[ccp4bb] AW: [ccp4bb] How to remove phospholipids bound to a cytoplasmic protein
Dear Lionel, In case of a lipid-binding protein, we were able to remove most (>90%) of the bound lipids with a lipidex column. This could be verified by DSF since the melting temperature of lipid-bound protein was ~6°C higher as the melting temperature of the free protein. The delipidated protein did crystallize but in the end we just replaced the bound lipid with our own compounds by adding these to the protein in the absence of the lipid. The first thing I would try in your case would be to set up crystallizations in the presence of a high (~10 mM) concentration of a natural ligand. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Lionel Gesendet: Donnerstag, 26. Juni 2014 18:16 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] How to remove phospholipids bound to a cytoplasmic protein Dear all, I would like to remove two phospholipids bound to (actually into) a cytoplasmic protein. The protein was expressed in E. coli and the crystal structure revealed the presence of two co-purified phospholipids (most probably DPPE). A web search gave me three methods to remove bound lipids: - 1-butanol liquid–liquid extraction - Lipidex 1000, VI column at 37°C - HIC (aka Hydrophobic Interaction Chromatography) on a Phenyl HP column in presence of 1M ammonium sulphate All are described in Velkov 2008 (http://www.sciencedirect.com/science/article/pii/S1570023208002390). I assume these delipidation methods would also work for phospholipid; and I am more tempting by the last one, the milder one. I would appreciate any comment or practical advice. Best regards, Lionel
[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] How to remove phospholipids bound to a cytoplasmic protein
PS: if you are working with a human protein: most human proteins are quite stable at 37°C (for obvious reasons). -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Schreuder, Herman R&D/DE Gesendet: Freitag, 27. Juni 2014 09:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] AW: [ccp4bb] How to remove phospholipids bound to a cytoplasmic protein Dear Lionel, In case of a lipid-binding protein, we were able to remove most (>90%) of the bound lipids with a lipidex column. This could be verified by DSF since the melting temperature of lipid-bound protein was ~6°C higher as the melting temperature of the free protein. The delipidated protein did crystallize but in the end we just replaced the bound lipid with our own compounds by adding these to the protein in the absence of the lipid. The first thing I would try in your case would be to set up crystallizations in the presence of a high (~10 mM) concentration of a natural ligand. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Lionel Gesendet: Donnerstag, 26. Juni 2014 18:16 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] How to remove phospholipids bound to a cytoplasmic protein Dear all, I would like to remove two phospholipids bound to (actually into) a cytoplasmic protein. The protein was expressed in E. coli and the crystal structure revealed the presence of two co-purified phospholipids (most probably DPPE). A web search gave me three methods to remove bound lipids: - 1-butanol liquid–liquid extraction - Lipidex 1000, VI column at 37°C - HIC (aka Hydrophobic Interaction Chromatography) on a Phenyl HP column in presence of 1M ammonium sulphate All are described in Velkov 2008 (http://www.sciencedirect.com/science/article/pii/S1570023208002390). I assume these delipidation methods would also work for phospholipid; and I am more tempting by the last one, the milder one. I would appreciate any comment or practical advice. Best regards, Lionel
[ccp4bb] Solvent channels
Hi, I'd like to do some soaking experiments with a relatively large molecule. Can someone suggest a program/method to display the solvent channels of a crystal? We have the crystal structure. I'd like to see if the channels are large enough to allow the molecule to travel to the hypothesized binding site. Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org
Re: [ccp4bb] Solvent channels
