[ccp4bb] on Rwork and Rfree
Dear All, After we refine the structure of the protein to satisfactory with satisfactory Rwork and Rfree, we pick water by phenix refine, and I find Rfree always increases slightly after the water picking refinment. Do you have nay idea to solve this problem or any comment? Cheers, Dialing
Re: [ccp4bb] Freezing crystal
Rationalising it completely may only be possible once you know the nature of the crystal contacts, i.e. when you have solved the structure. Until then it is mainly a matter of experimenting. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa H. Hsu Sent: Monday, February 06, 2012 11:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Freezing crystal Hi all Thanks for all the suggestions which I will try soon. How do the crystallization condition (PEG vs. salts like ammonium sulfate) affect the croyprotectant condition? Do factors like presence of low concentration of high molecular weight PEG ( 2000) mean PEG is better? Do buffers and salts in protein also important? Trying to rationalize it :) Theresa
Re: [ccp4bb] Fwd: tcl tk RHEL 6.2
Hi, I think your 64bit system lacks the 32bit loader. Try as root: yum install /lib/ld-linux.so.2 and be prepared to install a couple of 32bit libraries, as needed. HTH, Kay Original Message Subject: tcl tk RHEL 6.2 Date: Mon, 6 Feb 2012 10:17:22 -0600 From: Kenneth A. Satyshur satys...@wisc.edu I just installed ccp4 6.2.0 on a new RHEL6.2 system and I get this error. Anyone know how to fix this? I installed tcl and tk into the system but ccp4 seems to want to use its own version. [xray@paprika ~/Desktop]$ /home/xray/ccp4/ccp4-6.2.0/ccp4-6.2.0/bin/ccp4i: /home/xray/ccp4/ccp4-6.2.0/tcltkplusplus/bin/wish: /lib/ld-linux.so.2: bad ELF interpreter: No such file or directory /home/xray/ccp4/ccp4-6.2.0/ccp4-6.2.0/bin/ccp4i: line 4: /home/xray/ccp4/ccp4-6.2.0/tcltkplusplus/bin/wish: Success thanks kas -- Kenneth A. Satyshur, M.S.,Ph.D. Associate Scientist University of Wisconsin Madison, Wisconsin 53706 608-215-5207 -- Kay Diederichshttp://strucbio.biologie.uni-konstanz.de email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183 Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz This e-mail is digitally signed. If your e-mail client does not have the necessary capabilities, just ignore the attached signature smime.p7s. smime.p7s Description: S/MIME Cryptographic Signature
[ccp4bb] mutagenesis based on ligand-bound crystal structures
Dear colleagues, I am looking for a few example studies in which a complete mutagenesis of all residue-ligand interactions has been conducted to evaluate the energetic contributions of each individual interaction. Thank you. Best regards. -Chris --- Chris Ulens, Ph.D. Lab of Structural Neurobiology Department of Cellular and Molecular Medicine Campus Gasthuisberg, ON1 Herestraat 49, PB 601 B-3000 Leuven Belgium e chris.ul...@med.kuleuven.be t+32 16 330689 f+32 16 345699 w http://www.xtal.be
Re: [ccp4bb] mutagenesis based on ligand-bound crystal structures
Dear Chris A whole series of studies from the early to late 90's did his for the growth hormone-GHR interaction. Papers by Pearce, Cunningham, Clackson and Wells come to mind. Also a recent study on the IL13-IL13R interaction by Lupardus et al has covered most of the interaction epitope. Best regards Savvas On 07 Feb 2012, at 10:16, Chris Ulens chris.ul...@med.kuleuven.be wrote: Dear colleagues, I am looking for a few example studies in which a complete mutagenesis of all residue-ligand interactions has been conducted to evaluate the energetic contributions of each individual interaction. Thank you. Best regards. -Chris --- Chris Ulens, Ph.D. Lab of Structural Neurobiology Department of Cellular and Molecular Medicine Campus Gasthuisberg, ON1 Herestraat 49, PB 601 B-3000 Leuven Belgium e chris.ul...@med.kuleuven.be t+32 16 330689 f+32 16 345699 w http://www.xtal.be
Re: [ccp4bb] on Rwork and Rfree
On 02/07/2012 08:31 AM, Dialing Pretty wrote: Dear All, After we refine the structure of the protein to satisfactory with satisfactory Rwork and Rfree, we pick water by phenix refine, and I find Rfree always increases slightly after the water picking refinment. Do you have nay idea to solve this problem or any comment? Cheers, Dialing Well - it shouldnt! Check if there is some problem.. Eleanor
Re: [ccp4bb] on Rwork and Rfree
Hi Dialing, Most water picking tools are rather overenthusiastic and end up placing some waters at places where they should not be. This causes some overfitting and an increase of R-free. I'm hideously old-fashioned and recommend conservatively building waters by hand. There are some good validation tools that help get rid of excess water: centrifuge in PDB_REDO does the basic work; check/delete waters in Coot highlights other suspicious waters; WHAT_CHECK checks hydrogen bonding thoroughly, finds nonsense clusters of water and also finds possible ions. It must be noted that all these tools break down at very high resolution where you may get alternate waters. Fortunately, this problem doesn't occur very often. Cheers, Robbie From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dialing Pretty Sent: Tuesday, February 07, 2012 09:31 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] on Rwork and Rfree Dear All, After we refine the structure of the protein to satisfactory with satisfactory Rwork and Rfree, we pick water by phenix refine, and I find Rfree always increases slightly after the water picking refinment. Do you have nay idea to solve this problem or any comment? Cheers, Dialing
Re: [ccp4bb] shape complementarity between protein and DNA surface
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Deepak, the only atom type present in DNA and not in Proteins would be the Phosphorus. You can probably add this to sc_radii.lib yourself with the line ***P* 1.80 the radius stems from http://en.wikipedia.org/wiki/Phosphorus and the link to http://en.wikipedia.org/wiki/Van_der_Waals_radius provides you with the reference. The results will hardly differ if you modify this value within 100%, I guess. Cheers, Tim On 02/07/2012 04:19 AM, Deepak Thankappan Nair wrote: Dear All For calculation of the shape complementarity index using the Sc program between a protein and DNA surface, does anybody have a modified radii.lib file that includes information about DNA atoms? Also, is there any reference that has information regarding the atomic radii of DNA atoms? thanks Deepak - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPMPu1UxlJ7aRr7hoRAhAYAKDHJzkHfEPwcHO99FAV57zOrsYasACgvO/V CtUM1Z8x0NSl0cXK4A4tH0k= =H8Uk -END PGP SIGNATURE-
[ccp4bb] Fwd: TR: Permanent Scientist at beamline SWING
Hi, I am forwarding an announcement for a permanent scientist position in structural biology using SAXS at the synchrotron SOLEIL located in Paris suburbs, France. Please address all questions concerning the position to the responsible : Dr. Javier PEREZ pe...@synchrotron-soleil.fr Best, Louis R Original Message Subject:TR: Permanent Scientist at beamline SWING Date: Wed, 1 Feb 2012 14:40:04 + From: PEREZ Javier pe...@synchrotron-soleil.fr To: PEREZ Javier pe...@synchrotron-soleil.fr Dear User, Please find an announcement for permanent scientist position in structural biology at beamline SWING dedicated to Small Angle X-ray Scattering (SAXS)on the synchrotron SOLEIL. You'll find on the following link the conditions of the offer. http://www.synchrotron-soleil.fr/portal/page/portal/Soleil/OffresEmplois/Scientifique-ligne-SWING-CDI (click on the bristish flag if it is not displayed in english) Best regards, Javier Perez *Javier Pérez* Group leader Beamline SWING *Synchrotron SOLEIL* L'Orme des Merisiers Saint-Aubin - BP 48 91192 Gif sur Yvette Cedex France Tel : +33 (0)1 69 35 96 19 (office) +33 (0)1 69 35 97 53 (beamline) *javier.pe...@synchrotron-soleil.fr mailto:javier.pe...@synchrotron-soleil.fr*
[ccp4bb] On pKa of Aspartic acid
Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]
Hi CCP4 list, Thank you very much for additional messages and references. Here goes the image of the PCR product before digested and after digested and cleaned. http://ompldr.org/vY29jbA The results of the transformation of 3 microL (90 ng) of non-mutated/paretal plasmid gave hundreds of colonies; 3 microL of Dpn1 digested sample gave two colonies only; and transformation of 3 microL(90 ng) of cleaned product gave 14 colonies. So, if the amplification is not abundant, chances are that home made competent cell will not be transformed with the digested product. Don't want start another discussion but, is there any reason for differences in the transformation efficiency between the parental and the mutated (cleaned) plasmids? All the Best, Fred Em 03-02-2012 16:14, Fred escreveu: Hi CCP4 list, Thanks everyone who have answered my post concerning to mutagenesis. From quick reading most of the answers, the following seems to be a consensus: 1) Do not concentrate your PCR product; 2) Too much DNA and/or impurities like salts or whatever can inhibits transformation; 3) Purify your PCR product before transformation if possible or use 3 of 4 microL of it. This is more or less the amount of DNA showed in the uploaded image. Kind regards, Fred P.S.: I'll let you know the results.
