Re: [ccp4bb] Staining Crystals with comassie
Dear All, izit dye is a solution containing methylene blue that you could prepare in your lab. I usually prepare a solution of 0.05%w/v of dye in water and then I add a volume of dye solution equals to 10% of the volume of the drop containing the crystal to test. I prefer to add the dye solution in small portions (if the volume permits) every 2-3h in order to limit the shock due to the new solution on the crystal. You should remember that this test is not definitive: the dye is a cationic dye, that needs of anion counter part to bind the protein. Therefore, the dye is not able to colour all protein crystals: in addition, colouration is affect by pH of the crystallization condition, since low pH could increase the positive charge on the protein reducing its ability to bind the dye. You could try also glutaraldehyde as alternative. In order to perform this test, you should put the crystal into a low ionic strength buffered solution containing up to 2% glutaraldehyde. In this condition, formation of Schiff bases with the lysines and N-term residues occurs and the crystal become a yellow gel, while salt crystals dissolve and should not be coloured. To perform comassie crystal staining you should prepare a solution of 1% comassie in 40% MeOH and 10% Glacial Acetic Acid and add this solution as in methylene blue test. However, I rarely use this test because the use of MeOH and Glacial Acetic Acid causes the crystal dissolution. Only a final tip: obviously these tests enable you to distinguish between protein and salt, however they do not differentiate between protein in the crystal and protein in solution. For these reason, in some cases could be difficult to see the crystal colouration due to the low contrast with the colouration of the solution. Hence, I prefer to put the crystals to test in a new solution with the same formulation of the drop where the crystals have grown but without protein and perform here the dye test that I have chosen. In this way, you can easily see the colouration of the crystal without background effect. I hope I've helped you. Danilo On Tue, 15 Oct 2013 13:29:20 +0530, Swastik Phulera swastik.phul...@gmail.com wrote: Dear All, I am looking for a method to quickly differentiate between salt and protein crystals. I have been told thats its a popular alternative to the commercially available izit dye. I would appreciate if some one would share their comassie crystal staining protocol. Swastik
Re: [ccp4bb] Staining Crystals with comassie
Dear Swastik, izit dye is a solution containing methylene blue that you could prepare in your lab. I usually prepare a solution of 0.05%w/v of dye in water and then I add a volume of dye solution equals to 10% of the volume of the drop containing the crystal to test. I prefer to add the dye solution in small portions (if the volume permits) every 2-3h in order to limit the shock due to the new solution on the crystal. You should remember that this test is not definitive: the dye is a cationic dye, that needs of anion counter part to bind the protein. Therefore, the dye is not able to colour all protein crystals: in addition, colouration is affect by pH of the crystallization condition, since low pH could increase the positive charge on the protein reducing its ability to bind the dye. You could try also glutaraldehyde as alternative. In order to perform this test, you should put the crystal into a low ionic strength buffered solution containing up to 2% glutaraldehyde. In this condition, formation of Schiff bases with the lysines and N-term residues occurs and the crystal become a yellow gel, while salt crystals dissolve and should not be coloured. To perform comassie crystal staining you should prepare a solution of 1% comassie in 40% MeOH and 10% Glacial Acetic Acid and add this solution as in methylene blue test. However, I rarely use this test because the use of MeOH and Glacial Acetic Acid causes the crystal dissolution. Only a final tip: obviously these tests enable you to distinguish between protein and salt, however they do not differentiate between protein in the crystal and protein in solution. For these reason, in some cases could be difficult to see the crystal colouration due to the low contrast with the colouration of the solution. Hence, I prefer to put the crystals to test in a new solution with the same formulation of the drop where the crystals have grown but without protein and perform here the dye test that I have chosen. In this way, you can easily see the colouration of the crystal without background effect. I hope I've helped you. Danilo On Tue, 15 Oct 2013 13:29:20 +0530, Swastik Phulera swastik.phul...@gmail.com wrote: Dear All, I am looking for a method to quickly differentiate between salt and protein crystals. I have been told thats its a popular alternative to the commercially available izit dye. I would appreciate if some one would share their comassie crystal staining protocol. Swastik
Re: [ccp4bb] A case of perfect pseudomerehedral twinning?
