Re: [ccp4bb] Fo-Fo Difference Map
Dale, On Tue, May 4, 2010 at 12:19 AM, Dale Tronrud det...@uoxray.uoregon.edu wrote: The greater the difference in cell constants the greater the noise in the map. I think the high resolution cutoff for the maps should be 2 A delta/(A+delta) where A is the cell edge with the largest change, and delta is the amount of change (in Angstrom). Basically a 1A change for a 100A edge would require a 2A resolution limit. A 5A change would imply a 10A cutoff and a very boring map. I think this estimate may be a little over-optimistic. Crick Magdoff (1956, Acta Cryst.9, 901–908) calculated that for a 100Å cubic unit cell a 0.5% change in all the cell dimensions, or a 0.5° rotation of the molecule within the unit cell, would produce an average 15% change in intensity, equivalent to a 7.5% average change in F, for data to 3Å resolution. So your 1% change in cell at 2Å would give ~ 22.5% average change in F, just from the non-isomorphism effects. For an Fo-Fc map, given that measurement errors are typically ~ 4 or 5% in F, you need to have the average difference due the ligand or heavy atom at least 3 times the noise level at the bare minimum, say at least 12%, and indeed we see average differences (i.e. partial Fc) due to a soaked ligand in the range 10-20%. For an Fo-Fo map the noise level is multiplied by sqrt(2) since you have 2 lots of measurement errors, so you have 6-8% average experimental error in Fo-Fo. Now clearly if you have experimental errors of ~ 7% plus non-isomorphism errors of 22.5% and you're looking for a signal of 15% due to the ligand then you have a problem! It implies you need to reduce the non-isomorphism errors by a factor of at least 3, i.e. to = 0.3% change in cell at 2Å. Such a stringent requirement, which is virtually impossible to achieve for frozen/soaked crystals (freezing soaking can easily induce 1% and sometimes 5% change), is surely the reason that Fo-Fo maps never caught on! One objection to this that I've seen is that Fo-Fc differences include errors due to the model, say ~ 20% (assuming that's the R factor), which Fo-Fo differences clearly do not. However the point is that this 20% contribution from the model error is spread throughout the cell, not just at the ligand site, since it arises from co-ordinate B factor errors, disorder etc in all atoms, of which the ligand makes up a very small fraction, so the contribution to this from errors in the ligand parameters are likely to be negligible (adding the ligand has a negligible effect on the R factor!). In contrast the ~ 5% experimental error in Fo-Fc, or ~ 7% + non-isomorphism error for Fo-Fo, contribute roughly equally to the error in the density over the whole cell. Cheers -- Ian
[ccp4bb] Post-doctoral fellow/scientist position at Karolinska Institutet
Postdoctoral Position in Structural Enzymology of Bacterial Pathogens A postdoctoral position is available for a highly motivated individual at the Department of Medical Biochemistry Biophysics, Karolinska Institutet, Stockholm, Sweden. The candidate will join on-going multidisciplinary research aimed at the structural and functional characterization of proteins and protein complexes from major pathogens, in particular Pseudomonas aeruginosa (for more details see http://www.aeropath.eu/). The position is for two years, with the possibility of a one year extension. The project includes recombinant production of bacterial protein targets and their biochemical, biophysical, and structural characterization. The methods used will include e.g. protein crystallography, enzyme kinetics, and fragment based screening of small-molecule libraries. The successful candidate must have a doctoral degree, a strong commitment to science, and should possess good written and oral communication skills. A background in biochemistry, biophysics and/or protein crystallography is an advantage. The laboratory has access to state-of-the-art facilities for structural biology and biochemical research. A protein production and crystallization platform and a broad range of instruments for biophysical characterization of proteins protein complexes are available at the department. For further enquiries please contact Prof Gunter Schneider (phone: +46 (0) 8 5248 7675) or by e-mail (gunter.schnei...@ki.se). Interested candidates are encouraged to apply by sending curriculum vitae, a brief statement describing research experience, scientific interests and motivation and the names and e-mail addresses of at least two referees by e-mail to (gunter.schnei...@ki.se). Deadline for applications: june 15, 2010 --- Prof Gunter Schneider Department of Medical Biochemistry Biophysics Scheelevägen 2 Karolinska Institutet, S-171 77 Stockholm, Sweden Phone: +46 8 5248 7675 , mobile phone: 0733 342 877 FAX: +46-8-32 76 26 e-mail: gunter.schnei...@ki.se Home page: http://phillips.mbb.ki.se