Re: [ccp4bb] On pKa of Aspartic acid
Hi Deepak, Assuming that you have done the necessary things to measure the pKr of that particular Asp, I would say that the increase is advantageous for your enzyme. Enzyme catalysis often involves very subtle changes on the ionization state of the active site. But you need to be very careful in proposing a catalysis mechanism. b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? Are you sure that the water donates a proton? Was your resolution high enough to observe it? How did you measure that pKr, by the way? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? I would say that the dominant fraction of Asp is deprotonated. But as you can see in the papers below, the pKr of Asp can vary from 0.5 to 9.2 in folded proteins. d) Have similar increase in pKa values observed for aspartic acids before? there are some papers by Nick Pace's group: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2708032/?tool=pubmed http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679426/?tool=pubmed http://www.sciencedirect.com/science/article/pii/S002228360600934X Cheers, Clement -- On 2/7/12 8:48 PM, Deepak Oswal wrote: Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] Freezing crystal
One last thing--sometimes crystals can be frozen as is, particularly if you use mitegen mounts and get nearly all of the mother liquor off the crystals by dabbing the loop on the dry surface next to the drop several times. So simple it is always worth a try JPK On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij mjvanra...@cnb.csic.es wrote: Rationalising it completely may only be possible once you know the nature of the crystal contacts, i.e. when you have solved the structure. Until then it is mainly a matter of experimenting. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa H. Hsu Sent: Monday, February 06, 2012 11:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Freezing crystal Hi all Thanks for all the suggestions which I will try soon. How do the crystallization condition (PEG vs. salts like ammonium sulfate) affect the croyprotectant condition? Do factors like presence of low concentration of high molecular weight PEG ( 2000) mean PEG is better? Do buffers and salts in protein also important? Trying to rationalize it :) Theresa -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]
Hi Fred: For the mutated plasmids, it generates the nicked dna, so the transform efficiency will be lower compared to the parental plasmids. And that is the reason why people usually use super competent cell to transform these nicked plasmid. It seems ok to me to get 2 or 14 colonies from the nicked plasmid. You just need ONE. And usually for these mutagenesis PCR, the success rate is pretty high. I suggest you to sequence these colonies. And you can get it. Yu Xiaodi Date: Tue, 7 Feb 2012 10:17:43 -0200 From: ccp4bb.l...@gmail.com Subject: Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC] To: CCP4BB@JISCMAIL.AC.UK Hi CCP4 list, Thank you very much for additional messages and references. Here goes the image of the PCR product before digested and after digested and cleaned. http://ompldr.org/vY29jbA The results of the transformation of 3 microL (90 ng) of non-mutated/paretal plasmid gave hundreds of colonies; 3 microL of Dpn1 digested sample gave two colonies only; and transformation of 3 microL(90 ng) of cleaned product gave 14 colonies. So, if the amplification is not abundant, chances are that home made competent cell will not be transformed with the digested product. Don't want start another discussion but, is there any reason for differences in the transformation efficiency between the parental and the mutated (cleaned) plasmids? All the Best, Fred Em 03-02-2012 16:14, Fred escreveu: Hi CCP4 list, Thanks everyone who have answered my post concerning to mutagenesis. From quick reading most of the answers, the following seems to be a consensus: 1) Do not concentrate your PCR product; 2) Too much DNA and/or impurities like salts or whatever can inhibits transformation; 3) Purify your PCR product before transformation if possible or use 3 of 4 microL of it. This is more or less the amount of DNA showed in the uploaded image. Kind regards, Fred P.S.: I'll let you know the results.
Re: [ccp4bb] On pKa of Aspartic acid
Hi Deepak: I think it is common for the residues which participate catalysis to have a Pka deviated from the reality pKa value especially for acid/base catalysis (acid base titration assay can help you to figure out the way of catalysis). Usually the pKa values of these kind of critical residues are affected by their local environment and this character is related to the enzyme's working mechanism. I am sorry that I am not professional in enzyme, I cannot answer your questions for each questions. Yu Xiaodi Date: Tue, 7 Feb 2012 19:48:26 +0800 From: deepos...@gmail.com Subject: [ccp4bb] On pKa of Aspartic acid To: CCP4BB@JISCMAIL.AC.UK Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] On pKa of Aspartic acid
Residue pKa values in proteins are strongly affected by the local environment and can deviate far from the "norm". Of course, the higher the pKa of the residue, the stronger general base it will be. There is a significant thermodynamic/kinetic advantage in matching the pKa of a general base to the pKa of the proton that must be removed for catalysis. Aspartate frequently plays a role as a general base for assisting water in nucleophilic attack. I think it is unusual for water to act as a general acid (the pKa of water is 14-15, depending on how you calculate it), but if that is the case and there is compelling evidence that water is the proton donor in your reaction, it would be necessary for aspartic acid to be in its protonated form in order to assist a water-mediated protonation event. Raising the pKa of aspartic acid would allow a larger fraction of it to be in its protonated state at physiologically relevant pH values, although it would reduce the intrinsic effectiveness of Asp as a general acid. There should be a significant thermodynamic and kinetic advantage in having Asp participate directly in a general acid catalyzed reaction, rather than through a water molecule. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/7/2012 10:00 AM, Xiaodi Yu wrote: Hi Deepak: I think it is common for the residues which participate catalysis to have a Pka deviated from the reality pKa value especially for acid/base catalysis (acid base titration assay can help you to figure out the way of catalysis). Usually the pKa values of these kind of critical residues are affected by their local environment and this character is related to the enzyme's working mechanism. I am sorry that I am not professional in enzyme, I cannot answer your questions for each questions. Yu Xiaodi Date: Tue, 7 Feb 2012 19:48:26 +0800 From: deepos...@gmail.com Subject: [ccp4bb] On pKa of Aspartic acid To: CCP4BB@JISCMAIL.AC.UK Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] Freezing crystal
Something to add into this discussion is also go fro the tiny crystals versus the big ones. BIGGER is not always BETTER - in particular if you try to freeze directly out of your conditions without an additional cryo-protectant. And with small or tiny I mean 10 micron, whatever you are capable of mounting. It is also important to keep the amount of liquid volume around the crystal low, so rather use a loop in which you scoop the crystal up instead of having a large loop with lots of liquid. Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 leads to a quicker freeze of your material. If you have the option to anneal your crystal after testing it in the beam try it out and assess the success or damage, this will be very different depending on what cryo-additives you have around. Good luck, Jürgen On Feb 7, 2012, at 9:28 AM, Jacob Keller wrote: One last thing--sometimes crystals can be frozen as is, particularly if you use mitegen mounts and get nearly all of the mother liquor off the crystals by dabbing the loop on the dry surface next to the drop several times. So simple it is always worth a try JPK On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es wrote: Rationalising it completely may only be possible once you know the nature of the crystal contacts, i.e. when you have solved the structure. Until then it is mainly a matter of experimenting. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa H. Hsu Sent: Monday, February 06, 2012 11:00 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Freezing crystal Hi all Thanks for all the suggestions which I will try soon. How do the crystallization condition (PEG vs. salts like ammonium sulfate) affect the croyprotectant condition? Do factors like presence of low concentration of high molecular weight PEG ( 2000) mean PEG is better? Do buffers and salts in protein also important? Trying to rationalize it :) Theresa -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] on Rwork and Rfree
Dialing, After we refine the structure of the protein to satisfactory with satisfactory Rwork and Rfree, we pick water by phenix refine, and I find Rfree always increases slightly after the water picking refinment. Do you have nay idea to solve this problem or any comment? Just a reminder: there is phenixbb mailing list for Phenix related questions. Replying to your post: phenix.refine uses very sophisticated criteria for water update (which doen't mean there is no room for further improvement of course). If you believe it placed or removed a wrong water please submit a bug report to me that should include: 1) all input files (reflection data, PDB file with the model, CIF ligand and parameter files if any); 2) the command you used to run the program; 3) explain which waters you believe are wrong. Pavel.
