Re: [ccp4bb] I/sigmaI of 3.0 rule
Roberto, The reviewer's request is complete nonsense. The problem is how to best and politely respond so as not to prevent the paper from being accepted. Best would be to have educated editors who could simply tell you to ignore that request. Since this issue comes up quite often still, I think we all should come up with a canned response to such a request. One way to approach this issue is to avoid saying something like the structure has been refined to 2.2Å resolution, but instead say has been refined using data to a resolution of 2.2Å., or even has been refined using data with an I/sigmaI 1.5 (or whatever). Next could be to point out that even data with an I/sigmaI of 1 can contain information (I actually don't have a good reference for this, but I'm sure someone else can provide one'), and inclusion of such data can improve refinement stability and speed of convergence (not really important in a scientific sense, though). The point is that all of your data combined result in a structure with a certain resolution, pretty much no matter what high-resolution limits you choose (I/sigmaI of 0.5, 1.0, or 1.5). As long as you don't portrait your structure of having a resolution corresponding to the resolution of the high-resolution limit of your data, you should be fine. Now, requesting to toss out data with I/sigmaI of 3 simply reduces the resolution of your structure. You could calculate two electron density maps and show that your structure does indeed improve when including data with /sigmaI of 3. One criterion could be to use the optical resolution of the structure. Hope that helps. Best, MM On Mar 3, 2011, at 6:29 AM, Roberto Battistutta wrote: Dear all, I got a reviewer comment that indicate the need to refine the structures at an appropriate resolution (I/sigmaI of 3.0), and re-submit the revised coordinate files to the PDB for validation.. In the manuscript I present some crystal structures determined by molecular replacement using the same protein in a different space group as search model. Does anyone know the origin or the theoretical basis of this I/sigmaI 3.0 rule for an appropriate resolution? Thanks, Bye, Roberto. Roberto Battistutta Associate Professor Department of Chemistry University of Padua via Marzolo 1, 35131 Padova - ITALY tel. +39.049.8275265/67 fax. +39.049.8275239 roberto.battistu...@unipd.it www.chimica.unipd.it/roberto.battistutta/ VIMM (Venetian Institute of Molecular Medicine) via Orus 2, 35129 Padova - ITALY tel. +39.049.7923236 fax +39.049.7923250 www.vimm.it
Re: [ccp4bb] pH dependent conformational change
Daniel, You'll probably have to monitor pH changes through size changes of your protein, provided the structural changes will indeed cause size changes. You said easy, so that probably rules out Small-Angle X-Ray Scattering (SAXS), but that would be the highest-resolution method. You can try static and dynamic light scattering, analytical ultracentrifugation and fluorescence anisotropy. If you are really lucky, size exclusion chromatography might work too. And then there are the difficult ways... MM On Dec 6, 2010, at 11:59 AM, Daniel Jin wrote: Dear CCP4 colleagues, We have a protein that is composed of two domains connected by a short peptide linker. We have some indirect evidence showing that the two domains may somehow move against each other when exposed to different pH. It is unlikely to have any obvious secondary structure change since each domain behaves like a rigid body. I am wondering whether there is any “easy” way, biochemically or biophysically, to monitor the conformational changes in solution. Many thanks. As far as I know most of the pH sensing stories are linked to histidine residue. Can you point me to any references that show a different pH sensing mechanism (other than His)? Thanks. Best, Daniel --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edu
Re: [ccp4bb] partial hydrogen refinement.
We have a similar case. There is difference density, but only for some of the hydrogens (mostly methyl groups on Leu, Ile, Val, Ala). How does one decide which hydrogens to include in explicit refinement? The case in question has 0.99Å data (diffraction is significantly better, but data were collected to only 0.99Å). Sorry, first time refining at that kind of resolution. Thanks! MM On Nov 23, 2010, at 3:03 AM, George M. Sheldrick wrote: Even with SHELX, at 1.2A you should use a riding model for hydrogens and not refine them freely. SHELX has a useful facility (OMIT $H or OMIT followed by specific hydrogen atom names) to keep the hydrogen atoms in the atom list but not include their contributions to the structure factors. Then you can very easily see in a difference map (e.g. with COOT) if there is density near their expected positions. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-22582 On Tue, 23 Nov 2010, Ed Pozharski wrote: Not sure if all the sirs will concur (and it might be a good idea to ask madams also), but the answer is probably no. As far as protonation state goes (guess that is what you are after, not oxidation), a better strategy may be to look into the bond lengths between the appropriate heavy atoms that are affected by it. Make sure that you refine without restraints imposed on a particular bond and see if its length is closer to the one corresponding to protonated or deprotonated state. Cheers, Ed. On Mon, 2010-11-22 at 23:18 -0600, Kenneth Satyshur wrote: Sirs: We are attempting to refine hydrogens on a ligand (which is 100 % occupied) and has ~ 40 heavy atoms (CNO). The data is 1.2 A, 325 AA, 83335 data points in C2. We have refined aniso and with H riding along (Rf= 17, R = 15) in CCP4. Can we individually refine the protons on the ligand? Let them run free with the others along for the ride? Or will they just run away at this resolution? Can CCP4 even do this? Should we switch to Shelx or Phenix? It is important to find out what the oxidation state is for the ligand at the pH we crystallized the protein and complex. thanks --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@unc.edu
Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
Lake Wobegon!!! For those outside the US and/or otherwise not familiar with that small town, check out: http://en.wikipedia.org/wiki/Lake_Wobegon Lake Wobegon, where all the women are strong, all the men are good looking, and all the children are above average The best use of modern statistical concepts in a rebuttal (or in any paper, for that matter) I have seen in a long time! I totally support starting a collection of 'hilarious' reviewers' comments and rebuttals. Our resident KuK Hofkristallograf is probably correct in trying to establish first whether such Schmaeh is legal. If it is, let the flood gates burst! MM On Oct 28, 2010, at 8:40 PM, Zbyszek Otwinowski wrote: Feel free to use it as you wish. -- DUFF, Anthony wrote: I reckon you could share hypothetical review comments for educational purposes. -Original Message- From: CCP4 bulletin board on behalf of Bernhard Rupp (Hofkristallrat a.D.) Sent: Thu 10/28/2010 12:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) Why not double open review? If I have something reasonable to say, I should be able to sign it. Particularly if the publicly purported point of review is to make the manuscript better. And imagine what wonderful open hostility we would enjoy instead of all these hidden grudges! You would never have to preemptively condemn a paper on grounds of suspicion that it is from someone who might have reviewed you equally loathful earlier. You actually know that you are creaming the right bastard! A more serious question for the editors amongst us: Can I publish review comments or are they covered under some confidentiality rule? Some of these gems are quite worthy public entertainment. Best, BR -- Zbyszek Otwinowski UT Southwestern Medical Center 5323 Harry Hines Blvd., Dallas, TX 75390-8816 (214) 645 6385 (phone) (214) 645 6353 (fax) zbys...@work.swmed.edu REVIEW_CRITERIA.pdf
Re: [ccp4bb] Introducing PDBprints - salient, at-a-glance info about PDB entries
There are so many ways to address this issue. Perhaps the simplest would be to use a combination of dimming and thick, solid borders vs. dashed borders to distinguish the two states of the icons. Cheers! MM On Jul 15, 2010, at 9:46 AM, Kevin Cowtan wrote: Better still, I can let you see them though my eyes. Here's what the icons look like to me, and a link to Vizcheck, the tool I used to generate them: http://www.ysbl.york.ac.uk/~cowtan/colour/pdb/pdb.html http://www.vischeck.com/vischeck/vischeckImage.php Running this in various modes you should be able to pick colours which work for everyone, not just for me. Flip Hoedemaeker wrote: Yep, its green-blue vs grey... Bad choice I guess? Perhaps you can provide a set of examples that work for you? Flip On 7/15/2010 13:20, Kevin Cowtan wrote: Gerard DVD Kleywegt wrote: For a five-minute illustrated introduction to PDBprints (including instructions on how to include them in your own webpages) point your browser to: http://pdbe.org/pdbprints Good idea. But the icons for published/unpublished, protein present/protein absent, nucleotide present/nucleotide absent and ligand present/ligand absent look identical to me - I have to read the alt text. Is there some colour thing going on here which is invisible to protanopes? -- EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm --- Mischa Machius, PhD Director, Center for Structural Biology Assoc. Professor, Dept. of Pharmacology Member, Lineberger Comprehensive Cancer Center University of North Carolina 4079 Genetic Medicine CB#7365 120 Mason Farm Road Chapel Hill, NC 27599-7365, U.S.A. tel: +1-919-843-4485 fax: +1-919-966-5640 email: mach...@med.unc.edu
Re: [ccp4bb] degradation of protein durring freez thaw
A couple more thoughts: 1. thermodynamics says that proteins denature at low temperatures just as they do at high temperatures. 2. flash-cooling does away with some of what thermodynamics says (not an equilibrium process anymore) 3. Whether a given protein can be frozen needs to be experimentally demonstrated before accepting such a step. In many cases, the protein won't denature, but it may well aggregate. 4. In the particular case discussed here there is another aspect. Tris has a Delta(pH)/Delta(T) of -0.03. This means that freezing a protein at -80°C may well move the pH up by 3 units, which may or may not be tolerable. So it is important what Tom said earlier in the thread: spend some time working out the buffer. Cheers! MM On Apr 22, 2010, at 1:54 AM, Jhon Thomas wrote: Hello BB I apolozize an off topic query. I am working with small proetin-protein complex of 24kDa. I purify this N-terminal His-tagged complex through tylon resin in 20mM of Tris pH-8.0, 0.3M NaCl . After purification this protein complex are dialysed in 20mM tris pH=8.0.I am able to purify enough amount of protein for crystallisation, which can be concentrated upto 10mg per ml. Then i check the dgradation on the polyacrylamide gel after concentration of the protein. I donot see any degdation protein band on the gel. I store the protein at -80 in aliquotes of 100ul immedaitely after concentration in same buffer. protein concentartion are done at 4 degree by centrifugation. Next day before setting up the trays for crystallisation screening, protein solution concentration check is being done. it turns out that this complex has degraded and concentration is only 1-2 mg per ml. i would appreciate the suggestions to prevent the degradation of complex or How should i make it more stable? so, that i can proceed for the crystallisation. I would really appreciate the suggestions. Thanks in advance Thomas
Re: [ccp4bb] Eleven plausible phasing elements remain unused