Hi, It's not uncommon for pseudosymmetry to be found together with twinning, and the presence of pseudosymmetry perturbs the statistics used to test for twinning. In that circumstance, as Phil suggests, a really good way to see what is going on is to take the lower symmetry solution and see if it really obeys higher symmetry, but you can do that either with coordinates or calculated structure factors. Your NCS matrix specifies a 2-fold rotation around an axis that is about 1 degree off the x axis. Whether that 1 degree matters or not depends on how precisely the molecules are placed in the MR solution. If 30.8649 is precisely half of the a-cell edge, then this corresponds to a 2(1) screw axis, but whether or not that is crystallographic depends on whether the origin of that axis is in the right place relative to the 2(1) you're assuming is correct. Working all that out from coordinates can be a bit of a challenge, which will really have you hitting the books! The other way we've approached this kind of problem is to take the Fcalcs from an MR model (usually solved in P1 if possible to avoid making any assumptions about which symmetry operators are correct) and then use either pointless or xtriage to see if those Fcalcs obey higher symmetry. Another good approach is to use the zanuda program in the CCP4 suite, which is designed to answer questions about pseudosymmetry and other related problems. Good luck! Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +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 On 15 Oct 2013, at 22:31, Yarrow Madrona amadr...@uci.edu wrote: Thank you Dale, I will hit-the-books to better the rotation matrices. I am concluding from all of this that the space group is indeed P212121. So I still wonder why I have some outliers in the intensity stats for the two additional screw axis and why R and Rfree both drop by 5% when I apply a twin law to refinement in P21. Thanks for your help. -Yarrow Since Phil is no doubt in bed, I'll answer the easier part. Your second matrix is nearly the equivalent position (x,-y,-z). This is a two-fold rotation about the x axis. You also have a translation of about 31 A along x so if your A cell edge is about 62 A you have a 2_1 screw. Dale Tronrud On 10/15/2013 12:29 PM, Yarrow Madrona wrote: Hi Phil, Thanks for your help. I ran a Find-NCS routine in the phenix package. It came up with what I pasted below: I am assuming the the first rotation matrix is just the identity. I need to read more to understand rotation matrices but I think the second one should have only a single -1 to account for a possible perfect 2(1) screw axis between the two subunits in the P21 asymetric unit. I am not sure why there are two -1 values. I may be way off in my interpretation in which case I will go read some more. I will also try what you suggested. Thanks. -Yarrow NCS operator using PDB #1 new_operator rota_matrix1.0.0. rota_matrix0.1.0. rota_matrix0.0.1. tran_orth 0.0.0. center_orth 17.72011.4604 71.4860 RMSD = 0 (Is this the identity?) #2 new_operator rota_matrix0.9994 -0.02590.0250 rota_matrix -0.0260 -0.99970.0018 rota_matrix0.0249 -0.0025 -0.9997 tran_orth -30.8649 -11.9694 166.9271 Hello Yarrow, Since you have a refined molecular replacement solution I recommend using that rather than global intensity statistics. Obviously if you solve in P21 and it's really P212121 you should have twice the number of molecules in the asymmetric unit and one half of the P21 asymmetric unit should be identical to the other half. Since you've got decent resolution I think you can determine the real situation for yourself: one approach would be to test to see if you can symmetrize the P21 asymmetric unit so that the two halves are identical. You could do this via stiff NCS restraints (cartesian would be better than dihedral). After all the relative XYZs and even B-factors would be more or less identical if you've rescaled a P212121 crystal form in P21. If something violates the NCS than it can't really be P212121. Alternatively you can look for clear/obvious symmetry breaking between the two halves: different side-chain rotamers for surface side-chains for example. If you've got an ordered, systematic, difference in electron density between the two halves of the asymmetric unit in P21 then that's a basis for describing it as P21 rather than P212121. However if the two halves look nearly identical, down to equivalent
[ccp4bb] Deadline 20th October: Open Group Leader position at Grenoble Outstation of EMBL