Re: [ccp4bb] On pKa of Aspartic acid
As we know, the pKa of water is 15.7. Under pH 7.0, its protonation should be 50/50. In this case, we may need to consider water in two formats: H2O vs. H3O+ When we say water as acid, it usually stands for H3O+ in chemistry. In chemical equation, H+ represents H3O+. In enzyme catalysis, water as a general acid sounds reasonable under pH 7.0. In some famous paper, water has been concluded as the general base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need to be reconsidered. . Recently, I have read papers for pKa perturbation. I am also interested in the general base of Asp and Glu in enzyme catalysis. I will be very happy to read your paper in the future. Regards, Kevin Jin On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote: Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] On pKa of Aspartic acid
Check this review, for instance: Pace, C. et al. Protein Ionizable Groups: pK values and Their Contribution to Protein Stability and Solubility. J. Biol Chem. 284, 13285-13289 (May 15, 2009) Thierry From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaodi Yu Sent: Tuesday, February 07, 2012 10:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] On pKa of Aspartic acid Hi Deepak: I think it is common for the residues which participate catalysis to have a Pka deviated from the reality pKa value especially for acid/base catalysis (acid base titration assay can help you to figure out the way of catalysis). Usually the pKa values of these kind of critical residues are affected by their local environment and this character is related to the enzyme's working mechanism. I am sorry that I am not professional in enzyme, I cannot answer your questions for each questions. Yu Xiaodi Date: Tue, 7 Feb 2012 19:48:26 +0800 From: deepos...@gmail.com Subject: [ccp4bb] On pKa of Aspartic acid To: CCP4BB@JISCMAIL.AC.UK Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids' ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn't that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] On pKa of Aspartic acid
Oops, you meant catalytic residue. Check the following: Harris TK Turner GJ Structural basis of pertubed pKa values of catalytic groups in enzyme active sites IUBMB life, 53 85-98 (Feb 2002) Thierry From: Fischmann, Thierry Sent: Tuesday, February 07, 2012 10:59 AM To: 'Xiaodi Yu'; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] On pKa of Aspartic acid Check this review, for instance: Pace, C. et al. Protein Ionizable Groups: pK values and Their Contribution to Protein Stability and Solubility. J. Biol Chem. 284, 13285-13289 (May 15, 2009) Thierry From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaodi Yu Sent: Tuesday, February 07, 2012 10:00 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] On pKa of Aspartic acid Hi Deepak: I think it is common for the residues which participate catalysis to have a Pka deviated from the reality pKa value especially for acid/base catalysis (acid base titration assay can help you to figure out the way of catalysis). Usually the pKa values of these kind of critical residues are affected by their local environment and this character is related to the enzyme's working mechanism. I am sorry that I am not professional in enzyme, I cannot answer your questions for each questions. Yu Xiaodi Date: Tue, 7 Feb 2012 19:48:26 +0800 From: deepos...@gmail.com Subject: [ccp4bb] On pKa of Aspartic acid To: CCP4BB@JISCMAIL.AC.UK Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids' ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn't that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Freezing crystal
BIGGER is not always BETTER? Theoretically it should be better because you have more scattering matter. If it is not something has gone wrong in prior steps: Purification: You were less selective and picked up more heterogeneous protein. Crystallization: The bigger crystals grew under conditions that were less controlled because of changes in the state of protein supersaturation, precipitant or temperature. These inhomogeneities contributed to give you bigger, but not better crystals. Cryo-soak: Bigger crystals are more prone to be shocked when transfered to a less than optimal cryo-solution. This is a critical step, and crystals do not like too much the cryo-chemicals. To test this I tried lower concentrations of various cryo-compounds instead of a huge quantity of a single component and in most cases the mixture was better tollerated than the single components. Flash-freezing: Bigger crystals will cool more unevenly than small ones. A change from liquid nitrogen to liquid ethane could achieve faster cooling because of the greater heat capacity of the latter liquid. Large crystals are more difficult to handle than small ones but after experimenting with small ones we can build up a good experimental protocol so that the big crystals will give exceptionally good results. I compared small crystals on high intensity beamlines at the ESRF against large crystals on BM30 and the big crystals were statistically better. Unfortunately, it takes a lot of time and a certain amount of expertize to optimize the conditions. So ... although I strongly disagree with Jürgen, I will also advise to start with small ones. Enrico. On Tue, 07 Feb 2012 16:58:04 +0100, Bosch, Juergen jubo...@jhsph.edu wrote: Something to add into this discussion is also go fro the tiny crystals versus the big ones. BIGGER is not always BETTER - in particular if you try to freeze directly out of your conditions without an additional cryo-protectant. And with small or tiny I mean 10 micron, whatever you are capable of mounting. It is also important to keep the amount of liquid volume around the crystal low, so rather use a loop in which you scoop the crystal up instead of having a large loop with lots of liquid. Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 leads to a quicker freeze of your material. If you have the option to anneal your crystal after testing it in the beam try it out and assess the success or damage, this will be very different depending on what cryo-additives you have around. Good luck, Jürgen On Feb 7, 2012, at 9:28 AM, Jacob Keller wrote: One last thing--sometimes crystals can be frozen as is, particularly if you use mitegen mounts and get nearly all of the mother liquor off the crystals by dabbing the loop on the dry surface next to the drop several times. So simple it is always worth a try JPK On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es wrote: Rationalising it completely may only be possible once you know the nature of the crystal contacts, i.e. when you have solved the structure. Until then it is mainly a matter of experimenting. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa H. Hsu Sent: Monday, February 06, 2012 11:00 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Freezing crystal Hi all Thanks for all the suggestions which I will try soon. How do the crystallization condition (PEG vs. salts like ammonium sulfate) affect the croyprotectant condition? Do factors like presence of low concentration of high molecular weight PEG ( 2000) mean PEG is better? Do buffers and salts in protein also important? Trying to rationalize it :) Theresa -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/ -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1
Re: [ccp4bb] On pKa of Aspartic acid
Hi, you may also look into the papers of John A. Gerlt, who did a lot on protonabtraction reactions and the theory behind this. Esspecially the pKa disturbance and the match to the pkA of the substrate of the reaction. Best Wishes Christian Am Dienstag 07 Februar 2012 12:48:26 schrieb Deepak Oswal: Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] Freezing crystal
Hi Enrico, I was just looking at non-optimal cryo-conditions and the original posters starting point. Of course if you have a good cryo bigger is better for the reasons you write but if you have no clue how your crystals will perform then I'd rather go for small to be cautious and also have those around and not only the big ones which everybody mounts because they looks so nice. To be disappointed by big crystals is often not a surprise to me and if you have not tried small crystals from the same batch well then you missed 50% of your chances to solve s structure with the first light the crystals saw. Jürgen On Feb 7, 2012, at 11:51 AM, Enrico Stura wrote: BIGGER is not always BETTER? Theoretically it should be better because you have more scattering matter. If it is not something has gone wrong in prior steps: Purification: You were less selective and picked up more heterogeneous protein. Crystallization: The bigger crystals grew under conditions that were less controlled because of changes in the state of protein supersaturation, precipitant or temperature. These inhomogeneities contributed to give you bigger, but not better crystals. Cryo-soak: Bigger crystals are more prone to be shocked when transfered to a less than optimal cryo-solution. This is a critical step, and crystals do not like too much the cryo-chemicals. To test this I tried lower concentrations of various cryo-compounds instead of a huge quantity of a single component and in most cases the mixture was better tollerated than the single components. Flash-freezing: Bigger crystals will cool more unevenly than small ones. A change from liquid nitrogen to liquid ethane could achieve faster cooling because of the greater heat capacity of the latter liquid. Large crystals are more difficult to handle than small ones but after experimenting with small ones we can build up a good experimental protocol so that the big crystals will give exceptionally good results. I compared small crystals on high intensity beamlines at the ESRF against large crystals on BM30 and the big crystals were statistically better. Unfortunately, it takes a lot of time and a certain amount of expertize to optimize the conditions. So ... although I strongly disagree with Jürgen, I will also advise to start with small ones. Enrico. On Tue, 07 Feb 2012 16:58:04 +0100, Bosch, Juergen jubo...