Huber's empire in Martinsried had a cabinet with ~500 compounds, many of them synthesized by himself (occasionally blowing up a lab in the process...) that in fact contained thorium, hafnium, etc. compounds. Radioactive compounds were kept in a little lead box. I am not aware of any successful derivatization with the heavy atoms you mention, but it certainly wasn't for lack of trying... Some of us went through hundreds of trials to get phases. That was in the early to mid 90's. I just checked my own dissertation and found that I had indeed used dysprosium and hafnium. Since these were not successful I should probably write them up and publish in the Journal of Failed Crystallization Experiments. Cheers - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Apr 1, 2009, at 9:21 AM, Thomas Womack wrote: A perusal of the PDB reveals that the game of Periodic Table bingo still has eleven rounds to run: scandium, titanium, germanium, zirconium, niobium, neodymium, dysprosium, thulium, hafnium, bismuth and thorium remain absent from PDB entries. OK, many of these are elements that would rather be refractory oxides or jet-engine components than hexammines, and niobium chloride clusters don't seem to be as water-stable as Ta6Br14, but why have neodymium, dysprosium and thulium so consistently been left out there in the cold rather than admitted to the warmish embrace of carboxyl groups? There must somewhere be a protein with a site that cries out for ThCl2(2+), an unexpectedly water-stable cation. Tom
Re: [ccp4bb] two identical proteins in one asymmetric unit
Having dealt with quite a few cases of more than one molecule in the AU (including a couple of dreaded 12-meric assemblies... bleah), I am still looking for the best way to identify proper NCS operators for the myriad of potential combinations of fragments. As has been said, it is generally worthwhile to identify the equivalent portions of the molecules and appropriate NCS weights, not only for potentially finding something interesting in terms of biology, but also for doing the best possible refinement job. I therefore wish there were better tools for this purpose. Overall, I think this area has not received proper attention yet. But it should, because I have the feeling that the impact of a great set of tools would be immense. Eternally hopeful - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Mar 24, 2009, at 1:22 PM, Roger Rowlett wrote: I had a student solve a medium resolution (2.3 A) data set with (unfortunately) 12 identical protein chains in the asymmetric unit. To save a little time, and to take advantage of a large amount of potential averaging we used NCS to do the initial phase of the refinement. For 10 of the 12 chains, everything was hunky-dory. For the 11th and 12th chains, however, there was an extremely messy area of high-sigma difference map density that turned out to be a very interesting ligand-binding interaction. Releasing the symmetry constraints resulted in a very sharp map of the protein chain rearrangement and bound ligand in the two different chains. In general, even with homodimers and homotetramers in the ASU, we find that there are often subtle but significant differences in individual protein chains, especially around packing contacts and external loops of the protein. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar 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...@mail.colgate.edu Skrzypczak-Jankun, Ewa wrote: I have seen proteins refined as ‘the same’, modeled to an averaged map etc only to have one of them with much higher Bj because most likely they are NOT the same so watch out by treating them as ‘the same’ you are losing the very valuable information that you might be looking for Ewa Dr Ewa Skrzypczak-Jankun Associate Professor University of ToledoOffice: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Fairman Sent: Tuesday, March 24, 2009 11:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] two identical proteins in one asymmetric unit Sang Hoon, Each molecule in the asymmetric unit is most likely different. I work on a protein that crystallizes as a homodimer with 2 molecules per asymmetric unit and there are some differences between the two (eg: electron density visible for the 14 N-terminal residues in one molecule, but not the other). Cheers, Jim On Tue, Mar 24, 2009 at 11:03 AM, Folmer Fredslund folm...@gmail.com wrote: Dear Sang They are really different! And I guess you would probably want to use NCS restraints depending on your resolution. Regards, Folmer 2009/3/24 Sang Hoon Joo s...@duke.edu: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD. -- Sang Hoon Joo, PhD Postdoctoral Associate Duke University 239 Nanaline H. Duke Box 3711, DUMC Durham, NC 27710 -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu james.fair...@case.edu
Re: [ccp4bb] generating omit maps
On Dec 10, 2008, at 9:52 AM, Mark J. van Raaij wrote: as a small variation on this, I would first finish the protein, and then include ligands, working from larger to smaller (ATP = citrate = glycerol = sulphates = waters). Sometimes several waters (from automated solvent building) in place of a bona fide ligand (or a glycerol for example) refine eerily well and give reasonable maps... Automated solvent building that includes automatic refinement should probably be banned ;) I usually add solvent molecules before adding ligands. For one, the electron density usually improves when adding solvent, so that the interpretation of the ligands becomes easier. I would recommend to check every single solvent molecule, i.e., never use automatic solvent- modeling and refinement (!) routines blindly. Make sure to remove those solvent molecules that have clearly been placed into ligand density before doing the actual refinement. Another potential problem may have to be taken into consideration as well: depending on the resolution, it can happen that protein side chains are being moved into ligand density if it is not occupied by some atoms. In such cases, I use a mixed strategy derived from the approaches described in my first post. Best - MM On 10 Dec 2008, at 16:41, Mischa Machius wrote: Kathleen - The easiest way is to simply remove