -colin-fo /*/*/[F,SIGF] -colin-phifom-1 /*/*/[PHIC,FOM] \ -colin-phifom-2 /*/*/[PHI_ori,FOM_ori] Now, I did expect to see a reasonable map using the F- phasematch.Phi_fom.phi-phasematch.Phi_fom.fom combination that would resemble F-PHI-FOM, but superimposed onto F-PHIC-FOM(C). But the F- phasematch.Phi_fom.phi-phasematch.Phi_fom.fom map does not resemble the protein structure at all. It is slowly coming to me that I must be doing this the wrong way. Can anyone spot the problem? By the way, the crystal is cubic insulin (a=78 A.) I would be happy to send out the data, the phasematch.mtz is just 140 kB in size. Many thanks, Wolfram Tempel
