Re: [ccp4bb] monomer-dimer
Dear all, If I may add that I find the statement 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. a bit misleading. While concentration will not change where the monomer or the dimer appears in the elution volume, concentration will affect the monomer-dimer equilibrium during your gel-filtration run. Thus, I would say that concentration is a factor. If your dimer has a kD of ~100uM, and you inject it at a concentration of ~100uM, after getting diluted during gel-filtration (about ten-fold) it will appear 90% as a monomer ... The results of any analytical technique to determine stoichiometry are concentration dependent, and concentration is actually the major variable that needs to be considered to define the oligomerization state (in AUC this can be done nicely). And do not forget that the in-vitro oligomerization state does not necessarily imply the same for in vivo, so please do make mutants to prove it before submitting the paper ... A. On Aug 10, 2010, at 1:38, 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
Re: [ccp4bb] To view the 2mFo-DFc map generated by SIGMAA
Actually I think it needs the labels FWT PHWT I may be wrong but like REFMAC I think it outputs a ositive FWT and PHWT is either equal to PHIC or to PHHIC+180, depending on the sign og FWT = (2m|Fo| - D|Fc|) Eleanor Hailiang Zhang wrote: Dear Tim: This is also what I thought. Thanks! Best Regards, Hailiang Dear Hailiang, the man-page of sigmaa claims FWT (2m|Fo| - D|Fc|) exp(i AlphaCalc) Analogous to 2Fo-Fc map, FFT input: F1=FWT PHI=PHIC ^^ so I would leave out WCMB. Tim On Tue, Aug 10, 2010 at 01:33:11PM -0400, Hailiang Zhang wrote: Hi there: When I generate the mtz file by SIGMAA, and want to view the 2mFo-DFc map in coot, should I choose FWT PHIC WCMB combination or just FWT PHIC? I think the later is more reasonable and I did see somebody the former as well. Didn't see explicit instruction in SIGMAA document, and I appreciate for any hint. Best Regards, Hailiang -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] About using COOT
Dear all, My protein has two chains A B. The space gp is P212121. If I want to have a new PDB file containing the symmetry-equivalent molecule of B (x+1/2, -y+1/2, z+(0 -1 0)), and keep the chain A (x,y,z) unchanged. How can I do this COOT? many thanks for this help. stephen -- Dr. Stephen Sin-Yin Chui Research Assistant Professor, Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Tel: 22415814 (Office), 22415818 (X-ray Diffraction Laboratory)
Re: [ccp4bb] To view the 2mFo-DFc map generated by SIGMAA
Hi Eleanor, I hate to disagree with you, even partially, but it depends on whether or not you're doing phase combination with external phases. In the usual case with just model information, SIGMAA doesn't take the absolute value of 2mFo-DFc so it can be positive or negative and goes with the original PHIC. When phase combination is carried out, then the phase of the map coefficient is different from any of the input phases, and FWT has to be combined with PHFWT. There's a discussion in the documentation: http://www.ccp4.ac.uk/html/sigmaa.html#files. Regards, Randy On 11 Aug 2010, at 10:52, Eleanor Dodson wrote: Actually I think it needs the labels FWT PHWT I may be wrong but like REFMAC I think it outputs a ositive FWT and PHWT is either equal to PHIC or to PHHIC+180, depending on the sign og FWT = (2m|Fo| - D|Fc|) Eleanor Hailiang Zhang wrote: Dear Tim: This is also what I thought. Thanks! Best Regards, Hailiang Dear Hailiang, the man-page of sigmaa claims FWT (2m|Fo| - D|Fc|) exp(i AlphaCalc) Analogous to 2Fo-Fc map, FFT input: F1=FWT PHI=PHIC ^^ so I would leave out WCMB. Tim On Tue, Aug 10, 2010 at 01:33:11PM -0400, Hailiang Zhang wrote: Hi there: When I generate the mtz file by SIGMAA, and want to view the 2mFo-DFc map in coot, should I choose FWT PHIC WCMB combination or just FWT PHIC? I think the later is more reasonable and I did see somebody the former as well. Didn't see explicit instruction in SIGMAA document, and I appreciate for any hint. Best Regards, Hailiang -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
[ccp4bb] process data in R32
Hi, I have read all of the past messages about the confusion of H32 and R32 and I want to get the correct indexing sp that I can import the .sca file into CCP4.. I use DENZO and SCALEPACK in HKL2000. If you read the manual then it states that space group R32 should only be autoindexed by DENZO on a primitive lattice using the DENZO keyword H32. This indeed gives a primitive unit cell and not the hexagonal setting apparently demanded by CCP4 programs. What do you do next? It is possible in SCALEPACK to reindex the H32 primitive data into a rhombohedral hexagonal setting which SCALEPACK calls R32. Or course you cannot import this .sca file into CCP4. Why not? I can go into the .sca file and edit the header and change the R32 label to H32. Does this screw up the indexing? If anybody has successfully used DENZO/SCALEPACK/CCP4 in space group R32 then please tell me how to do it correctly. Cheers. Ray email ray-br...@att.net