the ligand from the coordinates and refine for a few cycles. Whether that is particularly meaningful is another question. Better would be to remove the ligand coordinates, shake the remaining coordinates (i.e., randomly displace them by a small amount), and then refine. Even better, perhaps, would be to calculate a simulated-annealing omit map, but AFAIK, you can't use CCP4 for that. IMHO, the best option is to not include the ligand in the model-building and refinement processes until all of the protein(s), solvent molecules, etc. have been properly modeled. I personally tend to include ligands only at the very end of the modeling/refinement process, unless there is really no ambiguity. This strategy will minimize any model bias from the ligand, and it will give you an omit map by default (until you actually include the ligand). Best - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Dec 10, 2008, at 9:30 AM, Kathleen Frey wrote: Hi Everyone, Can anyone tell me a relatively easy way to generate an omit density map for a ligand? I know that CNS can do this, but I was wondering if there's a CCP4 related program to generate omit maps. Thanks, Kathleen
Re: [ccp4bb] generating omit maps
Kathleen - The easiest way is to simply remove the ligand from the coordinates and refine for a few cycles. Whether that is particularly meaningful is another question. Better would be to remove the ligand coordinates, shake the remaining coordinates (i.e., randomly displace them by a small amount), and then refine. Even better, perhaps, would be to calculate a simulated-annealing omit map, but AFAIK, you can't use CCP4 for that. IMHO, the best option is to not include the ligand in the model-building and refinement processes until all of the protein(s), solvent molecules, etc. have been properly modeled. I personally tend to include ligands only at the very end of the modeling/refinement process, unless there is really no ambiguity. This strategy will minimize any model bias from the ligand, and it will give you an omit map by default (until you actually include the ligand). Best - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Dec 10, 2008, at 9:30 AM, Kathleen Frey wrote: Hi Everyone, Can anyone tell me a relatively easy way to generate an omit density map for a ligand? I know that CNS can do this, but I was wondering if there's a CCP4 related program to generate omit maps. Thanks, Kathleen
Re: [ccp4bb] suggestions for UV spectrometer
Tim - I would recommend a spectrometer that records entire spectra, instead of one that takes readings at just 280 nm. Contributions from light scattering can be very strong and can give results that deviate from the true value by a factor of two or more. One cannot detect scattering without recording spectra. The most severe case we have had was someone who thought the protein concentration was 10 mg/mL (based on 280) when in reality (after subtraction of the scattering contribution) it was only 4 mg/mL. Lots of other, less severe cases as well. Hope that helps. Best - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Dec 4, 2008, at 9:16 AM, Tim Gruene wrote: Dear all, we would like to purchase a UV spectrometer for measuring protein concentrations (280nm), and I would like to here your comments and especially recommendations. We don't need anything fancy, a small, fast device would be sufficient. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
Re: [ccp4bb] Crystallographic computing platform recommendations?
After having dealt, over the years, with several dozens of 'crystallographic computing platforms' and having setup and maintained quite a few myself, I would recommend to not be cheap. I would recommend to go with well supported hardware and OS. For linux, I would recommend a commercial solution, and the hardware could come from a vendor such as Dell. In our lab, we use Macs practically exclusively, except for a few legacy Linux boxes. I don't think it is worth saving a few hundred dollars when you end up spending/wasting so much time down the road assembling and fixing the machine as well as trying to keep up with the latest OS patches and drivers. I'd rather spend my time doing something else than being a computer support person. I realize I am not using the latest, greatest, pimped-out number-crunching monster, but a quad-core Mac is plenty sufficient. I like the fact that a refinement takes a few minutes longer, because that gives me time to fetch a cup of coffee or chat with a colleague. Just a thought. Best - MM Dear list, I haven't seen the crystallographic computing platform thread come up for a while, and I've got a chance to upgrade my desktop to a workstation, so I thought I'd ask the CCP4BB for advice on: 1. Mac vs. Linux (which flavor?) vs. Windows 2. Graphics cards 3. Displays 4. Processors - multiple processors, multiple cores? Speed? About half of what I do involves ~1.0 A X-ray structures - data processing, rebuilding in Coot, refinement, and so forth - so my current desktop (Optiplex GX745, Radeon X1300) machine drags on graphics sometimes. I don't seem to need stereo these days, for what it's worth. Anybody have suggestions or specs they'd like to share? Thanks in anticipation of your advice. Regards, Anna Gardberg Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] is it Ok to freeze