Hi, I'm not aware of a program with an option to display channels in crystals but if you use any of the currently available molecular display program and ask to display symmetry-related molecules + adjacent unit cells, it should give you a good enough idea of the spaces between molecules. Using programs for calculation of intermolecular distances would also be helpful here. Independently of the calculation, I would try soaking first and consult the calculations later (in the spirit of Rossmann's American method: shoot first ask later). Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Reza Khayat [rkha...@ccny.cuny.edu] Sent: Friday, June 27, 2014 2:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Solvent channels Hi, I'd like to do some soaking experiments with a relatively large molecule. Can someone suggest a program/method to display the solvent channels of a crystal? We have the crystal structure. I'd like to see if the channels are large enough to allow the molecule to travel to the hypothesized binding site. Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org
Re: [ccp4bb] seeking help about running SCALEPACK2mtz
It is very hard to diagnose this sort of error - I didnt think scalepack2mtz cared about data quality! There is more likely some format problem.. Is a sca file meant to have some sort of terminator? But best to attach the offending sca file so that experts can check it.. Eleanor On 26 June 2014 19:41, chen c wrote: > Dear CCP4BBers: > > I'e got a problem about data processing when running SCALEPACK2mtz. Hope > you can give me some advice on that. > > Here's my problem: > > I have an .sca file processed by HKL2000 about 4 years ago, I just ran the > Scalepack2mtz program in order to transform it into .mtz file. However, the > program keeps running. To ensure that the Scalepack2mtz program is ok, I > use another .sca file for verification and it turns out that this file for > verification is successfully transformed into .mtz file within seconds. > > I checked the .log file, the program keeps running when there are the > following messeages: > "Mean acentric moments I from input data: > /^2 = 2.360 (Expected = 2.000, Perfect Twin = 1.500) > /^3 = 9.187 (Expected value = 6.000, Perfect Twin = 3.000) > /^4 = 52.348 (Expected value = 24.000, Perfect Twin = 7.500) > Mean acentric moments I from anisotropically corrected data: > /^2 = 3.121 (Expected = 2.000, Perfect Twin = 1.500) > /^3 = 12.788 (Expected value = 6.000, Perfect Twin = 3.000) > /^4 = 57.952 (Expected value = 24.000, Perfect Twin = 7.500) > " > > does this means that the .sca file isn't processed properly? I also > checked the scale.log file, and there are the following messages: > > " Shell Summary of observation redundancies: > Lower Upper % of reflections with given No. of observations > limit limit 0 1 2 3 4 5-6 7-8 9-12 13-19 >19 > total > 50.00 5.60 5.9 6.1 18.0 15.1 26.9 19.7 8.3 0.0 0.0 > 0.0 94.1 >5.60 4.45 7.9 9.4 13.0 12.9 23.2 27.5 6.1 0.0 0.0 > 0.0 92.1 >4.45 3.88 19.2 5.6 13.3 13.5 20.4 21.9 6.0 0.0 0.0 > 0.0 80.8 >3.88 3.53 31.9 6.5 11.6 11.3 14.9 17.9 5.8 0.0 0.0 > 0.0 68.1 >3.53 3.28 2.4 4.7 11.3 15.3 23.5 34.1 8.7 0.0 0.0 > 0.0 97.6 >3.28 3.08 0.2 3.6 7.0 15.4 23.7 40.1 9.9 0.0 0.0 > 0.0 99.8 >3.08 2.93 0.4 4.4 8.7 16.8 21.1 41.3 7.2 0.0 0.0 > 0.0 99.6 >2.93 2.80 4.2 8.0 12.7 15.2 20.3 33.7 6.0 0.0 0.0 > 0.0 95.8 >2.80 2.69 15.4 9.1 14.0 13.8 19.2 25.2 3.3 0.0 0.0 > 0.0 84.6 >2.69 2.60 31.6 16.2 11.7 14.2 13.7 10.6 2.0 0.0 0.0 > 0.0 68.4 > All hkl 11.8 7.3 12.2 14.4 20.8 27.1 6.4 0.0 0.0 > 0.0 88.2 > " > This clearly indicates an ice ring problem in the data-collection > process. However, the ice ring problem shouldn't have caused the error > during the .sca to .mtz process. In fact, I have use it for > structure-solvement, and currently the two R factor is around 30%. > > However, the large deviation of /^3 or /^4 do means > something wrong. Would you tell me what exact mistakes did I make? Can I > use this .sca file for subsequent structure solving and refinement? > > Great thanks for your help > best regards > chen > > -- > >
Re: [ccp4bb] Solvent channels
Just a remark: diffusion is a slow and random-walk process. Particularly large molecules in viscous media (PEG anybody?) move (diffuse) slowly in solution. To simply extrapolate from the fact that the ligand is smaller than the solvent channels to the odds of the presence of a ligand is a risky proposition. Positive omit difference density after 'shoot first' as Boaz indicated is a much better indication. And shoot you probably will a lot. The little movie below shows how slowly even a small aromatic dye molecule soaks into a crystal. Total time 10 hrs. http://www.ruppweb.org/cryscam/lysozyme_dye_small.wmv The literally hundreds of empty ligand structures collected in Twilight attest to that fact. http://journals.iucr.org/d/issues/2013/02/00/issconts.html Best, BR Science is a way of trying not to fool yourself: The first principle is that you