Re: [ccp4bb] monomer-dimer
Thank you. Now I understand the difference. I thought there was separation. Maia Xuewu Zhang wrote: Hi Maia, I have seen your post regarding this before and I just want to point out that you may have confused AUC (analytical ultracentrifugation) with gradient-based ultra-centrifugation methods for separating macromolecules. AUC does not involve separation of different species in the sample. There are two types of AUCs: sedimentation velocity and sedimentation equilibrium. In sedimentation equilibrium experiments, the system reaches the equilibrium at the end, and the monomer/dimer ratio, Kd, etc parameters can be worked out by fitting the data to a model globally. The shape of the molecule does not matter. For starters: http://en.wikipedia.org/wiki/Ultracentrifuge Xuewu Zhang On Wed, Aug 11, 2010 at 10:37 AM, chern ch...@ualberta.ca mailto:ch...@ualberta.ca wrote: Hi Anastassis, We are back to the same argument that AUC is not a good method. As everyone knows, it's a dynamic equilibrium between monomers and dimers that exists before separation. Once you started separation in any method, the equilibrium is disturbed now in each separated band. That will cause re-equilibration and constant migration of newly formed dimers from the monomer band and newly formed monomers from the dimer band. The t(eq) is the re-equilibration time. Your method of separation of monomers and dimers should be quick enough before any re-equilibration occurs (t(sep)t(eq)). Otherwise, you get a mess and smearing of bands. Also, most conventional methods depend on shape etc. I find SEC is most convenient. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 2:38 PM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 22:04, chern wrote: Hi, Anastassis If you had just a monomer at the start time then t(eq) is the time to get to equilibrium with the dimer and vice versa. sorry to say but the definition of that time in a biophysical sense, is in my opinion equal to infinity and cannot be defined. I am being a bit pedantic here, but I am just saying that t(eq) cannot be defined, it can be approximated, and thus t(eq) is wrong to define. Why not talk about kD and kON and kOFF that have robust definitions based on kinetic properties and a physical meaning? When you separated the two bands (monomers and dimers) in AUC, and then the equilibrium is quickly established in each band again what's the point? So, to be successful in this method, you need to have t(eq) much lower than the separation run. Ideally, if you could separate monomers and dimers instantly and freeze them in the separated state, then you can have good estimate of the both fractions. I think this is clear. But, I disagree and I think what you say is wrong. The equilibrium is dynamic. Why do you insist there is a point in 'separation'? The monomer changes to a dimer and vise versa in a continuous fashion. All you can say is that in a given concentration the equilibrium is shifted towards one or the other form. But its a dynamic one. Even at a concentration which is 50-50 between two states, the molecules that are in one state or another are changing according to kinetic parameters that are characteristic for the complex. Even at 100% - lets say of a dimer - by your definition, (100% cannot exist since its reached asymptotically by any derivation about equilibriums) molecules will fall to monomer and will reassemble to a dimer rapidly. To be honest I think that talking about t(eq) is largely wrong in biophysical terms, since it does not exist. A. That's what I meant. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 11:45 AM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 19:30, chern wrote: Thank you for reply. 1.It will be nice to have mass-spec method for non-covalent complexes. Carol Robinson is doing these
Re: [ccp4bb] monomer-dimer