Re: [ccp4bb] Fo-Fo Difference Map
Ian's analysis is, as usual, much more sophisticated then my little back-of-the-envelope calculation. I would like to make one philosophical note: The non-isomorphism between the two crystals is not noise in the Fo-Fo map, it is the whole point. Sometimes the non-isomorphism is dominated by the direct effects of the presence of a compound binding to the protein (for instance) but other times by the large scale changes in orientation of the domains of the protein in response to that compound. If your protein undergoes large scale motion upon binding and you are interested in those motions then the Fo-Fo map will show them to you, even though the small changes directly caused by the compound might be difficult to discern. If the protein doesn't have domain motions then you are likely to see very interesting, specific, motions of side chains and solvent around the binding site. If you are not interested by the major non-isomorphism of your pair of crystals you will have to find some way to filter out the boring non-isomorphism from the interesting non-isomorphism. This will require a model and the potential for model bias. Very often this cannot be avoided. The variability of cell constants after freezing is a property of the protein, not intrinsic to the freezing process itself. With my favorite protein the cell constants are very consistent after freezing and I can calculate Fo-Fo maps that show great detail. Perhaps Fo-Fo maps would be more popular if they weren't so cumbersome to calculate in the CCP4 world. Dale Tronrud On 05/04/10 04:48, Ian Tickle wrote: Dale, On Tue, May 4, 2010 at 12:19 AM, Dale Tronrud det...@uoxray.uoregon.edu wrote: The greater the difference in cell constants the greater the noise in the map. I think the high resolution cutoff for the maps should be 2 A delta/(A+delta) where A is the cell edge with the largest change, and delta is the amount of change (in Angstrom). Basically a 1A change for a 100A edge would require a 2A resolution limit. A 5A change would imply a 10A cutoff and a very boring map. I think this estimate may be a little over-optimistic. Crick Magdoff (1956, Acta Cryst.9, 901–908) calculated that for a 100Å cubic unit cell a 0.5% change in all the cell dimensions, or a 0.5° rotation of the molecule within the unit cell, would produce an average 15% change in intensity, equivalent to a 7.5% average change in F, for data to 3Å resolution. So your 1% change in cell at 2Å would give ~ 22.5% average change in F, just from the non-isomorphism effects. For an Fo-Fc map, given that measurement errors are typically ~ 4 or 5% in F, you need to have the average difference due the ligand or heavy atom at least 3 times the noise level at the bare minimum, say at least 12%, and indeed we see average differences (i.e. partial Fc) due to a soaked ligand in the range 10-20%. For an Fo-Fo map the noise level is multiplied by sqrt(2) since you have 2 lots of measurement errors, so you have 6-8% average experimental error in Fo-Fo. Now clearly if you have experimental errors of ~ 7% plus non-isomorphism errors of 22.5% and you're looking for a signal of 15% due to the ligand then you have a problem! It implies you need to reduce the non-isomorphism errors by a factor of at least 3, i.e. to = 0.3% change in cell at 2Å. Such a stringent requirement, which is virtually impossible to achieve for frozen/soaked crystals (freezing soaking can easily induce 1% and sometimes 5% change), is surely the reason that Fo-Fo maps never caught on! One objection to this that I've seen is that Fo-Fc differences include errors due to the model, say ~ 20% (assuming that's the R factor), which Fo-Fo differences clearly do not. However the point is that this 20% contribution from the model error is spread throughout the cell, not just at the ligand site, since it arises from co-ordinate B factor errors, disorder etc in all atoms, of which the ligand makes up a very small fraction, so the contribution to this from errors in the ligand parameters are likely to be negligible (adding the ligand has a negligible effect on the R factor!). In contrast the ~ 5% experimental error in Fo-Fc, or ~ 7% + non-isomorphism error for Fo-Fo, contribute roughly equally to the error in the density over the whole cell. Cheers -- Ian