must not fool yourself - and you are the easiest person to fool. R. Feynman, 1974 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Boaz Shaanan Sent: Friday, June 27, 2014 2:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solvent channels Hi, I'm not aware of a program with an option to display channels in crystals but if you use any of the currently available molecular display program and ask to display symmetry-related molecules + adjacent unit cells, it should give you a good enough idea of the spaces between molecules. Using programs for calculation of intermolecular distances would also be helpful here. Independently of the calculation, I would try soaking first and consult the calculations later (in the spirit of Rossmann's American method: shoot first ask later). Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Reza Khayat [rkha...@ccny.cuny.edu] Sent: Friday, June 27, 2014 2:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Solvent channels Hi, I'd like to do some soaking experiments with a relatively large molecule. Can someone suggest a program/method to display the solvent channels of a crystal? We have the crystal structure. I'd like to see if the channels are large enough to allow the molecule to travel to the hypothesized binding site. Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org =
Re: [ccp4bb] Solvent channels
And yet halides--even iodide--permeate those same lysozyme crystals and others entirely in <30--60 sec. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp Sent: Friday, June 27, 2014 9:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solvent channels Just a remark: diffusion is a slow and random-walk process. Particularly large molecules in viscous media (PEG anybody?) move (diffuse) slowly in solution. To simply extrapolate from the fact that the ligand is smaller than the solvent channels to the odds of the presence of a ligand is a risky proposition. Positive omit difference density after 'shoot first' as Boaz indicated is a much better indication. And shoot you probably will a lot. The little movie below shows how slowly even a small aromatic dye molecule soaks into a crystal. Total time 10 hrs. http://www.ruppweb.org/cryscam/lysozyme_dye_small.wmv The literally hundreds of empty ligand structures collected in Twilight attest to that fact. http://journals.iucr.org/d/issues/2013/02/00/issconts.html Best, BR Science is a way of trying not to fool yourself: The first principle is that you must not fool yourself - and you are the easiest person to fool. R. Feynman, 1974 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Boaz Shaanan Sent: Friday, June 27, 2014 2:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solvent channels Hi, I'm not aware of a program with an option to display channels in crystals but if you use any of the currently available molecular display program and ask to display symmetry-related molecules + adjacent unit cells, it should give you a good enough idea of the spaces between molecules. Using programs for calculation of intermolecular distances would also be helpful here. Independently of the calculation, I would try soaking first and consult the calculations later (in the spirit of Rossmann's American method: shoot first ask later). Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Reza Khayat [rkha...@ccny.cuny.edu] Sent: Friday, June 27, 2014 2:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Solvent channels Hi, I'd like to do some soaking experiments with a relatively large molecule. Can someone suggest a program/method to display the solvent channels of a crystal? We have the crystal structure. I'd like to see if the channels are large enough to allow the molecule to travel to the hypothesized binding site. Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org =
[ccp4bb] AW: [ccp4bb] Solvent channels
To get a rough idea of the solvent channels, one could use coot. By displaying the symmetry molecules as Ca traces (an option hidden in the symmetry menu under "symmetry by molecule") one can set a large radius (100Å) and still rotate the display. A more accurate display can be obtained by generating a number of symmetry mates and reading them in pymol. Even in surface mode, pymol can handle quite a few complete protein molecules without getting excessively slow. However, it is just as important (or even more important) to examine whether the putative binding site is free and not involved in crystal contacts and whether enough room is available to accommodate the ligand. To be absolutely sure, the gold standard is of course still cocrystallization and with 96-well plates and crystallization robots it is not prohibitively difficult. I agree with Jacob Keller, in my experience, soaking is usually much faster than 10 hrs. unless some conformational change in the protein is necessary to let the ligand in the binding site. Nevertheless, we routinely soak overnight (24 hrs.). It is convenient and there is less risk that the structures end up in the twilight database. Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Bernhard Rupp Gesendet: Freitag, 27. Juni 2014 15:00 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Solvent channels Just a remark: diffusion is a slow and random-walk process. Particularly large molecules in viscous media (PEG anybody?) move (diffuse) slowly in solution. To simply extrapolate from the fact that the ligand is smaller than the solvent channels to the odds of the presence of a ligand is a risky proposition. Positive omit difference density after 'shoot first' as Boaz indicated is a much better indication. And shoot you probably will a lot. The little movie below shows how slowly even a small aromatic dye molecule soaks into a crystal. Total time 10 hrs. http://www.ruppweb.org/cryscam/lysozyme_dye_small.wmv The literally hundreds of empty ligand structures collected in Twilight attest to that fact. http://journals.iucr.org/d/issues/2013/02/00/issconts.html Best, BR Science is a way of trying not to fool yourself: The first principle is that you must not fool yourself - and you are the easiest person to fool. R. Feynman, 1974 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Boaz Shaanan Sent: Friday, June 27, 2014 2:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solvent channels Hi, I'm not aware of a program with an option to display channels in crystals but if you use any of the currently available molecular display program and ask to display symmetry-related molecules + adjacent unit cells, it should give you a good enough idea of the spaces between molecules. Using programs for calculation of intermolecular distances would also be helpful here. Independently of the calculation, I would try soaking first and consult the calculations later (in the spirit of Rossmann's American method: shoot first ask later). Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Reza Khayat [rkha...@ccny.cuny.edu] Sent: Friday, June 27, 2014 2:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Solvent channels Hi, I'd like to do some soaking experiments with a relatively large molecule. Can someone suggest a program/method to display the solvent channels of a crystal? We have the crystal structure. I'd like to see if the channels are large enough to allow the molecule to travel to the hypothesized binding site. Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org =
Re: [ccp4bb] Solvent channels
For small ion soaking for phasing purposes, partial occupancy is not a problem. For example, a few 1/2 occupied Iodines still can phase quite well. 1/2 a C is only 3 electrons, not that great. Add in higher displacement, and odds are that the ligand interpretation will become difficult. Particularly when the binding constants are poor, one will out of principle never reach full occupancy, which further exacerbates the weak density problem. Patience is definitely a virtue here. BR -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: Friday, June 27, 2014 3:07 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solvent channels And yet halides--even iodide--permeate those same lysozyme crystals and others entirely in <30--60 sec. JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp Sent: Friday, June 27, 2014 9:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solvent channels Just a remark: diffusion is a slow and random-walk process. Particularly large molecules in viscous media (PEG anybody?) move (diffuse) slowly in solution. To simply extrapolate from the fact that the ligand is smaller than the solvent channels to the odds of the presence of a ligand is a risky proposition. Positive omit difference density after 'shoot first' as Boaz indicated is a much better indication. And shoot you probably will a lot. The little movie below shows how slowly even a small aromatic dye molecule soaks into a crystal. Total time 10 hrs. http://www.ruppweb.org/cryscam/lysozyme_dye_small.wmv The literally hundreds of empty ligand structures collected in Twilight attest to that fact. http://journals.iucr.org/d/issues/2013/02/00/issconts.html Best, BR Science