Deadline of 20th October is approaching ! Dear All, The Grenoble Outstation of the European Molecular Biology Laboratory is seeking to recruit a Group Leader in Structural Biology of Complexes. The appointed Group Leader will be an ambitious structural biologist with an original multidisciplinary research programme oriented towards structure-function relationships of macromolecular complexes in eukaryotic systems. Particular, but not exclusive, areas of interest are host-pathogen interactions, RNA biology or computational biology/modelling. He/she will benefit from the world-class environment of the EMBL Grenoble Outstation within the Partnership for Structural Biology (www.psb-grenoble.eu) which gives access to integrated state-of-the-art structural biology technologies, including ESRF synchrotron X-ray beamlines for MX and SAXS, cryo-EM/tomography and NMR as well as protein expression screening, insect cell, biophysics, confocal microscopy and high-throughput crystallization platforms. Applicants should have a Ph.D. and at least 3 years post-doctoral experience and a strong record of achievement in structural, molecular or cell biology. Further information about research at EMBL Grenoble can be found on http://www.embl.fr/index.php Further information about the position can be found on http://www.embl.fr/aboutus/jobs/searchjobs/index.php?loc=4list=1 Stephen Cusack
Re: [ccp4bb] Staining Crystals with comassie
Hi Danilo and all, A little trick for the glutaraldehyde staining: you can hang a 1-2uL drop of 25% glutaraldehyde (or the most concentrated stock solution you can find) besides your crystal drop in the vapour diffusion chamber. The glutaraldehyde will get into the crystal drop via vapour diffusion. The color will normally show within 2hrs and become very intense overnight. It is also a gentle way of crosslinking the crystals (http://scripts.iucr.org/cgi-bin/paper?wb0066, and http://hamptonresearch.com/tip_detail.aspx?id=74, ). Care should be taken when handling aldehyde concentrates: do not breathe it, and do not let the vapour get in touch with your eyes. Waste can be inactivated by concentrated glycine solution. BTW, the acetic acid in the coommasie blue solution seems unnecessary in a crystal staining solution. The solution recipe seems to be taken from a gel staining solution. When staining polyacrylamide gels, the acid (oringinally HCl) is supposed to denature the proteins so that they do not diffuse in the gel. The MeOH is for solubilizing the commonly used coommassie R250. (Another thing: I strongly suggest to substitute the MeOH in PAGE staining and de-staining solutions with EtOH. EtOH works perfectly fine, without MeOH's poisonous effect on human. Our staining solution contains 20% EtOH and 20%HAc.) For staining crystals, we do not need to add the acetic acid. Also coommassie G250 is more soluble in water than the R250 by having methyl groups instead of ethyl groups. 0.5% coommassie G250 can be readily made in DMSO or 95% EtOH. Then this stock solution can be diluted with water or the mother liquor 10x-100x for the staining. Many crystals can tolerate up to 10% DMSO. Zhijie -Original Message- From: Danilo Belviso Sent: Wednesday, October 16, 2013 3:53 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Staining Crystals with comassie Dear All, izit dye is a solution containing methylene blue that you could prepare in your lab. I usually prepare a solution of 0.05%w/v of dye in water and then I add a volume of dye solution equals to 10% of the volume of the drop containing the crystal to test. I prefer to add the dye solution in small portions (if the volume permits) every 2-3h in order to limit the shock due to the new solution on the crystal. You should remember that this test is not definitive: the dye is a cationic dye, that needs of anion counter part to bind the protein. Therefore, the dye is not able to colour all protein crystals: in addition, colouration is affect by pH of the crystallization condition, since low pH could increase the positive charge on the protein reducing its ability to bind the dye. You could try also glutaraldehyde as alternative. In order to perform this test, you should put the crystal into a low ionic strength buffered solution containing up to 2% glutaraldehyde. In this condition, formation of Schiff bases with the lysines and N-term residues occurs and the crystal become a yellow gel, while salt crystals dissolve and should not be coloured. To perform comassie crystal staining you should prepare a solution of 1% comassie in 40% MeOH and 10% Glacial Acetic Acid and add this solution as in methylene blue test. However, I rarely use this test because