Hi ccp4bb Could you please send me some references with the sedimentation equilibrium calculations of Kd, monomer/dimer ratio etc. Maia Maia Cherney wrote: Thank you. Now I understand the difference. I thought there was separation. Maia Xuewu Zhang wrote: Hi Maia, I have seen your post regarding this before and I just want to point out that you may have confused AUC (analytical ultracentrifugation) with gradient-based ultra-centrifugation methods for separating macromolecules. AUC does not involve separation of different species in the sample. There are two types of AUCs: sedimentation velocity and sedimentation equilibrium. In sedimentation equilibrium experiments, the system reaches the equilibrium at the end, and the monomer/dimer ratio, Kd, etc parameters can be worked out by fitting the data to a model globally. The shape of the molecule does not matter. For starters: http://en.wikipedia.org/wiki/Ultracentrifuge Xuewu Zhang On Wed, Aug 11, 2010 at 10:37 AM, chern ch...@ualberta.ca mailto:ch...@ualberta.ca wrote: Hi Anastassis, We are back to the same argument that AUC is not a good method. As everyone knows, it's a dynamic equilibrium between monomers and dimers that exists before separation. Once you started separation in any method, the equilibrium is disturbed now in each separated band. That will cause re-equilibration and constant migration of newly formed dimers from the monomer band and newly formed monomers from the dimer band. The t(eq) is the re-equilibration time. Your method of separation of monomers and dimers should be quick enough before any re-equilibration occurs (t(sep)t(eq)). Otherwise, you get a mess and smearing of bands. Also, most conventional methods depend on shape etc. I find SEC is most convenient. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 2:38 PM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 22:04, chern wrote: Hi, Anastassis If you had just a monomer at the start time then t(eq) is the time to get to equilibrium with the dimer and vice versa. sorry to say but the definition of that time in a biophysical sense, is in my opinion equal to infinity and cannot be defined. I am being a bit pedantic here, but I am just saying that t(eq) cannot be defined, it can be approximated, and thus t(eq) is wrong to define. Why not talk about kD and kON and kOFF that have robust definitions based on kinetic properties and a physical meaning? When you separated the two bands (monomers and dimers) in AUC, and then the equilibrium is quickly established in each band again what's the point? So, to be successful in this method, you need to have t(eq) much lower than the separation run. Ideally, if you could separate monomers and dimers instantly and freeze them in the separated state, then you can have good estimate of the both fractions. I think this is clear. But, I disagree and I think what you say is wrong. The equilibrium is dynamic. Why do you insist there is a point in 'separation'? The monomer changes to a dimer and vise versa in a continuous fashion. All you can say is that in a given concentration the equilibrium is shifted towards one or the other form. But its a dynamic one. Even at a concentration which is 50-50 between two states, the molecules that are in one state or another are changing according to kinetic parameters that are characteristic for the complex. Even at 100% - lets say of a dimer - by your definition, (100% cannot exist since its reached asymptotically by any derivation about equilibriums) molecules will fall to monomer and will reassemble to a dimer rapidly. To be honest I think that talking about t(eq) is largely wrong in biophysical terms, since it does not exist. A. That's what I meant. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 11:45 AM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 19:30, chern wrote: Thank you for reply.
[ccp4bb] advice for processing data from multiple small wedges
I am having problems processing data from multiple wedges of 5-20 degrees each. I am working with data from microcrystals collected at a minibeam. I want to use the data for molecular replacement. I can usually only collect about 10-15 frames before the crystal is obviously radiation damaged. I need to scale and merge the data from multiple crystals. I typically get poor diffraction, maybe to 4A. I have been able to process the data with xds/xscale, but the final statistics are worse than I would expect. For example, I can see fairly strong spots out to 4A, but xds might say the data only goes to 5A. This might be accurate, but I would like to also try the data with mosflm and scala. I can usually get mosflm to autoindex if I give it 3+ frames, but the results are not as robust as with xds. Usually the residual is poor, 0.3 mm. When I try scala (tried multiple options) with just one wedge, it fails with too few reflections, no observations, or negative scales. xds says the crystals are fairly isomorphous, 1%. Symmetry is low, space group C2. Does anyone have some advice for processing this kind of data successfully?