[ccp4bb] Advice on Over-expressing and Purifying Metalloproteins
Dear all, I am trying to purify a metalloprotein (a hydrogenase) using affinity chromatography. I have produced two tagged versions of the enzyme: one with an N-terminal 6x histidine affinity tag, and the other with a C-terminal 6x his-tag. The tagged proteins are both tied to an IPTG-inducible promoter. When trying to express and purify the N-terminal tagged protein, I have found that almost all of the expressed protein goes into inclusion bodies when the culture is grown at 37 or at 30 degrees C. When the culture is grown at 20 degrees C, a small amount of protein can be found in the cell extract. Unfortunately, as the enzyme has several oxygen-sensitive metal clusters, we do not believe that the protein can be refolded from the inclusion bodies. Could you offer some advice on how to express and purify this protein and reduce the quantity of protein found in inclusion bodies? Thanks! and all the best, --Buz
[ccp4bb] Symposium Celebration of Greg Petsko and Dagmar Ringe
Dear CCP4bbers, On September 4, 1980, Prof. Dagmar Ringe and Prof. Greg Petsko of MIT entered into an official scientific collaboration, and the scientific community has never been the same. Greg and Dagmar have jointly trained over 120 graduate students and postdocs and countless undergraduates in the past 29 years, and many of you might have worked with a student, postdoc, PhD advisor or postdoc mentor who previously either trained in or collaborated with the Petsko-Ringe lab. Now, 30 years later, the members of the Petsko-Ringe lab are holding a symposium at Brandeis University in honor of their combined lifetimes of achievement. The symposium and celebration is titled, From Sequence to Consequence: Celebrating 30 Years of Science with Dagmar Ringe and Greg Petsko. Read more about the symposium at: http://www.bio.brandeis.edu/PRSymposium2010/ While the symposium is a lab reunion of sorts for all the former and current students, postdocs, staff and collaborators of the Petsko- Ringe lab, we invite everyone from the structural community to take part in the celebration by posting congratulatory messages for Greg Petsko and Dagmar Ringe on our online Message Board. Please post your HEARTY GREETINGS for Greg and Dagmar at: http://prsymposium2010.blogspot.com/2010/05/celebration-of-dynamic-duo.html Please could you circulate this email to colleagues who do not subscribe to CCP4BB. With Warm Regards, Members of the Petsko-Ringe Lab --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University
Re: [ccp4bb] Advice on Over-expressing and Purifying Metalloproteins
Buz, How big is the protein? How many metal binding sites are there? Do you know what metal it should bind? What is your media? I am assuming it is a zinc binding protein, some zinc binding protein will not fold without enough zinc in the media. Perhaps you should not rule out refolding. ray USF On May 4, 2010, at 5:11 PM, Buz Barstow wrote: Dear all, I am trying to purify a metalloprotein (a hydrogenase) using affinity chromatography. I have produced two tagged versions of the enzyme: one with an N-terminal 6x histidine affinity tag, and the other with a C-terminal 6x his-tag. The tagged proteins are both tied to an IPTG-inducible promoter. When trying to express and purify the N-terminal tagged protein, I have found that almost all of the expressed protein goes into inclusion bodies when the culture is grown at 37 or at 30 degrees C. When the culture is grown at 20 degrees C, a small amount of protein can be found in the cell extract. Unfortunately, as the enzyme has several oxygen-sensitive metal clusters, we do not believe that the protein can be refolded from the inclusion bodies. Could you offer some advice on how to express and purify this protein and reduce the quantity of protein found in inclusion bodies? Thanks! and all the best, --Buz
Re: [ccp4bb] Advice on Over-expressing and Purifying Metalloproteins
Buz, Have you tried putting on a solubility tag, such as GST? I've found some times using M9 to grow my proteins in can give more soluble protein, although yields are usually lower. Best, Peter
