Re: [ccp4bb] Negative electron Density for Zinc
I wish I could give a sensible explanation, but this feature is very common with heavy atoms. Possible problems. The default wavelength for all atomic scattering functions is Cu Ka - you can see the formula used and details in the file $CLIBD/atomsf.lib. The atomic scattering values are much the same for C N O at a range of wavelengths, but metals can have significant f' differences. You can correct this in REFMAC input - see documentation. The Zn is not fully occupied - this depends on how it is incorporated. Eleanor On 28 Aug 2012, at 18:44, Deepthi wrote: Hi everybody, I am working on a mutant protein structure which is 56 aminoacids long and solved the structure using SAD( Single wavelength anomalous dispersion) with zinc ions. We used SHELX software to locate the Zinc atoms and solved phases. We got the structure as a monomer. The resolution of the structure is 1.4 A0 . The problem with the structure is, when i calculate the structure factors in COOT it always shows negative electron density around ZINC atoms which have been located by SHELX. I initially thought it was because my COOT software was very old and it doesn't recognize the Zinc atoms( I am new to crystallography) . I calculated the structure factors using FFT from CCP4 and it shows the same. I don't understand the problem. It refines well in Refmac and B-factors also look very good. Anyone know what the problem is? Thank You in advance Deepthi -- Deepthi
[ccp4bb] off topic: reduced glutathione interfering with protein activity?
Hi all, I've been purifying my protein off a GST column and have noticed a massive difference in activity of my protein between a prep that was freed from the column via on column cleavage, and a prep that was eluted (20mM GSH) and then cleaved and further purified. I'm suspecting that the glutathione is somehow modifying/inhibiting my protein in some way, despite having removed the glutathione from the buffer via dialysis/ion exchange. I don't see anything out of the ordinary in my electron density that would suggest that glutathione has affected my protein in some way, but the huge difference seen in my activity assay suggests otherwise. My question is, has anyone else seen an effect from glutathione affecting their protein in some way? My second question is, what's the minimum amount of glutathione necessary to elute your protein from a column? Sorry for the off topic question and thanks for any responses, Peter
Re: [ccp4bb] off topic: reduced glutathione interfering with protein activity?
GSH will reduce your protein quite nicely - is your enzyme activity redox sensitive? --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On 8/29/12 5:56 PM, Peter Hsu hsuu...@u.washington.edu wrote: Hi all, I've been purifying my protein off a GST column and have noticed a massive difference in activity of my protein between a prep that was freed from the column via on column cleavage, and a prep that was eluted (20mM GSH) and then cleaved and further purified. I'm suspecting that the glutathione is somehow modifying/inhibiting my protein in some way, despite having removed the glutathione from the buffer via dialysis/ion exchange. I don't see anything out of the ordinary in my electron density that would suggest that glutathione has affected my protein in some way, but the huge difference seen in my activity assay suggests otherwise. My question is, has anyone else seen an effect from glutathione affecting their protein in some way? My second question is, what's the minimum amount of glutathione necessary to elute your protein from a column? Sorry for the off topic question and thanks for any responses, Peter
Re: [ccp4bb] off topic: reduced glutathione interfering with protein activity?
I don't think so since I purify in the presence of reducing agents (DTT/BME) and I got activity out of the prep that was released by on column cleavage. On the other hand, I don't usually add those fresh each time I use the buffers so it's entirely possible the reducing agents are nowhere near the initial reduced form they were in initially. On Wed, 29 Aug 2012, Antony Oliver wrote: GSH will reduce your protein quite nicely - is your enzyme activity redox sensitive? --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On 8/29/12 5:56 PM, Peter Hsu hsuu...@u.washington.edu wrote: Hi all, I've been purifying my protein off a GST column and have noticed a massive difference in activity of my protein between a prep that was freed from the column via on column cleavage, and a prep that was eluted (20mM GSH) and then cleaved and further purified. I'm suspecting that the glutathione is somehow modifying/inhibiting my protein in some way, despite having removed the glutathione from the buffer via dialysis/ion exchange. I don't see anything out of the ordinary in my electron density that would suggest that glutathione has affected my protein in some way, but the huge difference seen in my activity assay suggests otherwise. My question is, has anyone else seen an effect from glutathione affecting their protein in some way? My second question is, what's the minimum amount of glutathione necessary to elute your protein from a column? Sorry for the off topic question and thanks for any responses, Peter
Re: [ccp4bb] Jelly body refinement?