@jhsph.edumailto:jubo...@jhsph.edu wrote: Something to add into this discussion is also go fro the tiny crystals versus the big ones. BIGGER is not always BETTER - in particular if you try to freeze directly out of your conditions without an additional cryo-protectant. And with small or tiny I mean 10 micron, whatever you are capable of mounting. It is also important to keep the amount of liquid volume around the crystal low, so rather use a loop in which you scoop the crystal up instead of having a large loop with lots of liquid. Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 leads to a quicker freeze of your material. If you have the option to anneal your crystal after testing it in the beam try it out and assess the success or damage, this will be very different depending on what cryo-additives you have around. Good luck, Jürgen On Feb 7, 2012, at 9:28 AM, Jacob Keller wrote: One last thing--sometimes crystals can be frozen as is, particularly if you use mitegen mounts and get nearly all of the mother liquor off the crystals by dabbing the loop on the dry surface next to the drop several times. So simple it is always worth a try JPK On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es wrote: Rationalising it completely may only be possible once you know the nature of the crystal contacts, i.e. when you have solved the structure. Until then it is mainly a matter of experimenting. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Theresa H. Hsu Sent: Monday, February 06, 2012 11:00 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Freezing crystal Hi all Thanks for all the suggestions which I will try soon. How do the crystallization condition (PEG vs. salts like ammonium sulfate) affect the croyprotectant condition? Do factors like presence of low concentration of high molecular weight PEG ( 2000) mean PEG is better? Do buffers and salts in protein also important? Trying to rationalize it :) Theresa -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edumailto:j-kell...@northwestern.edumailto:j-kell...@northwestern.edu *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute
Re: [ccp4bb] On pKa of Aspartic acid
Is the water molecule in question coordinated to any other group(s)? -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kevin Jin Sent: Tuesday, February 07, 2012 10:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] On pKa of Aspartic acid As we know, the pKa of water is 15.7. Under pH 7.0, its protonation should be 50/50. In this case, we may need to consider water in two formats: H2O vs. H3O+ When we say water as acid, it usually stands for H3O+ in chemistry. In chemical equation, H+ represents H3O+. In enzyme catalysis, water as a general acid sounds reasonable under pH 7.0. In some famous paper, water has been concluded as the general base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need to be reconsidered. . Recently, I have read papers for pKa perturbation. I am also interested in the general base of Asp and Glu in enzyme catalysis. I will be very happy to read your paper in the future. Regards, Kevin Jin On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote: Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids' ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn't that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
[ccp4bb] Merging hi and low res data
We have a high and low res pass on a crystal that is going to around 1.2 A on the pilatus at Diamond. We tried to scale them together using the xds CORRECT files from the xia2 -3dii runs (gives best results as far as we can see comparing xia2.html files) through aimless via ccp4i interface. The combined dataset looks worse (based on merging statistics at lower resolutions) than the individual particular at low res.(Compared to scala xia2 runs). XDS does not record the pilatus as having overloads even on the high res pass using the correct detector parameters http://xds.mpimf-heidelberg.mpg.de/html_doc/detectors.html What I wanted to do was limit the resolution of the two runs ie to use low res between 100-2.5 beyond which completeness plummets and then the highres between 8 and 1.2. Aimless does not seem to allow me to do that at least by the ccp4i interface- is this then bad practice? I guess I could run a utility to remove the resolutions I don't want. The one effect that makes me wonder if the lowres is better data, and worth combining, ie there is some overload in the high res, is that it goes from 30-8 mean I/SIGI whereas the high res has I/SIGI of 15 in most of the low res shells and then slowly falls off to 2.0 at 1.2 angstrom. Am intending to carry on just using the hires pass. Any comments? I think Phil is in NZ and not looking at email hence putting this to the whole community -- Prof Nicholas H. Keep Executive Dean of School of Science Professor of Biomolecular Science Crystallography, Institute for Structural and Molecular Biology, Department of Biological Sciences Birkbeck, University of London, Malet Street, Bloomsbury LONDON WC1E 7HX email n.k...@mail.cryst.bbk.ac.uk Telephone 020-7631-6852 (Room G57 Office) 020-7631-6800 (Department Office) Fax 020-7631-6803 If you want to access me in person you have to come to the crystallography entrance and ring me or the department office from the internal phone by the door
Re: [ccp4bb] On pKa of Aspartic acid
Maybe you would also be interested in http://www.jinkai.org/AAD_history.html Regards, Kevin On Tue, Feb 7, 2012 at 8:52 AM, Christian Roth christian.r...@bbz.uni-leipzig.de wrote: Hi, you may also look into the papers of John A. Gerlt, who did a lot on protonabtraction reactions and the theory behind this. Esspecially the pKa disturbance and the match to the pkA of the substrate of the reaction. Best Wishes Christian Am Dienstag 07 Februar 2012 12:48:26 schrieb Deepak Oswal: Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] Freezing crystal
Dear Jürgen, Am 07.02.12 16:58, schrieb Bosch, Juergen: snip Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 leads to a quicker freeze of your material. /snip Are you sure? There was a publication by Warkentin et al. [1] about a cold gas layer above liquid nitrogen that reduces the expected cooling rate a lot! My very personal experience is, that cryo-cooling in the N2-stream worked better for me than in LN2 in a variety of projects - but the reason could just be me ;-) Best regards, Dirk. [1] Matthew Warkentin, Viatcheslav Berejnov, Naji S Husseini, and Robert E Thorne: Hyperquenching for protein cryocrystallography, J. Appl. Crystallogr., 39, 805-811 (2006) -- *** Dirk Kostrewa Gene Center Munich Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
Re: [ccp4bb] Freezing crystal
Hi Dirk, I remember a neat paper don't recall who wrote it. I think it was in Acta D where the authors made a tiny probe the size of an elongated crystal glued to a [/Advertisement on] Hampton loop [/Advertisement off]. The probe was a temperature sensor and they recorded the cooling rate under different methods. The winner as far as I recall was freezing in liquid propane for the lack of the missing gas layer, but the second best method was LN2. Propane for whatever reason has gone extinct in certain areas of the world :-) . I'll try to find that reference but perhaps somebody else on this highly educated board knows which paper I'm referring to. I want to say it was published around 2004-2006. Jürgen On Feb 7, 2012, at 11:12 AM, Dirk Kostrewa wrote: Dear Jürgen, Am 07.02.12 16:58, schrieb Bosch, Juergen: snip Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 leads to a quicker freeze of your material. /snip Are you sure? There was a publication by Warkentin et al. [1] about a cold gas layer above liquid nitrogen that reduces the expected cooling rate a lot! My very personal experience is, that cryo-cooling in the N2-stream worked better for me than in LN2 in a variety of projects - but the reason could just be me ;-) Best regards, Dirk. [1] Matthew Warkentin, Viatcheslav Berejnov, Naji S Husseini, and Robert E Thorne: Hyperquenching for protein cryocrystallography, J. Appl. Crystallogr., 39, 805-811 (2006) -- *** Dirk Kostrewa Gene Center Munich Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.demailto:kostr...@genzentrum.lmu.de WWW: www.genzentrum.lmu.de *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] On pKa of Aspartic acid
Hi Deepak, With regards observed pKa shifts, Prof. Ondrechen from Northeastern University has had a long interest in this field. http://www.northeastern.edu/org/wp/ Under the computational tools that she has developed a program called THEMATICS that allows you to predict the pka of titratable amino acids and she has been able to predict shifts. Though the server seems to be down at this point, here is the reference: Y. Wei, J. Ko, L.F. Murga, and M.J. Ondrechen, BMC Bioinformatics 8:119, (2007) From the commercial side, Dr. Spassov from Accelrys has also been working on tools that predict protein ionization. In his work, he has also been able to predict significant pka shifts for functionally relevant residues. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2578799/ Cheers, Francisco From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Deepak Oswal Sent: Tuesday, February 07, 2012 3:48 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] On pKa of Aspartic acid Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids' ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn't that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] Freezing crystal
Just a thought for those that mentioned propane and ethane, I would like to suggest that they try carbon tetrafluoride (CF4) instead. It certainly should be much safer. It melts at 90 K and boils at 145 K, so you know you are below 145 K if you see it as a liquid.