is a way of trying not to fool yourself: The first principle is that you must not fool yourself - and you are the easiest person to fool. R. Feynman, 1974 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Boaz Shaanan Sent: Friday, June 27, 2014 2:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Solvent channels Hi, I'm not aware of a program with an option to display channels in crystals but if you use any of the currently available molecular display program and ask to display symmetry-related molecules + adjacent unit cells, it should give you a good enough idea of the spaces between molecules. Using programs for calculation of intermolecular distances would also be helpful here. Independently of the calculation, I would try soaking first and consult the calculations later (in the spirit of Rossmann's American method: shoot first ask later). Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Reza Khayat [rkha...@ccny.cuny.edu] Sent: Friday, June 27, 2014 2:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Solvent channels Hi, I'd like to do some soaking experiments with a relatively large molecule. Can someone suggest a program/method to display the solvent channels of a crystal? We have the crystal structure. I'd like to see if the channels are large enough to allow the molecule to travel to the hypothesized binding site. Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org =
[ccp4bb] summertime reading
I recently reread an old science fiction book titled Highways in Hiding by George O. Smith. To my surprise there is some crystallography on page 58 (1967 Lancer book). This is not exactly a great book and I am sure Joe Ferrara would pan it. It is available on Amazon and there are a number of very inexpensive copies on used.addall.com if you are interested in buying it. Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 =
Re: [ccp4bb] Solvent channels
On 06/27/2014 06:33 AM, Bernhard Rupp wrote: > For small ion soaking for phasing purposes, partial occupancy is not a > problem. For example, a few 1/2 occupied Iodines still can phase quite well. > 1/2 a C is only 3 electrons, not that great. Add in higher displacement, and > odds are that the ligand interpretation will become difficult. Particularly > when the binding constants are poor, one will out of principle never reach > full occupancy, which further exacerbates the weak density problem. > Patience is definitely a virtue here. > > BR Here you are starting to mix equilibrium arguments with the previous kinetic arguments. If you have a weak binder you can always get full occupancy by adding enough of the compound - to determine how much, you must consider not only the binding constant but the number of binding sites in the crystal and the total volume of the drop containing your crystal. Time is not a factor. Halide ions and cryoprotectants are known to pervade crystals very rapidly, but they are usually added with "overwhelming force". Much more is added than is required to bind to every specific binding site in the crystal. The rate of diffusion, as mass flow, depends not only on viscosity but on the concentration of unbound molecules inside the crystal. When I was soaking an inhibitor into a crystal of Thermolysin I was having problems with the crystals falling apart. My belief was that the inhibitor caused a small change in cell constants and since the inhibitor first bound in a shell around the surface of the crystal strain was created and the crystal cracked. My solution was to add small aliquots of inhibitor with a long enough wait between to allow each batch to diffuse throughout the crystal. Despite waiting up to 6 hours between additions the crystals still cracked. This is when I realized that after the inhibitor bound in the outer shell of the crystal the remaining concentration of free inhibitor was one billionth (since the binding constant was nanomolar) that of the concentration of active sites and the remaining mass flow within the crystal was insignificant. Of course the next aliquot would rapidly diffuse through the occupied region of the crystal and be bound in the shell just below it, becoming trapped itself and increasing the strain. Your movie doesn't include any details of concentration of your dye, nor what its binding constant is to any sites in a protein nor any mention of kon or koff. The lack of information makes it very difficult to draw any conclusions from the experiment, but I believe the experience from many other molecules is that small molecules do move very rapidly through protein crystals, until they are caught by a binding site. I don't believe your movie represents typical diffusion of small molecules in a protein crystal. My interpretation of your movie is: 1) The dye rapidly diffuses into the crystal reaching a simple equilibrium where the concentration in the bulk solvent matches that of the outside solution. Since the protein excludes about half of the volume of the crystal the overall concentration is half that of the mother liquor and the color of the crystal is 1/2 as dark as the surrounding solution. 