Hi Gunnar, A couple of comments, to clarify a few of the similarities and dissimilarities between DEN and analogous technologies: According to your very nice paper from 2010, DEN refinement with gamma=0 gives a higher weight to external information, whilst gamma=1 ignores external information in favour of self-restraints. Thus, unless I am mistaken, isn't it gamma=1 that would be more analogous to jelly-body refinement? Both jelly-body and DEN with gamma=1 are similar in that they are both independent of explicit externally-derived information. Indeed, DEN with gamma in [0,1] is analogous, but not equivalent, to a combination of jelly-body (or self-restraints) and external reference structure restraints as implemented in REFMAC5. In fact, jelly-body is actually quite different to DEN with gamma=1. Since jelly-body restraints are not applied to the target function (or 1st derivative), the restrained atoms are allowed to move easily if there is evidence to suggested that they should, e.g. from the electron density, or from other (external) restraints. The principal purpose of jelly-body restraints is simply to act as a regulariser thus stabilise refinement, not to inhibit deformation of interatomic distances where appropriate. Jelly-body is only applied to the 2nd derivative simply due to the form of the function: X=(d-d_current)^2. Note that d_current is updated at each step, thus we always have d=d_current. Thus, X=0, X'=0, but X''!=0. This formulation makes sense - in the absence of any external prior knowledge, we shouldn't change the likelihood function or the gradient, as we want the minima to remain in the same place. However, we can reasonably change the 2nd derivative, and we would like to benefit from the decreased effective parameter-to-observation ratio from this regulariser. Hopefully, that explains why jelly-body is actually quite different to DEN with gamma=1. Importantly, note that with jelly-body d_current is updated/reset at each step, which means that the structure is indeed very deformable. The structure is allowed to move away from the start values - in fact, d_current at cycle n is not dependent on d_current at cycle 0. I believe this contrasts with DEN, unless kappa=1. In contrast with jelly-body, external restraints and local NCS restraints are applied to the target function. In order to allow the inter-atomic distances to exhibit large deviations from the prior information, the Geman-McClure robust estimator function is used instead of assuming least squares residuals (i.e. parameters are estimated using generalised M-estimators instead of the traditional maximum likelihood method). Consequently, when using jelly-body and external restraints, regions of structure that need to move far should be able to do so, whilst the regions that are happy should remain where they are (ideally with more stable refinement and less overfitting) . Hopefully that helps to clarify a few of the similarities and dissimilarities between DEN and the analogous technologies implemented in REFMAC5 to anyone who may find it useful! Regards Rob On 28 Aug 2012, at 20:23, Gunnar Schroeder wrote: Just a quick comment on low resolution refinement: The concept of Deformable Elastic Network (DEN) refinement is quite similar to jelly-body refinement in the special case of gamma=0, for which the network is not deformable. In contrast to jelly-body refinement, the DEN restraints are however actually applied to the target function (and the first derivative). For gamma0 the minimum of the elastic network potential is allowed to move and, thus, to deform the restraints (which changes their equilibrium distances). Some individual distances can deform more than others depending on the force they feel from the target function. This automatically discriminates between those regions in the structure that need to move far (and are allowed to do so) and those regions that are happy where they are (and remain restrained). DEN refinement is implemented in CNS (1.3) and now also in Phenix (=1.7.3). Cheers, Gunnar
Re: [ccp4bb] Plastic and glass plates
Theresa, The major advantage of the plastic plates is indeed ease of harvest. However, the plastic plates also tend to have some evaporation issues and eventually dry out after a few months, where as the glass plates basically last forever. On the other hand, protein crystals in LCP tend to form in the first several weeks after an experiment is set up so this usually isn't a problem. Some people prefer to set up initial screening experiments in the glass plates and then optimize and set up farm trays using the plastic plates where it's easier to harvest from (and cheaper). I've also set up everything in plastic trays from the start of a project to finish without any problems. If you're looking to perform LCP FRAP experiments, glass is the best way to go. Cheers, Jim On Wed, Aug 29, 2012 at 12:24 PM, Theresa Hsu theresah...@live.com wrote: Dear all Is there any pros and cons of using plastic plates for LCP crystallization? The glass is clearer but it is very difficult to open. Thank you. -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures http://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com
Re: [ccp4bb] off topic: reduced glutathione interfering with protein activity?
