Re: [ccp4bb] Bicarb at low pH
On 10/02/11 16:56, Jacob Keller wrote: Dear Crystallographers, I would like to soak my crystals in bicarbonate (a possible substrate), but the crystals have grown--and only grow--in pH 5.2-6.0, so the bicarb/CO2 will just keep evolving out of the solution and reliquishing its hydroxyls until the pH is elevated sufficiently out of range. Does anyone have a clever way of getting bicarb into these crystals? Grow them under CO2? Transfer them to higher pH, and hope for the best? Jacob Keller Is it the bicarb you are interested in, or the CO2? Domsic, et al were able to trap a carbon dioxide in carbonic anhydrase II by adding CO2 during a high pressure cryo-cooling experiment. J Biol Chem. 2008 November 7; 283(45): 30766--30771. doi: 10.1074/jbc.M805353200 http://dx.crossref.org/10.1074%2Fjbc.M805353200 -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Bicarb at low pH
Hi Jacob, high-pressure cryocrystallography methods may be useful in this case. I copied your question to Chae Un Kim here at MacCHESS and he forwards this suggestion: -- Hi Richard, I think pressure cryocooling might be useful. They may want to check the following reference. JF Domsic et al (2008), Entrapment of carbon dioxide in the active site of carbonic anhydrase II, J. Biol. Chem. 283, 30766-30771 Best regards, chaeun -- On Oct 2, 2011, at 6:22 PM, Roger Rowlett wrote: You can't change the pKa of CO2, which is 6.3. Any attempt to grow bicarb complexes below pH 7.5 will be problematic due to CO2 bubble formation, which may crack the crystal. What we do in these situations is to soak crystals in a cryo solution at a higher pH for as long as practical, then transfer to another, identical solution with bicarbonate fo li and soak. This can be tricky, because crystals may take a long time to pH equilibrate, crack, or dissolve. At pH 7.5 or above, CO2 formation at chemical equilibrium is minimal. At low pH, you can also consider using acetate as a bicarbonate analog. Roger Rowlett On Oct 2, 2011 4:56 PM, Jacob Keller j-kell...@fsm.northwestern.edumailto:j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, I would like to soak my crystals in bicarbonate (a possible substrate), but the crystals have grown--and only grow--in pH 5.2-6.0, so the bicarb/CO2 will just keep evolving out of the solution and reliquishing its hydroxyls until the pH is elevated sufficiently out of range. Does anyone have a clever way of getting bicarb into these crystals? Grow them under CO2? Transfer them to higher pH, and hope for the best? Jacob Keller
Re: [ccp4bb] Anomalous patterson not consistent with systematic extinctions
Further Qs. Do you have a noncryst translation parallel to the b axis (ctruncate will list any such translation..) If the b shift is 0.5 then the 0k0 absences will be present whether the spacegroup is P2 or P21. How many Xe sites do you expect? If there is only one then phasing is more difficult in monoclinic SGs - you have to break the centrosymmetry of the heavy atom distribution. Do you have native data without Xe? I always check for peaks which are consistent in the isomorphous and anomalous difference pattersons. The NCS symmetry will doubtless make the exptl phasing more complicated, but it should help you with averaging later! Eleanor On 10/03/2011 03:33 PM, Marta Ferraroni wrote: Dear all, We collected some Xenon derivatives of a protein (an heterotetramer α2β2)that seems to crystallize in P21 since the 0k0 reflections with k=2n+1 are not present. However in the anomalous Patterson we found strong peaks in the section v=0 yet none in the Harker section v=1/2. Furthermore we weren't able to solve the structure both in P2 and P21 using these derivatives with the most commonly used programs. The cell is 89 125 90 90 102 90 so a is approximately equal to c that could permit pseudomerohedral twinning albeit the tests (Padilla-Yeates and Britton) estimate a fraction of twinning around 0.05. Data cannot be scaled as C orthorhombic even if the data reduction programs (XDS and imosflm) assign a higher score to the related orthorhombic cell with dimensions 112 139 124 90.00 90.00 90.00. In the native Patterson map there are not strong peaks. According to the Matthews coefficient the asymmetric unit contains 2 heterotetramers and the self rotation function may indicate a 222 non crystallographic symmetry with one two-fold axis perpendicular to the crystallographic one (see figure attached). Our questions are: 1) why the anomalous Patterson is not consistent with the space group? 2) Is there the possibility that the NCS could hamper the determination of the correct space group and eventually determine a lower estimate of the twinning fraction? thanks in advance for your help Regards, Marta Ferraroni Dept. of Chemistry University of Florence Italy
Re: [ccp4bb] Anomalous patterson not consistent with systematic extinctions