Ha, everyone seems to be bragging about how far back cryo- crystallography really goes. In that vain, I'd like to mention that, in Martinsried, we had a room that was lined with insulated steel walls and that could be flushed with liquid nitrogen. It was requested (demanded, really...) by Robert Huber when the Max-Planck Institute was finalized in 1972 (I hope I got my history right). That room contained an entire diffraction system. Talk about crystal cooling... bah, way too dinky. Cool the entire room! Of course, it was a hazard to work in that room, and so - as far as I know - there was only one post-doc from India how ever used it. That room had an ante-room with two more generators plus detectors that could be cooled down to -20°C! Ah, the good old Wild West times of macromolecular crystallography... Cheers - MM On Jun 19, 2008, at 11:48 AM, Pietro Roversi wrote: Well everyone, talking of early applications of cryocooling to X-ray crystallography, what about Sten Samson's marvellous helium cryostat which was operational at Caltech since the end of the 1970s and used to reach temperatures around 20 K routinely , see for example: Proc Natl Acad Sci U S A. 1982 Jul;79(13):4040-4. Structure of a B-DNA dodecamer at 16 K. Drew HR, Samson S, Dickerson RE. That instrument (and its twin) are now both with Riccardo Destro in Milano. Ciao! Pietro -- Pietro Roversi Sir William Dunn School of Pathology, Oxford University South Parks Road, Oxford OX1 3RE, England UK Tel. 0044-1865-275385 Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] is it Ok to freeze
Sadly, I have never seen the room being used. Perhaps one of the 'older' Martinsrieder on the forum has seen it. MM On Jun 19, 2008, at 12:11 PM, Klaus Futterer wrote: ... room that was lined with insulated steel walls and that could be flushed with liquid nitrogen. I'm trying to picture this ... did you guys have some kind of LN2- proof SCUBA diving equipment to work in there? Klaus - Klaus Fütterer, Ph.D. School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: [EMAIL PROTECTED] Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/ klaus/ - On 19 Jun 2008, at 18:04, Mischa Machius wrote: Ha, everyone seems to be bragging about how far back cryo- crystallography really goes. In that vain, I'd like to mention that, in Martinsried, we had a room that was lined with insulated steel walls and that could be flushed with liquid nitrogen. It was requested (demanded, really...) by Robert Huber when the Max-Planck Institute was finalized in 1972 (I hope I got my history right). That room contained an entire diffraction system. Talk about crystal cooling... bah, way too dinky. Cool the entire room! Of course, it was a hazard to work in that room, and so - as far as I know - there was only one post-doc from India how ever used it. That room had an ante-room with two more generators plus detectors that could be cooled down to -20°C! Ah, the good old Wild West times of macromolecular crystallography... Cheers - MM On Jun 19, 2008, at 11:48 AM, Pietro Roversi wrote: Well everyone, talking of early applications of cryocooling to X-ray crystallography, what about Sten Samson's marvellous helium cryostat which was operational at Caltech since the end of the 1970s and used to reach temperatures around 20 K routinely , see for example: Proc Natl Acad Sci U S A. 1982 Jul;79(13):4040-4. Structure of a B-DNA dodecamer at 16 K. Drew HR, Samson S, Dickerson RE. That instrument (and its twin) are now both with Riccardo Destro in Milano. Ciao! Pietro -- Pietro Roversi Sir William Dunn School of Pathology, Oxford University South Parks Road, Oxford OX1 3RE, England UK Tel. 0044-1865-275385 Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] Activity of a mutant enzyme compared to wild type - puzzle
I assume you are talking about a sugar-binding enzyme ;) I have some aspects to consider in addition to what Artem raises. Many effects of a mutation are not recognizable in a static crystal structure or even in an NMR structure. For example, it is usually difficult to assess the thermodynamics of substrate binding, not to mention the kinetics. Multi-valent substrates usually display some sort of cooperativity for the binding process, which you might have affected by mutating one of the subsites. You might be able to obtain some hints from a Michaelis- Menten analysis of the mutant compared to the wild type, but that would only be a start. Your crystallographic result of a less occupied substrate-binding site for the mutant serves as a hint as well, but such results are hardly conclusive. You will have to follow up with more rigorous methods, such as ITC (thermodynamics of binding) and time-resolved methods (kinetics of binding). One example of an effect of a mutation that is usually not recognizable in a crystal structure has to do with substrate guiding. In this case, the mutation has changed the surface of the protein, thus affecting how well the multi-valent substrate can approach and wiggle itself into the binding site. Once in the binding site, it is structurally virtually indistinguishable from the wild-type. Ah, the nightmares of interpreting crystal structures in terms of biology! Good luck! Best - MM On Jun 11, 2008, at 7:21 PM, Narayanan Ramasubbu wrote: Dear all: I have a single residue mutant whose enzyme activity is about 50% of the wild type. Interestingly, the mutation is in a region that involves a secondary site but not the active site. The two structures with or without ligands fit well (0.18 A) and the metal binding and cofactor binding sites are all preserved in the mutant. The one difference noticed is that the ligand does not fill the active site (partially occupied subsites) unlike the wild type where all the subsites are occupied. Water structure around the actives site residues are identical. I looked at the electrostatics and both surfaces look similar (not an expert). There are some residues whose sides chains show some positional disorder and these residues are at the edges of the active site. The resolution of the both data sets are 1.5A. The mutant enzyme was derived by MR. One another possibility that I want to look at is to compare the compactness of the two enzyme structures. What is the best way to compare that? I am wondering whether the breathing that was mentioned for some enzymes might be playing a role in the mutant enzyme. Also, I would appreciate comments on other possible explanations for this unusual (?) behavior. Thanks a lot Subbu Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
[ccp4bb] TLS, B factors, phenix and refmac
