[ccp4bb] on Rwork and Rfree

2012-02-07 Thread Dialing Pretty
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,  

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Mark J van Raaij
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

Re: [ccp4bb] Fwd: tcl tk RHEL 6.2

2012-02-07 Thread Kay Diederichs
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.

[ccp4bb] mutagenesis based on ligand-bound crystal structures

2012-02-07 Thread Chris Ulens
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

Re: [ccp4bb] mutagenesis based on ligand-bound crystal structures

2012-02-07 Thread Savvas Savvides
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.

Re: [ccp4bb] on Rwork and Rfree

2012-02-07 Thread Eleanor Dodson
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

Re: [ccp4bb] on Rwork and Rfree

2012-02-07 Thread Robbie Joosten
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

Re: [ccp4bb] shape complementarity between protein and DNA surface

2012-02-07 Thread Tim Gruene
-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

[ccp4bb] Fwd: TR: Permanent Scientist at beamline SWING

2012-02-07 Thread Louis RENAULT
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,

[ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread 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

Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]

2012-02-07 Thread Fred
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Clement Angkawidjaja
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Jacob Keller
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

Re: [ccp4bb] [RESUME] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]

2012-02-07 Thread Xiaodi Yu
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Xiaodi Yu
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Roger Rowlett
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Bosch, Juergen
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

Re: [ccp4bb] on Rwork and Rfree

2012-02-07 Thread Pavel Afonine
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:

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Kevin Jin
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Fischmann, Thierry
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]

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Fischmann, Thierry
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Enrico Stura
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Christian Roth
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Bosch, Juergen
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Hong Zhang
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

[ccp4bb] Merging hi and low res data

2012-02-07 Thread Nicholas Keep
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.

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Kevin Jin
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Dirk Kostrewa
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Bosch, Juergen
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Francisco Hernandez-Guzman
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Jim Pflugrath
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

2012-02-07 Thread David Schuller
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Enrico Stura
BIGGER is almost always BETTER if you spend the time to do your experiment well, and in particular freezing (flash-cooling) your crystal after an appropriate soak in a good cryo-mixture that is respectful of your crystal. Thierry, X-ray absorption, varies with the wavelength, and by going

Re: [ccp4bb] Freezing crystal (Liquid Propane Crystal Prep)

2012-02-07 Thread Steven Herron
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

[ccp4bb] Staff scientist position at MacCHESS

2012-02-07 Thread Marian Szebenyi
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Zachary Wood
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+).

Re: [ccp4bb] Merging hi and low res data

2012-02-07 Thread Kay Diederichs
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Kevin Jin
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,

[ccp4bb] Invisible Reference on Pubmed?

2012-02-07 Thread Jacob Keller
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Roger Rowlett
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

Re: [ccp4bb] On pKa of Aspartic acid

2012-02-07 Thread Horacio Botti
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

Re: [ccp4bb] Invisible Reference on Pubmed?

2012-02-07 Thread Matthew Franklin
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

[ccp4bb] Generating parameters/cif files for macrocyclic ligands

2012-02-07 Thread Joel Tyndall
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

Re: [ccp4bb] No diffraction

2012-02-07 Thread Jesse
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:

Re: [ccp4bb] Invisible Reference on Pubmed?

2012-02-07 Thread Jacob Keller
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

Re: [ccp4bb] SAXS workshop

2012-02-07 Thread Richard Gillilan
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

[ccp4bb] shape complementarity

2012-02-07 Thread Francois Berenger
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

Re: [ccp4bb] Invisible Reference on Pubmed?

2012-02-07 Thread John Newitt
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Francis E Reyes
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Jacob Keller
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

Re: [ccp4bb] 2

2012-02-07 Thread Krystle Williams McLaughlin
...a weight is lifted off my shoulders http://www.flipperkiste.de/work.link.php?jgyahooID=65l2

Re: [ccp4bb] shape complementarity

2012-02-07 Thread Mike Lawrence
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

Re: [ccp4bb] shape complementarity

2012-02-07 Thread Francois Berenger
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

[ccp4bb] My hotmail address was hacked!

2012-02-07 Thread Krystle Williams McLaughlin
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

Re: [ccp4bb] Freezing crystal

2012-02-07 Thread Edward A. Berry
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

[ccp4bb] pH optimisation for crystallisation

2012-02-07 Thread 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

Re: [ccp4bb] pH optimisation for crystallisation

2012-02-07 Thread VAN RAAIJ , MARK JOHAN
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