Re: [ccp4bb] Freezing crystal
On 02/07/12 11:12, Dirk Kostrewa wrote: Dear Jürgen, Am 07.02.12 16:58, schrieb Bosch, Juergen: snip Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 leads to a quicker freeze of your material. /snip Are you sure? There was a publication by Warkentin et al. [1] about a cold gas layer above liquid nitrogen that reduces the expected cooling rate a lot! My very personal experience is, that cryo-cooling in the N2-stream worked better for me than in LN2 in a variety of projects - but the reason could just be me ;-) Yes, Warkentin, et al found the cold gas layer affects cooling rate - but they were able to overcome it by blowing air across the surface. Your point is taken that complications such as this affect the cooling rate much more than a simple liquid vs. stream choice. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Freezing crystal (Liquid Propane Crystal Prep)
Jürgen Quote: Propane for whatever reason has gone extinct in certain areas of the world :-) . I went to SSRL (Stanford) with a colleague who wanted to use liquid propane. We had to go through a mound of paper work to get permission bring propane on site and set up the experiments. I don't blame SSRL for their safety policy, but I can clearly understand why liquid propane is not commonly used. If you don't think it is much of a danger, you might enjoy: http://www.stupidvideos.com/video/stunts/propane_tank/#2974 You might also enjoy: http://www.stupidvideos.com/video/stunts/C4_Propane_Explosion/#175408 Note: We did not bring any C-4 to SSRL:) Steve On 2/7/2012 10:50 AM, Bosch, Juergen wrote: Hi Dirk, I remember a neat paper don't recall who wrote it. I think it was in Acta D where the authors made a tiny probe the size of an elongated crystal glued to a [/Advertisement on] Hampton loop [/Advertisement off]. The probe was a temperature sensor and they recorded the cooling rate under different methods. The winner as far as I recall was freezing in liquid propane for the lack of the missing gas layer, but the second best method was LN2. Propane for whatever reason has gone extinct in certain areas of the world :-) . I'll try to find that reference but perhaps somebody else on this highly educated board knows which paper I'm referring to. I want to say it was published around 2004-2006. Jürgen On Feb 7, 2012, at 11:12 AM, Dirk Kostrewa wrote: Dear Jürgen, Am 07.02.12 16:58, schrieb Bosch, Juergen: snip Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 leads to a quicker freeze of your material. /snip Are you sure? There was a publication by Warkentin et al. [1] about a cold gas layer above liquid nitrogen that reduces the expected cooling rate a lot! My very personal experience is, that cryo-cooling in the N2-stream worked better for me than in LN2 in a variety of projects - but the reason could just be me ;-) Best regards, Dirk. [1] Matthew Warkentin, Viatcheslav Berejnov, Naji S Husseini, and Robert E Thorne: Hyperquenching for protein cryocrystallography, J. Appl. Crystallogr., 39, 805-811 (2006) -- *** Dirk Kostrewa Gene Center Munich Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail:kostr...@genzentrum.lmu.de mailto:kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/ *TODAY*/(Beta) /*.*Powered by Yahoo! Funny moment on NFL champ's plane http://sports.yahoo.com/blogs/nfl-shutdown-corner/york-giants-celebrate-super-bowl-victory-airplane-sing-195044076.html;_ylc=X3oDMTFjM2oycTY1BF9TAzk1NDAxMDAyNwRwa2cDaWQtMTY2ODI5MwRzeWlkA2k3NGhoZWRfMDAy A video shot by one of the Giants shows teammates celebrating on the flight home from the Super Bowl. Privacy Policy http://info.yahoo.com/privacy/us/yahoo/webbeacons/details.html
[ccp4bb] Staff scientist position at MacCHESS
This job was posted earlier, but there is an update to the how-to-apply instructions. The Macromolecular Diffraction Facility of the Cornell High-Energy Synchrotron Source (MacCHESS) has an opening for a Staff Scientist (Research Associate) to pursue the development of novel techniques in x-ray scattering as applied to structural biology, and to support users at MacCHESS. There will also be opportunities to pursue projects in structural biology, using current crystallographic and SAXS methods. Research areas of particular interest include structure solution from multiple crystals, use of Laue diffraction, BioSAXS, microfluidics, and user interfaces for beamline operation. A Ph.D. in structural biology, biophysics, or a related field, and at least 3 years of experience beyond the degree in a relevant field is required. A solid publication record is essential, and experience working at a synchrotron facility is highly desirable. Excellent communication skills are a must, including fluency in the English language. Appointments are nominally for three years with the possibility for renewal, subject to mutual satisfaction and the availability of funds. Located on an Ivy League university campus in picturesque upstate New York, the Cornell High-Energy Synchrotron Source (CHESS) serves a world-wide user base of structural biologists, chemists, physicists, and engineers. MacCHESS is an NIH-supported National Resource providing support for structural biology at CHESS. MacCHESS is a heavily team-oriented environment. Applications should be submitted at http://academicjobsonline.org/ (posting no. 1422) and should include a cover letter, a CV, a list of publications, and a detailed summary of research experience and interests. Applicants must arrange to have at least three letters of recommendation sent, as per instructions on the academicjobsonline website. The job is available immediately. Cornell is an equal opportunity, affirmative action educator and employer.
Re: [ccp4bb] On pKa of Aspartic acid
Hi Kevin, Hate to point this out, but under pH 7.0, the protonation state of water is not 50:50, and it is not a good acid. The H30+ concentration of pure water is 10^-7 Molar. In pure water (assuming 55.5 M) only 1:555,000,000 water molecules is in the protonated, charged state (H3O+). This is why when an enzyme uses water in its mechanism as a nucleophile, base, or acid, there is usually an acid/base catalyst or metal that protonates or deprotonates the water to 'activate it'. Best regards, Z *** Zachary A. Wood, Ph.D. Assistant Professor Department of Biochemistry Molecular Biology University of Georgia Life Sciences Building, Rm A426B 120 Green Street Athens, GA 30602-7229 Office: 706-583-0304 Lab:706-583-0303 FAX: 706-542-1738 *** On Feb 7, 2012, at 11:22 AM, Kevin Jin wrote: As we know, the pKa of water is 15.7. Under pH 7.0, its protonation should be 50/50. In this case, we may need to consider water in two formats: H2O vs. H3O+ When we say water as acid, it usually stands for H3O+ in chemistry. In chemical equation, H+ represents H3O+. In enzyme catalysis, water as a general acid sounds reasonable under pH 7.0. In some famous paper, water has been concluded as the general base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need to be reconsidered. . Recently, I have read papers for pKa perturbation. I am also interested in the general base of Asp and Glu in enzyme catalysis. I will be very happy to read your paper in the future. Regards, Kevin Jin On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote: Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] Merging hi and low res data
Hi, from your description I do not quite understand a few points: - what is the evidence that there are overloads? The Pilatus pixels have 20bits (meaning max contents of a pixel = 1048575) , and the OVERLOAD parameter is set to 1048500 (slightly below that). Do you actually see pixels with a contents of 1048575, but INTEGRATE and CORRECT do not report any overloads? That could indicate a bug in XDS, and I'd appreciate to receive a frame with overloads. - could it be that the hi and the low resolution run do not scale well because they correspond to alternative indexing modes? - this can happen in spacegroups P3x, P4x, P6x and a couple of others. In that case you can use the XDS_ASCII.HKL of one run as a REFERENCE_DATA_SET for the other run, and run the CORRECT job - it will pick a consistent indexing. - did you collect the hi res first, and the low res after that? If so, bad scaling is to be expected, because the crystal has a lot of radiation damage in the second run, resulting in elevated R values. It is much better to do the low res run first, producing very little radiation damage due to the much lower dose required. best, Kay
Re: [ccp4bb] On pKa of Aspartic acid
Oops, It should be: [H3O+]/[OH-]= 50/50 Kw = [H3O+][OH-], pH = pKa +log ([OH-]/[H2O]) H3O+ concentration of pure water is 10^-7 mol/L total H+ = 55.5M * 10^-7 = 5.55* 10^-6 mole. Is this right? Regards, Kevin On Tue, Feb 7, 2012 at 12:13 PM, Zachary Wood z...@bmb.uga.edu wrote: Hi Kevin, Hate to point this out, but under pH 7.0, the protonation state of water is not 50:50, and it is not a good acid. The H30+ concentration of pure water is 10^-7 Molar. In pure water (assuming 55.5 M) only 1:555,000,000 water molecules is in the protonated, charged state (H3O+). This is why when an enzyme uses water in its mechanism as a nucleophile, base, or acid, there is usually an acid/base catalyst or metal that protonates or deprotonates the water to 'activate it'. Best regards, Z *** Zachary A. Wood, Ph.D. Assistant Professor Department of Biochemistry Molecular Biology University of Georgia Life Sciences Building, Rm A426B 120 Green Street Athens, GA 30602-7229 Office: 706-583-0304 Lab: 706-583-0303 FAX: 706-542-1738 *** On Feb 7, 2012, at 11:22 AM, Kevin Jin wrote: As we know, the pKa of water is 15.7. Under pH 7.0, its protonation should be 50/50. In this case, we may need to consider water in two formats: H2O vs. H3O+ When we say water as acid, it usually stands for H3O+ in chemistry. In chemical equation, H+ represents H3O+. In enzyme catalysis, water as a general acid sounds reasonable under pH 7.0. In some famous paper, water has been concluded as the general base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need to be reconsidered. . Recently, I have read papers for pKa perturbation. I am also interested in the general base of Asp and Glu in enzyme catalysis. I will be very happy to read your paper in the future. Regards, Kevin Jin On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote: Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
[ccp4bb] Invisible Reference on Pubmed?