Hi - Prompted by the recent discussions on B values, TLS refinement and differences between Phenix and refmac, we looked into these matters in more detail. We found that the crux of the problem lies in the fact that TLS and B value refinements are usually decoupled. We have developed a formalism that rolls both TLS and B value refinement into one. Phenix and refmac were modified to carry out the calculations, and the outputs from both programs were made compatible to allow proper comparison of the results. We found that the stability of the refinements is now vastly improved. More importantly, however, due to the reduced number of parameters, these calculations can be carried out to resolutions of 7 Å with meaningful representations of indiviual, anisotropic atomic displacement parameters. This low-resolution limit required reformulating the calculation of Wilson B values, but that is only a minor aspect of our treatment that can be neglected. The new, combined procedure for the simultaneous refinement of TLS/B is called 'TBS' refinement, reflecting all required components: Translation, Bibation, Screw. Interestingly, the ‘T’ component is fairly insensitive to input parameters, whereas the overall quality of the refinement is greatly dependent on the ‘B’ component. The more emphasis is put on ‘B’, the more convincing the results. There is a limit, though. At very high levels of ‘B’, the so-called ‘bibacity limit’, the refinement becomes very unstable, leading to inversion in severe cases. Seasoned crystallographers familiar with the concepts can successfully push the procedure to quite high 'B limits', whereas less experienced practitioners should follow the protocols very carefully. Please contact us for any details. Best - MM Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] differences between Rsym and Rmerge
OK, that brings us back to a more substantial question: is any of these R values actually suitable to judge the quality of a given dataset? Instead of introducing novel R factors, one could also simply ignore them altogether, make sure that the error models have been properly chosen and look at I/sigma(I) as the main criterion. [QUOTE ]If anyone then still wants to present low R factors, one can always divide by 2, if necessary. [/QUOTE] Best - MM On Jan 18, 2008, at 1:02 PM, Salameh, Mohd A., Ph.D. wrote: Thank you all, it was very, very helpful discussion. However, I collected crystal data and the Rmerge overall was very high around 0.17 at 2.6A resolution and I'm wondering what is the acceptable value (range) of R-merge that worth the time to continue processing! Very anxious to hear your thoughts. Thanks, M Mohammed A. Salameh, Ph.D. Mayo Clinic Cancer Center Griffin Cancer Research Building 4500 San Pablo Road Jacksonville, FL 32224 Tel:(904) 953-0046 Fax:(904) 953-0277 [EMAIL PROTECTED] -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Chris Putnam Sent: Friday, January 18, 2008 1:21 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] differences between Rsym and Rmerge On Friday 18 January 2008 09:30:06 am Ethan A Merritt wrote: Rmerge is an average over replicate measurements of the intensity for identical [hkl]. Rsym is an average over the measurements for all symmetry equivalent reflections. In the presence of anomalous scattering, Rsym will be higher than Rmerge because the Bijvoet pairs, although symmetry related, do not have identical intensities. One might logically report two values for Rsym, one which averages over the Bijvoet-paired reflections and one which does not. This has been an eye-opening discussion for me. I've been really surprised that there's been such a diversity of opinion about what these common terms ought to refer to, and the fact that my understanding was wrong. I always thought that Rsym was an average over all symmetry equivalent reflections from the same crystal (including Bijvoet pairs) and Rmerge was properly restricted to cases of multi-crystal averaging. (My versions of Table 1's from single crystals have used Rsym rather than Rmerge.) I wonder if the problem here is that the terms have become overloaded (and hence non-specific). In that sense Rmerge is a particularly unfortunate name as every R that we're discussing is a really a merge of some sort or another. (In the most naive sense, Rmerge might be thought to be the R for whatever variation of reflection merging the experimenter chooses to do.) One possible solution would be to push the community towards a new set of terms with clearly defined meanings (and whose names would be used explicitly by new releases of MOSFLM, HKL2000, etc. and changes for new entries in the PDB). If new terms were to be adopted, they ought to specifically distinguish between single crystal and multi-crystal merging. I see three such R values that might be useful (I've arbitrarily chosen names to distinguish them from each other and the older terms): Rhkl - R of identical hkl's Rrot - R of symmetry-related hkls, but not Bijvoet pairs (rot coming from the concept that all symmetry-related reflections can be found via rotations in reciprocal space and the fact that sym has already been used) RBijvoet - R of symmetry-related and Bijvoet-related hkls (including reflections related by both rotations and an inversion center in reciprocal space) Rhkl,multi - multi-crystal version of Rhkl Rrot,multi - muti-crystal version of Rrot RBijvoet,multi - multi-crystal version of RBijvoet The downside of adopting new names is that it makes the previous literature obsolete, but I wonder if the older terms were ambiguous enough that that's not such a problem. -- Christopher Putnam, Ph.D. Assistant Investigator Ludwig Institute For Cancer Research Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] ideal bond length and bong angel
Simply use a bong for a while, and you will hear the angels sing :) It's amazing to see how this thread devolved, within two or three posts, into a discussion about drug abuse and totalitarian political regimes... Good way to start the day :) On Jan 9, 2008, at 8:35 AM, Jim Pflugrath wrote: Bong angels are probably ideal already! Please explain, so that I can teach this to my students. :) Jim Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