the use of MeOH and Glacial Acetic Acid causes the crystal dissolution. Only a final tip: obviously these tests enable you to distinguish between protein and salt, however they do not differentiate between protein in the crystal and protein in solution. For these reason, in some cases could be difficult to see the crystal colouration due to the low contrast with the colouration of the solution. Hence, I prefer to put the crystals to test in a new solution with the same formulation of the drop where the crystals have grown but without protein and perform here the dye test that I have chosen. In this way, you can easily see the colouration of the crystal without background effect. I hope I've helped you. Danilo On Tue, 15 Oct 2013 13:29:20 +0530, Swastik Phulera swastik.phul...@gmail.com wrote: Dear All, I am looking for a method to quickly differentiate between salt and protein crystals. I have been told thats its a popular alternative to the commercially available izit dye. I would appreciate if some one would share their comassie crystal staining protocol. Swastik
Re: [ccp4bb] Staining Crystals with comassie
There are many caveats to using glutaraldehyde on crystals, either for fixing crystals or for staining them. First, I would not hang a 1-2uL drop of 25% glutaraldehyde in the vapour diffusion chamber, but add enough glutaraldehyde into the reservoir to make it 0.5-1.0 % (a 1:25 or 1:50 dilution; a 1% solution is 100 mM). Not only will it be just as effective, the reservoir becomes the control: if the reservoir turns yellow, you have free amines in the system (Tris, ammonium, etc.). Second, the yellow color, which is due to Schiff's base formation, is harder to see in warm light (color temperature, not the temperature of the stage) when you are looking at small or thin crystals. Use cool, white lights (like LEDs). Finally, keeps some buffer around that is suitable for solubilizing the protein. If you are not sure about the color change, just add 10 uL of buffer to the crystals and watch if they dissolve. If they don't when treated with glutaraldehyde, they are protein crystals. As Zhijie said, be careful with handling glutaraldehyde. It is highly volatile and dangerous. If you smell it (and the sweet smell will be obvious), it is fixing you. Keep a waste bottle half-full with 1 M ammonium sulfate, not glycine (too expensive), then just dump any glutaraldehyde waste into it. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Oct 16, 2013, at 6:45 AM, Zhijie Li wrote: Hi Danilo and all, A little trick for the glutaraldehyde staining: you can hang a 1-2uL drop of 25% glutaraldehyde (or the most concentrated stock solution you can find) besides your crystal drop in the vapour diffusion chamber. The glutaraldehyde will get into the crystal drop via vapour diffusion. The color will normally show within 2hrs and become very intense overnight. It is also a gentle way of crosslinking the crystals (http://scripts.iucr.org/cgi-bin/paper?wb0066, and http://hamptonresearch.com/tip_detail.aspx?id=74, ). Care should be taken when handling aldehyde concentrates: do not breathe it, and do not let the vapour get in touch with your eyes. Waste can be inactivated by concentrated glycine solution. BTW, the acetic acid in the coommasie blue solution seems unnecessary in a crystal staining solution. The solution recipe seems to be taken from a gel staining solution. When staining polyacrylamide gels, the acid (oringinally HCl) is supposed to denature the proteins so that they do not diffuse in the gel. The MeOH is for solubilizing the commonly used coommassie R250. (Another thing: I strongly suggest to substitute the MeOH in PAGE staining and de-staining solutions with EtOH. EtOH works perfectly fine, without MeOH's poisonous effect on human. Our staining solution contains 20% EtOH and 20%HAc.) For staining crystals, we do not need to add the acetic acid. Also coommassie G250 is more soluble in water than the R250 by having methyl groups instead of ethyl groups. 