2) With a slow kon, the dye molecules within the crystal start binding specifically to the protein. Since the dye is aromatic it probably has to dig deep into the protein to find a binding site and this takes time. As dye is removed from the bulk solvent it is rapidly replaced by diffusion from outside the crystal, and the crystal begins to darken, eventually becoming darker than the surrounding liquid. The speed of binding is controlling the kinetics not diffusion. Dale Tronrud > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Keller, Jacob > Sent: Friday, June 27, 2014 3:07 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Solvent channels > > And yet halides--even iodide--permeate those same lysozyme crystals and > others entirely in <30--60 sec. > > JPK > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Bernhard Rupp > Sent: Friday, June 27, 2014 9:00 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Solvent channels > > Just a remark: diffusion is a slow and random-walk process. Particularly > large molecules in viscous media (PEG anybody?) move (diffuse) slowly in > solution. To simply extrapolate from the fact that the ligand is smaller > than the solvent channels to the odds of the presence of a ligand is a risky > proposition. Positive omit difference density after 'shoot first' as Boaz > indicated is a much better indication. And shoot you probably will a lot. > > The little movie below shows how slowly even a small aromatic dye molecule > soaks into a crystal. Total time 10 hrs. > > http://www.ruppweb.org/cryscam/lysozyme_dye_small.wmv > > The literally hundreds of empty li
[ccp4bb] New Version of the Protein Geometry Database Now Available
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Protein Geometry Database Server V 1.0 http://pgd.science.oregonstate.edu/ Developed by Andy Karplus' laboratory at Oregon State University We are pleased to announce the availability of an enhanced version of the Protein Geometry Database (PGD) web service, originally announced in Berkholz et al (2010) Nucleic Acids Research 38, D320-5. This server allows you to explore the many backbone and side chain conformations that exist in the PDB as well as the protein geometry (lengths and angles) that occur in those conformations. This service is ideal for finding instances of particular conformations or peculiar bond lengths or angles. It is also quite adept at identifying sets of fragments that can then be examined for systematic variation in "ideal" geometry. The expanded PGD now includes all conformational and covalent geometry information not just for the backbone but also for the sidechains. There are three basic operations available: selecting a set of fragments via a delimited search, analyzing the geometry of those fragments, and dumping the results to your computer for more specialized analysis. To control bias in statistical analyses due to the variable number of entries with the same or similar sequence, the database contains only the highest quality model in each sequence cluster as identified by the Pisces server from Roland Dunbrack's lab. Two settings, 90% and 25% sequence identity, are available. Currently, at the 90% sequence identity level there are 16,000 chains and at the 25% level this drops to about 11,000 chains. You can filter a search based on the quality of the model as indicated by resolution and R values. A search can also be filtered based on DSSP secondary structure, amino acid type, the phi/psi/omega angles and bond lengths, angles, and chi angles. For example, you can find all cysteine residues in the center of three-residue peptide fragments (i.e. not at a peptide terminus), in beta sheet, with both peptide bonds trans, and CB-SG length greater than 1.85 A from models with resolution better than 1.5 A. By the way, in the "no more than 25% sequence identity" category there are 25 of them. Once you have a set of results, you can create 2D plots showing the relationships of up to three features (i.e. bond