Why not just go to P1, then build up the symmetry? Is completeness low? JPK On Mon, Oct 3, 2011 at 10:14 AM, Eleanor Dodson c...@ysbl.york.ac.uk wrote: Further Qs. Do you have a noncryst translation parallel to the b axis (ctruncate will list any such translation..) If the b shift is 0.5 then the 0k0 absences will be present whether the spacegroup is P2 or P21. How many Xe sites do you expect? If there is only one then phasing is more difficult in monoclinic SGs - you have to break the centrosymmetry of the heavy atom distribution. Do you have native data without Xe? I always check for peaks which are consistent in the isomorphous and anomalous difference pattersons. The NCS symmetry will doubtless make the exptl phasing more complicated, but it should help you with averaging later! Eleanor On 10/03/2011 03:33 PM, Marta Ferraroni wrote: Dear all, We collected some Xenon derivatives of a protein (an heterotetramer α2β2)that seems to crystallize in P21 since the 0k0 reflections with k=2n+1 are not present. However in the anomalous Patterson we found strong peaks in the section v=0 yet none in the Harker section v=1/2. Furthermore we weren't able to solve the structure both in P2 and P21 using these derivatives with the most commonly used programs. The cell is 89 125 90 90 102 90 so a is approximately equal to c that could permit pseudomerohedral twinning albeit the tests (Padilla-Yeates and Britton) estimate a fraction of twinning around 0.05. Data cannot be scaled as C orthorhombic even if the data reduction programs (XDS and imosflm) assign a higher score to the related orthorhombic cell with dimensions 112 139 124 90.00 90.00 90.00. In the native Patterson map there are not strong peaks. According to the Matthews coefficient the asymmetric unit contains 2 heterotetramers and the self rotation function may indicate a 222 non crystallographic symmetry with one two-fold axis perpendicular to the crystallographic one (see figure attached). Our questions are: 1) why the anomalous Patterson is not consistent with the space group? 2) Is there the possibility that the NCS could hamper the determination of the correct space group and eventually determine a lower estimate of the twinning fraction? thanks in advance for your help Regards, Marta Ferraroni Dept. of Chemistry University of Florence Italy -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Bicarb at low pH
On Sun, 2011-10-02 at 15:56 -0500, Jacob Keller wrote: Transfer them to higher pH, and hope for the best? Consider crosslinking your crystals with glutaraldehyde. They will then became virtually insoluble, although it's possible that you may lose diffraction at elevated pH. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] Bicarb at low pH
Hi Jacob, Once I had an issue with introuding CO2 into crystals and I was able to get it bound by putting a small piece of dry ice into the well solution and keeping the drop under CO2 pressure using nextal trays with screw cap. Please note in this case CO2 didn't get bound by using pressure cell. This could imply that CO2 gets incorporated into these crystals during crystal growth. regards, Mathews From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller [j-kell...@fsm.northwestern.edu] Sent: Sunday, October 02, 2011 1:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Bicarb at low pH Dear Crystallographers, I would like to soak my crystals in bicarbonate (a possible substrate), but the crystals have grown--and only grow--in pH 5.2-6.0, so the bicarb/CO2 will just keep evolving out of the solution and reliquishing its hydroxyls until the pH is elevated sufficiently out of range. Does anyone have a clever way of getting bicarb into these crystals? Grow them under CO2? Transfer them to higher pH, and hope for the best? Jacob Keller
[ccp4bb] Calculate real-space R-factor/corr coeff for ligand
Hi, I am trying to calculate real-space R-factors and correlation coefficients for an array of different ligand conformations to find out which fits best in experimental density. So far, I have been trying to use Overlapmap in CCP4 6.1.2 to do this, by correlating maps by residue and selecting the list a real-space R-factor option. I would like to compare a map with the ligand omitted to maps calculated with each ligand conformer. I am supplying Overlapmap with a refmac mtz file calculated without ligand in the model for map 1 and a pdb file that contains both protein and ligand coordinates to calculate map 2. However, I’m confused about the output. For the protein, which I know is well-defined and modeled correctly in the density, I see mostly reasonable correlation coefficients, ~0.9, but the real-space R-factor values are all over the place and range from zero to hundreds. For example, for one residue the correlation coefficient is 0.8309 with an R-factor of 210.333. I am very confused about how to interpret these values. Has anyone else tried to use Overlap for a similar purpose and could give suggestions as to what I’m doing wrong? Thanks! Brigitte
[ccp4bb] Off topic: Beryllium chloride
Sorry for the very off topic and dumb question, but does anyone know if BeCl2 needs to be prepared fresh for use (making BeF3) or can it be stored as a solution stock at room temperature/frozen? Thanks, Peter
Re: [ccp4bb] Off topic: Beryllium chloride
Hi Peter, Both BeF3(-) and AlF4(-) need to be made fresh, or used from frozen. It should not be stored at room temperature. While using, you may wish to keep it on ice. If you are using Vanadate, that should be fresh (Methods in Enzymology). Also kindly note that unlike AlF4(-), which is a single species; BeF3(-) is a mixture of BeF3.(H2O)-, BeF2(OH)- etc. It can make a difference in kinetics of binding (usually a two step binding is observed). Look at old ATPase and GTPase papers such as: Characterization of the aluminum and beryllium fluoride species which activate transducin. Analysis of the binding and dissociation kinetics.http://www.ncbi.nlm.nih.gov/pubmed/1551879 Antonny B, Chabre M. J Biol Chem. 1992 Apr 5;267(10):6710-8. PMID: 1551879 Cheers, Partha On Tue, Oct 4, 2011 at 5:05 AM, Peter Hsu hsuu...@u.washington.edu wrote: Sorry for the very off topic and dumb question, but does anyone know if BeCl2 needs to be prepared fresh for use (making BeF3) or can it be stored as a solution stock at room temperature/frozen? Thanks, Peter