0.5% coommassie G250 can be readily made in DMSO or 95% EtOH. Then this stock solution can be diluted with water or the mother liquor 10x-100x for the staining. Many crystals can tolerate up to 10% DMSO. Zhijie -Original Message- From: Danilo Belviso Sent: Wednesday, October 16, 2013 3:53 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Staining Crystals with comassie Dear All, izit dye is a solution containing methylene blue that you could prepare in your lab. I usually prepare a solution of 0.05%w/v of dye in water and then I add a volume of dye solution equals to 10% of the volume of the drop containing the crystal to test. I prefer to add the dye solution in small portions (if the volume permits) every 2-3h in order to limit the shock due to the new solution on the crystal. You should remember that this test is not definitive: the dye is a cationic dye, that needs of anion counter part to bind the protein. Therefore, the dye is not able to colour all protein crystals: in addition, colouration is affect by pH of the crystallization condition, since low pH could increase the positive charge on the protein reducing its ability to bind the dye. You could try also glutaraldehyde as alternative. In order to perform this test, you should put the crystal into a low ionic strength buffered solution containing up to 2% glutaraldehyde. In this condition, formation of Schiff bases with the lysines and N-term residues occurs and the crystal become a yellow gel, while salt crystals dissolve and should not be coloured. To perform comassie crystal staining you should prepare a solution
[ccp4bb] CCP4 Release 6.4.0 follow-up
Dear CCP4 users, It has been reported to us shortly after 6.4.0 release last week, that the software was dysfunctional for some users. The reason was found to be the clash between Qt framework distributed with CCP4 6.4.0 and the one installed locally. This problem is now fixed, and new CCP4 distribution packages are available from our web-site: http://www.ccp4.ac.uk/download/ In order to identify whether your CCP4 setup is affected by the bug, please check if any of the symptoms below applies: - ccp4i shows permanently Network off? in low-right corner - greyed, dysfunctional Manage Updates button in ccp4i - even if Manage Updates button is functional, CCP4 updater does not start - QtRView result viewer does not start - other Qt-based applications, such as qtpisa and viewhkl, do not start or do not work properly If you find your setup affected by the bug, or have any doubts, we advise you to re-install 6.4.0 now. CCP4 setups with working updater will be synchronised with today's changes through the corresponding update soon. CCP4 Core Group apologises for any inconvenience and would like to encourage all users to report any problems with CCP4 software that they have encountered. Sincerely yours, Eugene Krissinel -- Scanned by iCritical.
Re: [ccp4bb] A case of perfect pseudomerehedral twinning?
Thanks Randy, The a cell edge is 56.118, so not exactly half of 30.86. I am currently refining using NCS cartesian restraints as Phil suggested. Then I will visually inspect the model as well as compare b-factors. Thanks for your suggestions, I will look into them. -Yarrow Hi, It's not uncommon for pseudosymmetry to be found together with twinning, and the presence of pseudosymmetry perturbs the statistics used to test for twinning. In that circumstance, as Phil suggests, a really good way to see what is going on is to take the lower symmetry solution and see if it really obeys higher symmetry, but you can do that either with coordinates or calculated structure factors. Your NCS matrix specifies a 2-fold rotation around an axis that is about 1 degree off the x axis. Whether that 1 degree matters or not depends on how precisely the molecules are placed in the MR solution. If 30.8649 is precisely half of the a-cell edge, then this corresponds to a 2(1) screw axis, but whether or not that is crystallographic depends on whether the origin of that axis is in the right place relative to the 2(1) you're assuming is correct. Working all that out from coordinates can be a bit of a challenge, which will really have you hitting the books! The other way we've approached this kind of problem is to take the Fcalcs from an MR model (usually solved in P1 if possible to avoid making any assumptions about which symmetry operators are correct) and then use either pointless or xtriage to see if those Fcalcs obey higher symmetry. Another good approach is to use the zanuda program in the CCP4 suite, which is designed to answer questions about pseudosymmetry and other related problems. Good luck! Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 15 Oct 2013, at 22:31, Yarrow Madrona amadr...