lengths, bond angles, or conformational angles). For instance, you can look at how a given feature varies with phi and psi using a phi(i)/psi(i) plot. Or, you can just as easily look at the variation with psi(i)/phi(i+1), or even the relationships between any selected bond angles. As one example, it is instructive to perform a default search and plot NCaCb vs NCaC colored based on CbCaC. As this search is restricted to just the highest resolution models, you can see the justification for chiral volume restraints. Finally, all of your results can be downloaded for your own analysis. Development of the PGD continues. If you have worked with the site and have any ideas and suggestions for how to improvement it, please drop us a line. The publication describing the PGD is: Berkholz, D.S., Krenesky, P.B., Davidson, J.R., & Karplus, P.A. (2010) Protein Geometry Database: A flexible engine to explore backbone conformations and their relationships with covalent geometry. Nucleic Acids Res. 38, D320-5. Also, some examples of published analyses enabled by earlier versions of the PGD are listed here:. Berkholz, D.S., Shapovalov, M.V., Dunbrack, R.L.J. & Karplus, P.A. (2009). Conformation dependence of backbone geometry in proteins. Structure 17, 1316-1325. Hollingsworth, S.A., Berkholz, D.S. & Karplus, P.A. (2009). On the occurrence of linear groups in proteins. Protein Science 18, 1321-1325 Hollingsworth, S.A. & Karplus, P. A. (2010). Review: A fresh look at the Ramachandran plot and the occurrence of standard structures in proteins. BioMolecular Concepts 1, 271-283. Berkholz, D.S., Driggers, C.M., Shapovalov, M.V., Dunbrack, R.L., Jr. & Karplus P.A. (2012) Nonplanar peptide bonds in proteins are common and conserved but not biased toward active sites. Proc Natl Acad Sci U S A. 109, 449-53. Dale Tronrud & P. Andrew Karplus Department of Biochemistry and Biophysics Oregon State University -BEGIN PGP SIGNATURE- Version: GnuPG v2.0.22 (MingW32) Comment: Using GnuPG with Thunderbird - http://www.enigmail.net/ iEYEARECAAYFAlOt044ACgkQU5C0gGfAG12aZACdG68Vyjw7JJimw0ofMZrJQLu8 B1IAn0Q5xx8ptRosgMXswdYmdjNKyFkA =D63d -END PGP SIGNATURE-
Re: [ccp4bb] New Version of the Protein Geometry Database Now Available
I have wanted for some time to search for catalytic-triad-like configurations by defining three Ca-Cb bonds from known catalytic triads, then searching the pdb for matches, but have not thought of a quick and/or easy way to do this--can your software do this sort of thing, or is there some other software which could be used for this? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dale Tronrud Sent: Friday, June 27, 2014 4:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] New Version of the Protein Geometry Database Now Available -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Protein Geometry Database Server V 1.0 http://pgd.science.oregonstate.edu/ Developed by Andy Karplus' laboratory at Oregon State University We are pleased to announce the availability of an enhanced version of the Protein Geometry Database (PGD) web service, originally announced in Berkholz et al (2010) Nucleic Acids Research 38, D320-5. This server allows you to explore the many backbone and side chain conformations that exist in the PDB as well as the protein geometry (lengths and angles) that occur in those conformations. This service is ideal for finding instances of particular conformations or peculiar bond lengths or angles. It is also quite adept at identifying sets of fragments that can then be examined for systematic variation in "ideal" geometry. The expanded PGD now includes all conformational and covalent geometry information not just for the backbone but also for the sidechains. There are three basic operations available: selecting a set of fragments via a delimited search, analyzing the geometry of those fragments, and dumping the results to your computer for more specialized analysis. To control bias in statistical analyses due to the variable number of entries with the same or similar sequence, the database contains only the highest quality model in each sequence cluster as identified by the Pisces server from Roland Dunbrack's lab. Two settings, 90% and 25% sequence identity, are available. Currently, at the 90% sequence identity level there are 16,000 chains and at the 25% level this drops to about 11,000 chains. You can filter a search based on the quality of the model as indicated by resolution and R values. A search can also be filtered based on DSSP secondary structure, amino acid type, the