Re: [ccp4bb] monomer-dimer
Hi Maia, this review and website might be a good place to start: http://analyticalultracentrifugation.com/images/AUCinProteinScience.pdf http://analyticalultracentrifugation.com/default.htm Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 Hi ccp4bb Could you please send me some references with the sedimentation equilibrium calculations of Kd, monomer/dimer ratio etc. Maia Maia Cherney wrote: Thank you. Now I understand the difference. I thought there was separation. Maia Xuewu Zhang wrote: Hi Maia, I have seen your post regarding this before and I just want to point out that you may have confused AUC (analytical ultracentrifugation) with gradient-based ultra-centrifugation methods for separating macromolecules. AUC does not involve separation of different species in the sample. There are two types of AUCs: sedimentation velocity and sedimentation equilibrium. In sedimentation equilibrium experiments, the system reaches the equilibrium at the end, and the monomer/dimer ratio, Kd, etc parameters can be worked out by fitting the data to a model globally. The shape of the molecule does not matter. For starters: http://en.wikipedia.org/wiki/Ultracentrifuge Xuewu Zhang On Wed, Aug 11, 2010 at 10:37 AM, chern ch...@ualberta.ca mailto:ch...@ualberta.ca wrote: Hi Anastassis, We are back to the same argument that AUC is not a good method. As everyone knows, it's a dynamic equilibrium between monomers and dimers that exists before separation. Once you started separation in any method, the equilibrium is disturbed now in each separated band. That will cause re-equilibration and constant migration of newly formed dimers from the monomer band and newly formed monomers from the dimer band. The t(eq) is the re-equilibration time. Your method of separation of monomers and dimers should be quick enough before any re-equilibration occurs (t(sep)t(eq)). Otherwise, you get a mess and smearing of bands. Also, most conventional methods depend on shape etc. I find SEC is most convenient. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 2:38 PM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 22:04, chern wrote: Hi, Anastassis If you had just a monomer at the start time then t(eq) is the time to get to equilibrium with the dimer and vice versa. sorry to say but the definition of that time in a biophysical sense, is in my opinion equal to infinity and cannot be defined. I am being a bit pedantic here, but I am just saying that t(eq) cannot be defined, it can be approximated, and thus t(eq) is wrong to define. Why not talk about kD and kON and kOFF that have robust definitions based on kinetic properties and a physical meaning? When you separated the two bands (monomers and dimers) in AUC, and then the equilibrium is quickly established in each band again what's the point? So, to be successful in this method, you need to have t(eq) much lower than the separation run. Ideally, if you could separate monomers and dimers instantly and freeze them in the separated state, then you can have good estimate of the both fractions. I think this is clear. But, I disagree and I think what you say is wrong. The equilibrium is dynamic. Why do you insist there is a point in 'separation'? The monomer changes to a dimer and vise versa in a continuous fashion. All you can say is that in a given concentration the equilibrium is shifted towards one or the other form. But its a dynamic one. Even at a concentration which is 50-50 between two states, the molecules that are in one state or another are changing according to kinetic parameters that are characteristic for the complex. Even at 100% - lets say of a dimer - by your definition, (100% cannot exist since its reached asymptotically by any derivation about equilibriums) molecules will fall to monomer and will reassemble to a dimer rapidly. To be honest I think that talking about t(eq) is largely wrong in biophysical terms, since it does not exist. A. That's what I meant.
Re: [ccp4bb] monomer-dimer
I agree completely with Anastassis that the equilibrium will be effected by changing the concentration of the sample during gel filtration, however I wanted to point out that the elution volumes of the two species are independent of their populations. I apologize if I was misleading. Mike - Original Message - From: Anastassis Perrakis a.perra...@nki.nl To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, August 11, 2010 2:10:16 AM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] monomer-dimer Dear all, If I may add that I find the statement 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. a bit misleading. While concentration will not change where the monomer or the dimer appears in the elution volume, concentration will affect the monomer-dimer equilibrium during your gel-filtration run. Thus, I would say that concentration is a factor. If your dimer has a kD of ~100uM, and you inject it at a concentration of ~100uM, after getting diluted during gel-filtration (about ten-fold) it will appear 90% as a monomer ... The results of any analytical technique to determine stoichiometry are concentration dependent, and concentration is actually the major variable that needs to be considered to define the oligomerization state (in AUC this can be done nicely). And do not forget that the in-vitro oligomerization state does not necessarily imply the same for in vivo, so please do make mutants to prove it before submitting the paper ... A. On Aug 10, 2010, at 1:38, 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
[ccp4bb] calculating solvent volume from molecular surfaces
Does anyone know of software that will segment a unit cell into volume internal/external to a calculated molecular surface ? thanks! Alastair Fyfe