@uci.edu wrote: Thank you Dale, I will hit-the-books to better the rotation matrices. I am concluding from all of this that the space group is indeed P212121. So I still wonder why I have some outliers in the intensity stats for the two additional screw axis and why R and Rfree both drop by 5% when I apply a twin law to refinement in P21. Thanks for your help. -Yarrow Since Phil is no doubt in bed, I'll answer the easier part. Your second matrix is nearly the equivalent position (x,-y,-z). This is a two-fold rotation about the x axis. You also have a translation of about 31 A along x so if your A cell edge is about 62 A you have a 2_1 screw. Dale Tronrud On 10/15/2013 12:29 PM, Yarrow Madrona wrote: Hi Phil, Thanks for your help. I ran a Find-NCS routine in the phenix package. It came up with what I pasted below: I am assuming the the first rotation matrix is just the identity. I need to read more to understand rotation matrices but I think the second one should have only a single -1 to account for a possible perfect 2(1) screw axis between the two subunits in the P21 asymetric unit. I am not sure why there are two -1 values. I may be way off in my interpretation in which case I will go read some more. I will also try what you suggested. Thanks. -Yarrow NCS operator using PDB #1 new_operator rota_matrix1.0.0. rota_matrix0.1.0. rota_matrix0.0.1. tran_orth 0.0.0. center_orth 17.72011.4604 71.4860 RMSD = 0 (Is this the identity?) #2 new_operator rota_matrix0.9994 -0.02590.0250 rota_matrix -0.0260 -0.99970.0018 rota_matrix0.0249 -0.0025 -0.9997 tran_orth -30.8649 -11.9694 166.9271 Hello Yarrow, Since you have a refined molecular replacement solution I recommend using that rather than global intensity statistics. Obviously if you solve in P21 and it's really P212121 you should have twice the number of molecules in the asymmetric unit and one half of the P21 asymmetric unit should be identical to the other half. Since you've got decent resolution I think you can determine the real situation for yourself: one approach would be to test to see if you can symmetrize the P21 asymmetric unit so that the two halves are identical. You could do this via stiff NCS restraints (cartesian would be better than dihedral). After all the relative XYZs and even B-factors would be more or less identical if you've rescaled a P212121 crystal form in P21. If something violates the NCS than it can't really be P212121. Alternatively you can look for clear/obvious symmetry breaking between the two halves: different side-chain rotamers for surface side-chains for example. If you've got an ordered, systematic, difference in electron
[ccp4bb] DNA interaction 2D plot software
Hello everyone, I would like to display the interactions of a protein dsDNA complex in a simplified 2D plot, similar to what LIGPLOT does for protein ligand interactions. In many articles you find interactions displayed in such a way but as far as I know those are hand-made. In my experience LIGPLOT itself is suboptimal if there are too many interactions to display Thanks a lot in advance for your suggestions. Eike
Re: [ccp4bb] DNA interaction 2D plot software
Hello, you can try Nucplot. http://www.ebi.ac.uk/thornton-srv/software/NUCPLOT/ Very usefull and tunnable to find and show dsDNA/protein complex. Hope to help. Nicolas Le 16/10/13 21:23, Eike Schulz a écrit : Hello everyone, I would like to display the interactions of a protein dsDNA complex in a simplified 2D plot, similar to what LIGPLOT does for protein ligand interactions. In many articles you find interactions displayed in such a way but as far as I know those are hand-made. In my experience LIGPLOT itself is suboptimal if there are too many interactions to display … Thanks a lot in advance for your suggestions. Eike
[ccp4bb] Problems with key bindings in coot0.7.2 CCP4 install
Hi all, I recently installed CCP4 6.4 under Mac OS X 10.8.5 and now have problems using key bindings in the Coot version (0.7.2) that comes with CCP4. Although most of the standard key bindings still work, some of them (e.g. 'o' for other NCS chain) and most of the user-defined key bindings do not work. The key bindings file seems to be correctly loaded by Coot and the correct keys are listed in 'Extensions - Settings - Key bindings' and the linked functions work when I click on the buttons in that list. The terminal shows the following message when I hit one of the keys that are not working: (skip-to-next-ncs-chain 'forward) ((safe_scheme_command) Error in proc: key: unbound-variable args: (#f Unbound variable: ~S (skip-to-next-ncs-chain) #f)) Everything works correctly in a separately installed Coot v0.7.1 with the same .coot.py file. Any suggestions on what the problem might be? Thanks for the help Bernhard Bernhard C. Lechtenberg, PhD Postdoctoral Fellow Riedl Lab Sanford-Burnham Medical Research Institute 10901 North Torrey Pines Road La Jolla, CA 92037, USA Phone: 858.646.3100 x 4216 Email: blechtenb...@sanfordburnham.orgmailto:blechtenb...@sanfordburnham.org