Dear CCP4BB, this is perhaps my most egregious off-topic post, but can anyone explain why the following reference is not findable in PubMed? I can get it from the ACS website, but not on PubMed or elsewhere. The journal is on PubMed--is it perhaps because it's funded by ExxonMobil? Very strange... Jacob Article A Study of the Separation Principle in Size Exclusion Chromatography AbstractFull Text HTMLHi-Res PDF[140 KB]PDF w/ Links[240 KB]FiguresCiting Articles Thomas Sun* Baytown Polymers Center, ExxonMobil Chemical Company, 5200 Bayway Dr., Baytown, Texas 77520-2101 Ronald R. Chance, William W. Graessley,† and David J. Lohse Corporate Strategic Research, ExxonMobil Research and Engineering, Annandale, New Jersey 08801 Macromolecules, 2004, 37 (11), pp 4304–4312 DOI: 10.1021/ma030586k Publication Date (Web): April 29, 2004 Copyright © 2004 American Chemical Society -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] On pKa of Aspartic acid
No. Kw = [H3O+][OH-] = 1 x 10^-14 at 25 deg C. So at pH 7.0, you have 10^-7 M each at equilibrium no matter how you slice it or whatever else is in solution. If equilibrium [H3O+] goes up [OH-] goes down commensurately. The "pKa" of water as an acid is based on Kw and water's effective concentration of 55 M in pure water. This "pKa" is used to compare the instrinsic acidity of water to other weak acids. Water is an exceptionally weak acid or base. ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/7/2012 3:42 PM, Kevin Jin wrote: Oops, It should be: [H3O+]/[OH-]= 50/50 Kw = [H3O+][OH-], pH = pKa +log ([OH-]/[H2O]) H3O+ concentration of pure water is 10^-7 mol/L total H+ = 55.5M * 10^-7 = 5.55* 10^-6 mole. Is this right? Regards, Kevin On Tue, Feb 7, 2012 at 12:13 PM, Zachary Wood z...@bmb.uga.edu wrote: Hi Kevin, Hate to point this out, but under pH 7.0, the protonation state of water is not 50:50, and it is not a good acid. The H30+ concentration of pure water is 10^-7 Molar. In pure water (assuming 55.5 M) only 1:555,000,000 water molecules is in the protonated, charged state (H3O+). This is why when an enzyme uses water in its mechanism as a nucleophile, base, or acid, there is usually an acid/base catalyst or metal that protonates or deprotonates the water to 'activate it'. Best regards, Z *** Zachary A. Wood, Ph.D. Assistant Professor Department of Biochemistry Molecular Biology University of Georgia Life Sciences Building, Rm A426B 120 Green Street Athens, GA 30602-7229 Office: 706-583-0304 Lab: 706-583-0303 FAX: 706-542-1738 *** On Feb 7, 2012, at 11:22 AM, Kevin Jin wrote: As we know, the pKa of water is 15.7. Under pH 7.0, its protonation should be 50/50. In this case, we may need to consider water in two formats: H2O vs. H3O+ When we say water as acid, it usually stands for H3O+ in chemistry. In chemical equation, H+ represents H3O+. In enzyme catalysis, water as a general acid sounds reasonable under pH 7.0. In some famous paper, water has been concluded as the general base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need to be reconsidered. . Recently, I have read papers for pKa perturbation. I am also interested in the general base of Asp and Glu in enzyme catalysis. I will be very happy to read your paper in the future. Regards, Kevin Jin On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com wrote: Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal
Re: [ccp4bb] On pKa of Aspartic acid
Dear all, for further discussion I believe that using the 0-14 pH scale assumes water activity of pure water, something that is surely not matched in the surface or pocket of a protein, so keeping this in mind I always prefer to speak about apparent pKa of a group if talking about a non solvent-exposed group. Horacio 2012/2/7 Roger Rowlett rrowl...@colgate.edu No. Kw = [H3O+][OH-] = 1 x 10^-14 at 25 deg C. So at pH 7.0, you have 10^-7 M each at equilibrium no matter how you slice it or whatever else is in solution. If equilibrium [H3O+] goes up [OH-] goes down commensurately. The pKa of water as an acid is based on Kw and water's effective concentration of 55 M in pure water. This pKa is used to compare the instrinsic acidity of water to other weak acids. Water is an exceptionally weak acid or base. ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/7/2012 3:42 PM, Kevin Jin wrote: Oops, It should be: [H3O+]/[OH-]= 50/50 Kw = [H3O+][OH-], pH = pKa +log ([OH-]/[H2O]) H3O+ concentration of pure water is 10^-7 mol/L total H+ = 55.5M * 10^-7 = 5.55* 10^-6 mole. Is this right? Regards, Kevin On Tue, Feb 7, 2012 at 12:13 PM, Zachary Wood z...@bmb.uga.edu z...@bmb.uga.edu wrote: Hi Kevin, Hate to point this out, but under pH 7.0, the protonation state of water is not 50:50, and it is not a good acid. The H30+ concentration of pure water is 10^-7 Molar. In pure water (assuming 55.5 M) only 1:555,000,000 water molecules is in the protonated, charged state (H3O+). This is why when an enzyme uses water in its mechanism as a nucleophile, base, or acid, there is usually an acid/base catalyst or metal that protonates or deprotonates the water to 'activate it'. Best regards, Z *** Zachary A. Wood, Ph.D. Assistant Professor Department of Biochemistry Molecular Biology University of Georgia Life Sciences Building, Rm A426B 120 Green Street Athens, GA 30602-7229 Office: 706-583-0304 Lab:706-583-0303 FAX: 706-542-1738 *** On Feb 7, 2012, at 11:22 AM, Kevin Jin wrote: As we know, the pKa of water is 15.7. Under pH 7.0, its protonation should be 50/50. In this case, we may need to consider water in two formats: H2O vs. H3O+ When we say water as acid, it usually stands for H3O+ in chemistry. In chemical equation, H+ represents H3O+. In enzyme catalysis, water as a general acid sounds reasonable under pH 7.0. In some famous paper, water has been concluded as the general base (pKa 15.7) to deprotonate an alpha hydrogen (pKa ~ 22) or a hydrogen from a sp3 hybridized carbon (pKa ~36). This logic may need to be reconsidered. . Recently, I have read papers for pKa perturbation. I am also interested in the general base of Asp and Glu in enzyme catalysis. I will be very happy to read your paper in the future. Regards, Kevin Jin On Tue, Feb 7, 2012 at 3:48 AM, Deepak Oswal deepos...@gmail.com deepos...@gmail.com wrote: Dear colleagues, We have solved the crystal structure of a human enzyme. The pKa of a catalytically critical aspartic acid has increased to 6.44. It is hydrogen bonded (2.8 Angstroms) to a water molecule that is supposed to donate a proton during the catalysis. Can anybody help me a) interpret the significance of this increase in pKa of the aspartic acid from 3.8 to 6.44 in context with the catalysis? Is this advantageous or detrimental? b) How is pKa related to an amino acids’ ability to force a water molecule to donate a proton? c) At pH 7.4, the aspartic acid would be de-protonated irrespective of whether the pKa is 3.8 or 6.44; isn’t that true? d) Have similar increase in pKa values observed for aspartic acids before? I would be grateful if anybody could explain or comment on the above queries. Deepak Oswal -- Horacio Botti PhD MD Protein Crystallography Unit Institut Pasteur of Montevideo
Re: [ccp4bb] Invisible Reference on Pubmed?