[ccp4bb] Coot 0.4 on Mac OS X 10.4
Y'all - I was wondering if anyone had a precompiled, stand-alone binary (Universal or Intel) of coot 0.4 for Mac OS X 10.4 that is not dependent on Fink (or anything else, for that matter)? Thanks so much. Best - MM Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] Coot 0.4 on Mac OS X 10.4
On Dec 20, 2007, at 10:26 AM, Juergen Bosch wrote: Wrong board, I see it as one unified world of structural biology. I have stopped distinguishing between the boards and preceding messages with Off topic... Pretty much anyone who is subscribed to cootbb is also subscribed ccp4bb. Anyway, the 10.5 version doesn't work on 10.4 (at least on my machine). Cheers - MM here's what Bill posted to the cootbb yesterday: Hope that solves your problem, Juergen forwarded email: Hi Folks: In response to the numerous death threats, I've tried to make a stand- alone coot for OS X 10.5 intel. http://tinyurl.com/24mchk It might work on 10.4 intel as well. I have not tried it. Everything you need is sequestered in /usr/local/coot Peace and Joy, Bill Mischa Machius wrote: Y'all - I was wondering if anyone had a precompiled, stand-alone binary (Universal or Intel) of coot 0.4 for Mac OS X 10.4 that is not dependent on Fink (or anything else, for that matter)? Thanks so much. Best - MM - --- Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 -- Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
[ccp4bb] Depositing Raw Data
from our labs. The aim is that publication occurs simultaneously with release in PDB as well as raw diffraction data on our website. We hope to house as much of our data as possible, as well as data from other Australian labs, but obviously the potential dataset will be huge, so we are trying to develop, and make available freely to the community, software tools that allow others to easily setup their own repositories. After brief discussion with PDB the plan is that PDB include links from coordinates/SF's to the raw data using a simple handle that can be incorporated into a URL. We would hope that we can convince the journals that raw data must be made available at the time of publication, in the same way as coordinates and structure factors. Of course, we realise that there will be many hurdles along the way but we are convinced that simply making the raw data available ASAP is a 'good thing'. We are happy to share more details of our IT plans with the CCP4BB, such that they can be improved, and look forward to hearing feedback cheers Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] The importance of USING our validation tools
I don't think archiving images would be that expensive. For one, I have found that most formats can be compressed quite substantially using simple, standard procedures like bzip2. If optimized, raw images won't take up that much space. Also, initially, only those images that have been used to obtain phases and to refine finally deposited structures could be archived. If the average structure takes up 20GB of space, 5,000 structures would be 1TB, which fits on a single hard drive for less than $400. If the community thinks this is a worthwhile endeavor, money should be available from granting agencies to establish a central repository (e.g., at the RCSB). Imagine what could be done with as little as $50,000. For large detectors, binning could be used, but giving current hard drive prices and future developments, that won't be necessary. Best - MM On Aug 16, 2007, at 9:13 AM, Phil Evans wrote: What do you count as raw data? Rawest are the images - everything beyond that is modellling - but archiving images is _expensive_! Unmerged intensities are probably more manageable Phil On 16 Aug 2007, at 15:05, Ashley Buckle wrote: Dear Randy These are very valid points, and I'm so glad you've taken the important step of initiating this. For now I'd like to respond to one of them, as it concerns something I and colleagues in Australia are doing: The more information that is available, the easier it will be to detect fabrication (because it is harder to make up more information convincingly). For instance, if the diffraction data are deposited, we can check for consistency with the known properties of real macromolecular crystals, e.g. that they contain disordered solvent and not vacuum. As Tassos Perrakis has discovered, there are characteristic ways in which the standard deviations depend on the intensities and the resolution. If unmerged data are deposited, there will probably be evidence of radiation damage, weak effects from intrinsic anomalous scatterers, etc. Raw images are probably even harder to simulate convincingly. After the recent Science retractions we realised that its about time raw data was made available. So, we have set about creating the necessary IT and software to do this for our diffraction data, and are encouraging Australian colleagues to do the same. We are about a week away from launching a web-accessible repository for our recently published (eg deposited in PDB) data, and this should coincide with an upcoming publication describing a new structure from our labs. The aim is that publication occurs simultaneously with release in PDB as well as raw diffraction data on our website. We hope to house as much of our data as possible, as well as data from other Australian labs, but obviously the potential dataset will be huge, so we are trying to develop, and make available freely to the community, software tools that allow others to easily setup their own repositories. After brief discussion with PDB the plan is that PDB include links from coordinates/SF's to the raw data using a simple handle that can be incorporated into a URL. We would hope that we can convince the journals that raw data must be made available at the time of publication, in the same way as coordinates and structure factors. Of course, we realise that there will be many hurdles along the way but we are convinced that simply making the raw data available ASAP is a 'good thing'. We are happy to share more details of our IT plans with the CCP4BB, such that they can be improved, and look forward to hearing feedback cheers Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] The importance of USING our validation tools