Peter, Yes, in our hands it can eluted completely. Usually 20 ml of elution buffer per 2 ml of GS4B resin (GE healthcare, fast flow) is enough . We use 30 ml open-columns for purification - each column has 2 ml of resin. Vitali On Wed, Aug 29, 2012 at 3:02 PM, hsuu...@u.washington.edu wrote: Hi Vitali, I usually prep my GST elution buffers fresh, and make sure to pH it after dissolving the powder. So I don't think that's the problem, but good to know that you can use as low as 5mM GSH. Do you know if you knock off most of the protein from the resin at that concentration of GSH? Thanks, Peter On Wed, 29 Aug 2012, Vitali Stanevich wrote: Peter, Such high concentration of GSH may change the pH according to our experience. We usually use 50 mM Tris pH=8.0, 5 mM GSH, 3 mM DTT - so that you can load the sample on ion-exchange column after elution. If your protein is not stable without NaCl - you should add it also. Vitali On Wed, Aug 29, 2012 at 12:42 PM, Peter Hsu hsuu...@u.washington.edu wrote: I don't think so since I purify in the presence of reducing agents (DTT/BME) and I got activity out of the prep that was released by on column cleavage. On the other hand, I don't usually add those fresh each time I use the buffers so it's entirely possible the reducing agents are nowhere near the initial reduced form they were in initially. On Wed, 29 Aug 2012, Antony Oliver wrote: GSH will reduce your protein quite nicely - is your enzyme activity redox sensitive? --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On 8/29/12 5:56 PM, Peter Hsu hsuu...@u.washington.edu wrote: Hi all, I've been purifying my protein off a GST column and have noticed a massive difference in activity of my protein between a prep that was freed from the column via on column cleavage, and a prep that was eluted (20mM GSH) and then cleaved and further purified. I'm suspecting that the glutathione is somehow modifying/inhibiting my protein in some way, despite having removed the glutathione from the buffer via dialysis/ion exchange. I don't see anything out of the ordinary in my electron density that would suggest that glutathione has affected my protein in some way, but the huge difference seen in my activity assay suggests otherwise. My question is, has anyone else seen an effect from glutathione affecting their protein in some way? My second question is, what's the minimum amount of glutathione necessary to elute your protein from a column? Sorry for the off topic question and thanks for any responses, Peter
[ccp4bb] Pittsburgh Diffraction Conference 2012
The 70th Pittsburgh Diffraction Conference will be held at SSRL between September 30th and October 2nd 2012 and will feature 2 full days of lectures and poster presentations. The conference starts with a welcome reception on September 30 and there will be a banquet on the evening of October 1st. The conference will precede the 2012 SSRL/LCLS Users' Meeting and Workshops http://www-conf.slac.stanford.edu/ssrl-lcls/2012/ October 3rd - 6th. Session topic include: New Science with Femtosecond Diffraction Exciting Macromolecular Structures New Ideas in Crystallography, and Hybrid Methods in Macromolecular Crystallography There will be a Keynote Address to begin the Conference on Monday October 1st by Prof Brian Kobilka (Stanford University) on G protein coupled receptors (GPCRs). All speakers and session topics can be found at this link: http://www.pittdifsoc.org/PDC_2012/schedule.htm Registration is now open (US$100 or US$20 for students and postdocs) and for those who register at least 2 weeks in advance you will receive a free ticket to the banquet. There will be an award for the best student poster so get your abstracts in nice and early. The abstract deadline has been extended to September 15. Please visit the website below to register and submit your abstract. http://www.pittdifsoc.org/PDC_2012/index.htm If you have not made your hotel reservations yet and would like to stay at the SLAC Guest House, please reserve your room soon. The SLAC Guest House will only hold the remaining rooms that have been blocked for the PDC and Users' Meeting through this week. Please specify 'Users12' or 'PDS 2012' to take advantage of discount rates for SLAC guests. Visit this link for more information about accommodation options: http://www.pittdifsoc.org/PDC_2012/accommodations.htm. - Clyde A. Smith, Ph.D. Senior Staff Scientist Stanford University Stanford Synchrotron Radiation Lab, MS 99 2575 Sand Hill Rd Menlo Park, CA 94025 USA ph (650)926-8544 fax (650)926-3292 cell (650)714-6001 smb.slac.stanford.edu