On 2/7/12 4:02 PM, Jacob Keller wrote: Dear CCP4BB, this is perhaps my most egregious off-topic post, but can anyone explain why the following reference is not findable in PubMed? I can get it from the ACS website, but not on PubMed or elsewhere. The journal is on PubMed--is it perhaps because it's funded by ExxonMobil? Very strange... Jacob Hi Jacob - PubMed isn't universal - its goal is to cover the biomedical literature, so articles on the margins, like organic chemistry, materials science (even crystallography!) aren't fully covered. If you look at PubMed's total coverage of the journal Macromolecules, you'll see that it's extremely patchy: lots of articles from 1977, then almost nothing until a whole fleet of articles in 1998, then almost nothing again until 2007. Your article must have fallen into one of these coverage holes. Either that, or it's THE MAN suppressing the research needed to cure cancer and the common cold, and build a car that runs on water... :) - Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165
[ccp4bb] Generating parameters/cif files for macrocyclic ligands
Hi folks, I have an intriguing problem. I'm trying to generate a cif file for a macrocyclic peptide (of the likes in pdb1d4khttp://www.rcsb.org/pdb/explore/explore.do?structureId=1d4k). They are cyclic tripeptides units. I can generate a pdb or mol2 file easily. I have used PRODRG to generate a .cif file and Coot read thjis in nicely. However, as it is cyclic one cannot adjust the dihedral angles. I have previously done this using CNS where you can break the tricyclic peptide into residues and generate parameters to specify bonds/links between the residues (which allows this kind of movement). I can't come up with a way to do this without using CNS. I have looked ta J-ligand which allows for one link between two separate residues which precludes a macrocycle. I have looked at sketcher within CCP4 which reads the pdb files but I don't believe this can be done here. Within Coot I can refine the whole ligand but not certain components. Any suggestions greatly appreciated . ( I may stick to coot refinement with fixed atoms at this stage) Regards Joel _ Joel Tyndall, PhD Senior Lecturer in Medicinal Chemistry National School of Pharmacy University of Otago PO Box 56 Dunedin 9054 New Zealand Skype: jtyndall http://www.researcherid.com/rid/C-2803-2008 Pukeka Matua Te Kura Taiwhanga Putaiao Te Whare Wananga o Otago Pouaka Poutapeta 56 Otepoti 9054 Aotearoa Ph / Waea +64 3 4797293 Fax / Waeawhakaahua +64 3 4797034
Re: [ccp4bb] No diffraction
This might be obvious, but make sure you washed the crystals thoroughly before dissolving them for SDS-PAGE or mass spec -Jesse On Thu, Jan 26, 2012 at 10:48 AM, Katherine Sippel katherine.sip...@gmail.com wrote: Might I suggest consulting the CCP4 user community wiki on the topic: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality Good luck, Katherine On Thu, Jan 26, 2012 at 9:33 AM, Theresa H. Hsu theresah...@live.com wrote: Dear crystallographers I have a protein of 90 kDa forming dimers. Crystals formed with microbatch and vapor diffusion method in 24 hours but no diffraction at home source. Dissolved crystals was confirmed to be the protein with mass spec. Any suggestions to improve diffraction would be welcome. Thanking you in advance. Theresa
Re: [ccp4bb] Invisible Reference on Pubmed?
Well, perhaps it is because size exclusion chromatography is used so little in the life sciences؟, and who really cares how it works anyway?؟ JPK On Tue, Feb 7, 2012 at 3:18 PM, Matthew Franklin mfrank...@nysbc.org wrote: On 2/7/12 4:02 PM, Jacob Keller wrote: Dear CCP4BB, this is perhaps my most egregious off-topic post, but can anyone explain why the following reference is not findable in PubMed? I can get it from the ACS website, but not on PubMed or elsewhere. The journal is on PubMed--is it perhaps because it's funded by ExxonMobil? Very strange... Jacob Hi Jacob - PubMed isn't universal - its goal is to cover the biomedical literature, so articles on the margins, like organic chemistry, materials science (even crystallography!) aren't fully covered. If you look at PubMed's total coverage of the journal Macromolecules, you'll see that it's extremely patchy: lots of articles from 1977, then almost nothing until a whole fleet of articles in 1998, then almost nothing again until 2007. Your article must have fallen into one of these coverage holes. Either that, or it's THE MAN suppressing the research needed to cure cancer and the common cold, and build a car that runs on water... :) - Matt -- Matthew Franklin, Ph. D. Senior Research Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (646) 275-7165 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] SAXS workshop
The MacCHESS BioSAXS Essentials minicourse filled up just a few days after we made the announcement over a week ago. Hopefully the overflow will find their way to Brookhaven and other places. To date, there has NOT been a lot of interest among potential organizers in holding such a course at the annual American Crystallographic Association meeting. Given the intense interest in the subject, I would like to know if any of you CCP4 readers would be interested in attending a lecture-only BioSAXS HOWTO workshop at the ACA meeting in Hawaii 2013. Please be realistic, I know many would LIKE to go ... but would you actually be able to go? I would also be interested in hearing from any interested potential organizers. (Please also keep in mind that SAS2012 is in Australia Nov 2012, so that may make a difference in your travel plans). Richard Gillilan chair-elect ACA SAXS SIG MacCHESS Cornell University Ithaca, NY On Feb 6, 2012, at 1:06 PM, Robert Sweet wrote: And here's one from Brookhaven -- app. deadline is three weeks away: http://workshops.ps.bnl.gov/default.aspx?w=SAXSMar2012 http://www.chess.cornell.edu/BioSAXS%20course/index.htm Getting Started in Biological Small-Angle X-ray Solution Scattering Feb 24-26, 2012 Students will have the opportunity to collect data on CHESS beamlines using protein standards and/or their own samples. -- = Robert M. Sweet E-Dress: sw...@bnl.gov Group Leader, PXRR: Macromolecular ^ (that's L Crystallography Research Resource at NSLSnot 1) http://px.nsls.bnl.gov/ Biology Dept Brookhaven Nat'l Lab. Phones: Upton, NY 11973631 344 3401 (Office) U.S.A. 631 344 2741 (Facsimile) =
[ccp4bb] shape complementarity
Hello, After following the discussion on [ccp4bb] shape complementarity between protein and DNA surface, is there someone here able to explain simply what the SC software of CCP4 is calculating? I mean, is there some intuitive/easy to understand explanation of what SC is calculating? I know I should read the corresponding paper, but I'd like someone to enlighten me before so I have better chances of understanding the article. Thanks, Francois.
Re: [ccp4bb] Invisible Reference on Pubmed?