phi/psi/omega angles and bond lengths, angles, and chi angles. For example, you can find all cysteine residues in the center of three-residue peptide fragments (i.e. not at a peptide terminus), in beta sheet, with both peptide bonds trans, and CB-SG length greater than 1.85 A from models with resolution better than 1.5 A. By the way, in the "no more than 25% sequence identity" category there are 25 of them. Once you have a set of results, you can create 2D plots showing the relationships of up to three features (i.e. bond lengths, bond angles, or conformational angles). For instance, you can look at how a given feature varies with phi and psi using a phi(i)/psi(i) plot. Or, you can just as easily look at the variation with psi(i)/phi(i+1), or even the relationships between any selected bond angles. As one example, it is instructive to perform a default search and plot NCaCb vs NCaC colored based on CbCaC. As this search is restricted to just the highest resolution models, you can see the justification for chiral volume restraints. Finally, all of your results can be downloaded for your own analysis. Development of the PGD continues. If you have worked with the site and have any ideas and suggestions for how to improvement it, please drop us a line. The publication describing the PGD is: Berkholz, D.S., Krenesky, P.B., Davidson, J.R., & Karplus, P.A. (2010) Protein Geometry Database: A flexible engine to explore backbone conformations and their relationships with covalent geometry. Nucleic Acids Res. 38, D320-5. Also, some examples of published analyses enabled by earlier versions of the PGD are listed here:. Berkholz, D.S., Shapovalov, M.V., Dunbrack, R.L.J. & Karplus, P.A. (2009). Conformation dependence of backbone geometry in proteins. Structure 17, 1316-1325. Hollingsworth, S.A., Berkholz, D.S. & Karplus, P.A. (2009). On the occurrence of linear groups in proteins. Protein Science 18, 1321-1325 Hollingsworth, S.A. & Karplus, P. A. (2010). Review: A fresh look at the Ramachandran plot and the occurrence of standard structures in proteins. BioMolecular Concepts 1, 271-283. Berkholz, D.S., Driggers, C.M., Shapovalov, M.V., Dunbrack, R.L., Jr. & Karplus P.A. (2012) Nonplanar peptide bonds in proteins are common and conserved but not biased toward active sites. Proc Natl Acad Sci U S A. 109, 449-53. Dale Tronrud & P. Andrew Karplus Department of Biochemistry and Biophysics Oregon State University -BEGIN PGP SIGNATURE- Version: GnuPG v2.0.22 (
Re: [ccp4bb] Solvent channels
You can use CAVER but you would have to make all the symmetry mates as one chain in order to fool it. Still better to just do the experiment I think. Either it will work or it won't, regardless of what any software tells you. Just a wild idea : ) On Fri, Jun 27, 2014 at 5:06 PM, Yarrow Madrona wrote: > Hi Reza, > > CAVER is a great tool for this. There is a web version. You can also > download it to customize and run it in the command line. There is also a > Pymol CAVER plug in that works very well. I have even used it to analyze MD > trajectories. You can find it here: http://www.caver.cz/ > > -Yarrow > > > On Fri, Jun 27, 2014 at 4:00 AM, Reza Khayat > wrote: > >> Hi, >> >> I'd like to do some soaking experiments with a relatively large molecule. >> Can >> someone suggest a program/method to display the solvent channels of a >> crystal? We have the crystal structure. I'd like to see if the channels >> are large >> enough to allow the molecule to travel to the hypothesized binding site. >> Thanks. >> >> Best wishes, >> Reza >> >> Reza Khayat, PhD >> Assistant Professor >> The City College of New York >> Department of Chemistry, MR-1135 >> 160 Convent Avenue >> New York, NY 10031 >> Tel. (212) 650-6070 >> www.khayatlab.org >> > >
Re: [ccp4bb] solvent exposed
Hey Jeff, Why not try the command line version of CAVER. It is easily adjustable and provides nice figures of solvent accessibility for Pymol. It also prints out a ton of stats in the log files if you want numbers. -Yarrow On Wed, Jun 25, 2014 at 1:10 PM, Jeff Holden wrote: > Experts, > > I would like to compare the substrate binding site of two homologous > proteins. Based on crystal structures it is clear that the substrate > binding site of protein A is more solvent exposed then protein B. > > Is there a way to measure the solvent exposure of the substrate in protein > A and B? Or perhaps you have an additional suggestion for making a > structural comparison (besides what seems obvious...noting the differences > in non-covalent interactions)? > > Thanks, > Jeff >