Hmm - I think I miscalculated, by a factor of 100 even!... need more coffee. In any case, I still think it would be doable. Best - MM On Aug 16, 2007, at 9:30 AM, Mischa Machius wrote: I don't think archiving images would be that expensive. For one, I have found that most formats can be compressed quite substantially using simple, standard procedures like bzip2. If optimized, raw images won't take up that much space. Also, initially, only those images that have been used to obtain phases and to refine finally deposited structures could be archived. If the average structure takes up 20GB of space, 5,000 structures would be 1TB, which fits on a single hard drive for less than $400. If the community thinks this is a worthwhile endeavor, money should be available from granting agencies to establish a central repository (e.g., at the RCSB). Imagine what could be done with as little as $50,000. For large detectors, binning could be used, but giving current hard drive prices and future developments, that won't be necessary. Best - MM On Aug 16, 2007, at 9:13 AM, Phil Evans wrote: What do you count as raw data? Rawest are the images - everything beyond that is modellling - but archiving images is _expensive_! Unmerged intensities are probably more manageable Phil On 16 Aug 2007, at 15:05, Ashley Buckle wrote: Dear Randy These are very valid points, and I'm so glad you've taken the important step of initiating this. For now I'd like to respond to one of them, as it concerns something I and colleagues in Australia are doing: The more information that is available, the easier it will be to detect fabrication (because it is harder to make up more information convincingly). For instance, if the diffraction data are deposited, we can check for consistency with the known properties of real macromolecular crystals, e.g. that they contain disordered solvent and not vacuum. As Tassos Perrakis has discovered, there are characteristic ways in which the standard deviations depend on the intensities and the resolution. If unmerged data are deposited, there will probably be evidence of radiation damage, weak effects from intrinsic anomalous scatterers, etc. Raw images are probably even harder to simulate convincingly. After the recent Science retractions we realised that its about time raw data was made available. So, we have set about creating the necessary IT and software to do this for our diffraction data, and are encouraging Australian colleagues to do the same. We are about a week away from launching a web-accessible repository for our recently published (eg deposited in PDB) data, and this should coincide with an upcoming publication describing a new structure from our labs. The aim is that publication occurs simultaneously with release in PDB as well as raw diffraction data on our website. We hope to house as much of our data as possible, as well as data from other Australian labs, but obviously the potential dataset will be huge, so we are trying to develop, and make available freely to the community, software tools that allow others to easily setup their own repositories. After brief discussion with PDB the plan is that PDB include links from coordinates/SF's to the raw data using a simple handle that can be incorporated into a URL. We would hope that we can convince the journals that raw data must be made available at the time of publication, in the same way as coordinates and structure factors. Of course, we realise that there will be many hurdles along the way but we are convinced that simply making the raw data available ASAP is a 'good thing'. We are happy to share more details of our IT plans with the CCP4BB, such that they can be improved, and look forward to hearing feedback cheers -- -- Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] The importance of USING our validation tools
Due to these recent, highly publicized irregularities and ample (snide) remarks I hear about them from non-crystallographers, I am wondering if the trust in macromolecular crystallography is beginning to erode. It is often very difficult even for experts to distinguish fake or wishful thinking from reality. Non-crystallographers will have no chance at all and will consequently not rely on our results as much as we are convinced they could and should. If that is indeed the case, something needs to be done, and rather sooner than later. Best - MM Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] off-topic Apple computer question
drives failed within a few weeks of one another, making me wonder if they were really built by Ford. The second one came back from Apple today with a snotty message saying that the third-party memory had caused the problem and that they will refuse to do a repair if we ever send them a computer in the future with a third- party memory chip in it. This strikes me as absolute horse-hockey, but then again, maybe I am not aware of something I should be. Thanks. Bill Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
Re: [ccp4bb] off-topic Apple computer question
We have a similar deal here, but my understanding is that if the actual cost of parts exceeds $329, you get charged the difference. Since it was a hard drive, I didn't get to test the hypothesis. If the repair for a 2 year old laptop is much more than that, there is no point in doing the repair. I just inquired: it's a flat rate of $329 in all cases, except when the LCD panel needs to be replaced. Cheers - MM Mischa Machius, PhD Associate Professor UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353