This looks like one of those journals not routinely indexed by the NLM. There are some issues in Medline, but not all. I only found one article from 2004. http://www.ncbi.nlm.nih.gov/nlmcatalog/365316 John Sent from my iPad On Feb 7, 2012, at 4:02 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear CCP4BB, this is perhaps my most egregious off-topic post, but can anyone explain why the following reference is not findable in PubMed? I can get it from the ACS website, but not on PubMed or elsewhere. The journal is on PubMed--is it perhaps because it's funded by ExxonMobil? Very strange... Jacob Article A Study of the Separation Principle in Size Exclusion Chromatography AbstractFull Text HTMLHi-Res PDF[140 KB]PDF w/ Links[240 KB]FiguresCiting Articles Thomas Sun* Baytown Polymers Center, ExxonMobil Chemical Company, 5200 Bayway Dr., Baytown, Texas 77520-2101 Ronald R. Chance, William W. Graessley,† and David J. Lohse Corporate Strategic Research, ExxonMobil Research and Engineering, Annandale, New Jersey 08801 Macromolecules, 2004, 37 (11), pp 4304–4312 DOI: 10.1021/ma030586k Publication Date (Web): April 29, 2004 Copyright © 2004 American Chemical Society -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Freezing crystal
I'm going with Jurgen on this one. http://img27.imageshack.us/img27/3232/pastedgraphic1.png Sad was the day when I mounted this puppy and it shot to 8-10A. Room temperature. And messing around with cryos didn't help either. Can't remember the size, but I think I had scooped it with a 0.8 mm loop. I should've mounted it on a ring and given it to my wife. And that's not chromatic artifact, the ligand was red. On a side note, I had a very small crystal embedded in a chunk of ice at the end of a 0.025 mm loop. Couldn't even see it on the very nice on-axis cameras at the ALS. I shot blindly into the ice.. .it diffracted to about 1.8A (and the ice wasn't bad at all) F On Feb 7, 2012, at 9:52 AM, Bosch, Juergen wrote: Hi Enrico, I was just looking at non-optimal cryo-conditions and the original posters starting point. Of course if you have a good cryo bigger is better for the reasons you write but if you have no clue how your crystals will perform then I'd rather go for small to be cautious and also have those around and not only the big ones which everybody mounts because they looks so nice. To be disappointed by big crystals is often not a surprise to me and if you have not tried small crystals from the same batch well then you missed 50% of your chances to solve s structure with the first light the crystals saw. Jürgen - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Freezing crystal
Sad was the day when I mounted this puppy and it shot to 8-10A. Room temperature. And messing around with cryos didn't help either. But if that puppy had been smaller, it might have diffracted even worse, and just think if that little icy crystal had been bigger... JPK Can't remember the size, but I think I had scooped it with a 0.8 mm loop. I should've mounted it on a ring and given it to my wife. And that's not chromatic artifact, the ligand was red. On a side note, I had a very small crystal embedded in a chunk of ice at the end of a 0.025 mm loop. Couldn't even see it on the very nice on-axis cameras at the ALS. I shot blindly into the ice.. .it diffracted to about 1.8A (and the ice wasn't bad at all) F On Feb 7, 2012, at 9:52 AM, Bosch, Juergen wrote: Hi Enrico, I was just looking at non-optimal cryo-conditions and the original posters starting point. Of course if you have a good cryo bigger is better for the reasons you write but if you have no clue how your crystals will perform then I'd rather go for small to be cautious and also have those around and not only the big ones which everybody mounts because they looks so nice. To be disappointed by big crystals is often not a surprise to me and if you have not tried small crystals from the same batch well then you missed 50% of your chances to solve s structure with the first light the crystals saw. Jürgen - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] 2
...a weight is lifted off my shoulders http://www.flipperkiste.de/work.link.php?jgyahooID=65l2
Re: [ccp4bb] shape complementarity
Hi Francois Here's a one-liner. The major concept behind the Sc coefficient is that it measures the extent to which, on average, the normal vectors between closest-neighbour opposing points within the molecular interface are antiparallel. Sc=1 implies that the surfaces fit exactly, all such vectors are perfectly antiparallel. Heuristically, Sc values of 0.86 are about as good as protein-protein interfaces get (see Nature. 2005 435, pp773-8). Values below 0.65 indicate relatively poor shape complementarity. Use of a normal vector -based metric is considered superior to a distance-based metric, though Sc does have a distance-based weight applied to the normal dot products. Critical to this calculation is that the boundary of the buried molecular interface has to be discarded from the measure, as this region is intrinsically geometrically divergent. Sc is thus computed across only that part of the buried surface that might be expected to be shape complementarity, which makes it somewhat ill-suited to smaller interfaces. All these details are in the JMB paper, which, unfortunately, there is no substitute for reading :-) sincerely Mike Mike Lawrence, PhD Associate Professor and WEHI Fellow Division of Structural Biology Walter and Eliza Hall Institute of Medical Research 1G Royal Parade, Parkville Victoria 3052, AUSTRALIA Tel. 61-3-9345-2693 Fax 61-3-9345-2686 Email: lawre...@wehi.edu.au On 08/02/2012, at 12:08 PM, Francois Berenger wrote: Hello, After following the discussion on [ccp4bb] shape complementarity between protein and DNA surface, is there someone here able to explain simply what the SC software of CCP4 is calculating? I mean, is there some intuitive/easy to understand explanation of what SC is calculating? I know I should read the corresponding paper, but I'd like someone to enlighten me before so I have better chances of understanding the article. Thanks, Francois. __ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. __
Re: [ccp4bb] shape complementarity
On 02/08/2012 12:47 PM, Mike Lawrence wrote: Hi Francois Here's a one-liner. The major concept behind the Sc coefficient is that it measures the extent to which, on average, the normal vectors between closest-neighbour opposing points within the molecular interface are antiparallel. Sc=1 implies that the surfaces fit exactly, all such vectors are perfectly antiparallel. Heuristically, Sc values of 0.86 are about as good as protein-protein interfaces get (see Nature. 2005 435, pp773-8). Values below 0.65 indicate relatively poor shape complementarity. Use of a normal vector -based metric is considered superior to a distance-based metric, though Sc does have a distance-based weight applied to the normal dot products. Critical to this calculation is that the boundary of the buried molecular interface has to be discarded from the measure, as this region is intrinsically geometrically divergent. Sc is thus computed across only that part of the buried surface that might be expected to be shape complementarity, which makes it somewhat ill-suited to smaller interfaces. All these details are in the JMB paper, which, unfortunately, there is no substitute for reading :-) Thanks a lot for this very nice answer. I will definitely read your JMB article. Best regards, Francois.
[ccp4bb] My hotmail address was hacked!
Hi all,Please don't click on any links in emails from me, my hotmail password was compromised and it emailed my entire address book malicious links.Hope everyone is well!Krystle -- Krystle J. McLaughlin, Ph.D. SPIRE Postdoctoral Fellow Redinbo Group, Department of Chemistry University of North Carolina at Chapel Hill
Re: [ccp4bb] Freezing crystal
Bosch, Juergen wrote: Hi Dirk, I remember a neat paper don't recall who wrote it. I think it was in Acta D where the authors made a tiny probe the size of an elongated crystal glued to a [/Advertisement on] Hampton loop [/Advertisement off]. The probe was a temperature sensor and they recorded the cooling rate under different methods. The winner as far as I recall was freezing in liquid propane for the lack of the missing gas layer, but the second best method was LN2. Propane for whatever reason has gone extinct in certain areas of the world :-) . I'll try to find that reference but perhaps somebody else on this highly educated board knows which paper I'm referring to. I want to say it was published around 2004-2006. Not highly educated, but I remember hearing Haken Hope talk about this experiment at a workshop at SSRL- cooling a thermocouple or thermister in the cold stream vs in LN2. Maybe described here: Cryocrystallography of biological macromolecules: a generally applicable method. Hope H. Acta Crystallogr B. 1988 Feb 1;44 ( Pt 1):22-6. BTW I don't thing the greater cooling rate with ethane/propane is due to greater heat capacity so much as the fact that LN2 is used at it's boiling point: heat capacity is irrelevant since it can't absorb any more heat as a liquid, latent heat of vaporization is the reelevant parameter, but once it is vaporized the gas has low heat capacity and thermal conductivity. The liquid hydrocarbons are prepared by chilling to around their freezing point (hydrocarbon slush) so can absorb a lot of heat before any gas forms. Jürgen On Feb 7, 2012, at 11:12 AM, Dirk Kostrewa wrote: Dear Jürgen, Am 07.02.12 16:58, schrieb Bosch, Juergen: snip Then one last remark, LN2 versus cryo-stream freeze. Dipping in LN2 leads to a quicker freeze of your material. /snip Are you sure? There was a publication by Warkentin et al. [1] about a cold gas layer above liquid nitrogen that reduces the expected cooling rate a lot! My very personal experience is, that cryo-cooling in the N2-stream worked better for me than in LN2 in a variety of projects - but the reason could just be me ;-) Best regards, Dirk. [1] Matthew Warkentin, Viatcheslav Berejnov, Naji S Husseini, and Robert E Thorne: Hyperquenching for protein cryocrystallography, J. Appl. Crystallogr., 39, 805-811 (2006) -- *** Dirk Kostrewa Gene Center Munich Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax: +49-89-2180-76999 E-mail:kostr...@genzentrum.lmu.de mailto:kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de *** .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
[ccp4bb] pH optimisation for crystallisation
Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are- 1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)? 2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards, sreetama
Re: [ccp4bb] pH optimisation for crystallisation
Dear Sreetama, First of all, there are no hard-and-fast rules for successful crystallisation, try changing as many different variables as possible and go with what works. Having said that, yes, next I would go for a grid optimisation varying the pH in 0.2 or 0.5 units over as wide a range as the buffer can reasonable tolerate at the same molarity, and try different precipitant concentrations on the other axis. If you have enough protein, try plates at different temperatures as well, and different protein concentrations (in multidrop wells you can do this in the same experiment). A very important variable is the protein preparation itself, prepare more protein regularly and try to improve on purity and concentration. Mark Quoting sreetama das: Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are- 1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)? 2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards, sreetama Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es