[ccp4bb] Post-doctoral position at Umeå University, Sweden
Dear All, A two year post-doctoral position (stipend) is available in the laboratory of Karina Persson at Umea University, Sweden. The project will involve structural characterizations of adhesins and associated proteins from Gram-positive bacteria using X-ray crystallographic methods. The ideal applicant has extensive experience in macromolecular crystallography, protein molecular biology, biochemistry, crystallization and structure determination. Closing date is May 02, 2012. I am looking forward to receiving your application! /Karina Persson For detailed information and application instructions, please visit http://www.umu.se/english/about-umu/news-events/grants/223-944-12 For informal enquiries please contact karina.pers...@chem.umu.se -- Dr. Karina Persson Umea Centre for Molecular Research, UCMR Centre for Chemical Biology, KBC Department of Chemistry Umea University, Linnaeus vag 6, SE-901 87 Umea, Sweden Tel: +46-(0)90-786-5926 e-mail: karina.pers...@chem.umu.semailto:uwe.sa...@chem.umu.se
Re: [ccp4bb] COOT Real Space Refinement keyboard shortcut??
Dear Chris Are you trying to use the standard key binding a refine with auto-zone? Mine does not work whilst others such as m and n for zooming out and in do. For a I get the following message in the terminal: (graphics-general-key-press-hook 97) Key 97 not found in (scheme) key bindings Any ideas on how to fix this, anyone? I am using Coot 0.6.2 on CentOS 2.6.32-71.29.1.el6.x86_64 x86_64 Regards Petra -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Christopher Browning Sent: 27 March 2012 15:45 To: ccp4bb Subject: [ccp4bb] COOT Real Space Refinement keyboard shortcut?? Hi, I was wondering whether there was a way to assign a keyboard shortcut to the real space refine button in COOT. Atleast this way one does not have to keep looking back to where the real space refine button is to click it if you want to carry out the refinement. Cheers, Chris -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40
Re: [ccp4bb] Yeast plasmid DNA miniprep problem
Hi Zhen, I also had problems with low amounts of plasmid from yeast using 5 ml o/n cultures. But when increasing the culture volume it worked: Large-scale (0.51 l for obtaining preparative plasmid amounts) overnight yeast cultures were harvested, washed in cold water once, and then resuspended in Zymolyase incubation buffer (0.1 M sodium phosphate buffer pH 7.4, 2 mM DTT, and 510 mg/ml of Zymolyase with 1.2 M sorbitol). The yeast cell wall was digested for 30 min at 37°C, cells were then lysed by alkaline lysis as (described in: Singh MV, Weil PA. A method for plasmid purification directly from yeast. Anal Biochem.2002;307:1317). 0.51 µl of this preparation was used to transform chemically competent E. coli. Cheers Phil Von: Zhang, Zhen zhen_zh...@dfci.harvard.edu Antworten an: Zhang, Zhen zhen_zh...@dfci.harvard.edu Datum: Thu, 12 Apr 2012 15:32:27 + An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Yeast plasmid DNA miniprep problem Sorry for the non-ccp4 question. I am having trouble to miniprep plasmid from yeast. The plasmid is pCTCON2 and the yeast strain is EBY100. I am using Zymoprep Yeast Plasmid Miniprep II kit. I grow the culture in SDCAA medium to OD0.3 and spin down 1.5ml cells. The pellet was resuspended in solution 1 supplemented with Zymolyase and was incubated for 1 hr. I followed the manual exactly for the rest steps. However, I have never been able to recover any plasmid. There is nothing or smear in the agarose gel and there is no colony after transformation to DH5alpha after one overnight incubation at 37. The existence of the plasmid in the yeast has been confirmed by the ability to grow in the nutrition deficient medium SDCAA and the expression of plasmid protein in SGCAA medium was observed by FACS. I am wondering if someone here can shine some light on this issue. Any thoughts or suggestions are welcome. Thank you. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.
Re: [ccp4bb] COOT Real Space Refinement keyboard shortcut??
Are you trying to use the standard key binding a refine with auto-zone? Mine does not work whilst others such as m and n for zooming out and in do. For a I get the following message in the terminal: (graphics-general-key-press-hook 97) Key 97 not found in (scheme) key bindings Any ideas on how to fix this, anyone? The activation order is: 1) click Real Space Refine icon 2) click atom at in the residue at the centre of range (and centre of screen, typically) 3) press a (i.e. the A key) This was built into Coot pre-user-defined-key-bindings. I rarely, if ever, use it now that there is the t short-cut. In fact, as I write this, I think that I might well take it away so that A can be used for something else instead. Paul.
[ccp4bb] Postdoctoral position in DNA replication/repair at the LMB, Cambridge
Dear all, We have a postdoctoral position available for a talented and highly motivated individual to undertake single molecule microscopy studies on bacterial DNA replication and DNA repair. The lab focuses on the structural aspects of DNA replication and translesion synthesis DNA repair, using X-ray crystallography, SAXS and electron microscopy, in combination with different biochemical and biophysical techniques. Our institute is provided with state of the art microscopy equipment and a large community of scientist using single molecule microscopy. Closing date: 22 April 2012 For a full description see: www.nature.com/naturejobs/science/jobs/253545-Career-Development-Fellow Meindert -- ** Meindert H. Lamers, PhD Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, CB2 0QH United Kingdom tel +44 (0)1223 402401 fax +44 (0)1223 213556 web: http://www2.mrc-lmb.cam.ac.uk/groups/mlamers/ **
[ccp4bb] wwPDB and CCDC
I remember reading somewhere (or maybe at a seminar presentation?) of an agreement between the wwPDB and the CCDC. I can't find a reference to it on the web - is there such a thing? Thanks (even lmgtfy answers happily received), Paul.
Re: [ccp4bb] wwPDB and CCDC
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Paul, you are not referring to Gerard Bricogne's announcement for the grade-server, are you? http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg25770.html If not - what type of agreement do you have in mind? Tim On 04/13/12 15:14, Paul Emsley wrote: I remember reading somewhere (or maybe at a seminar presentation?) of an agreement between the wwPDB and the CCDC. I can't find a reference to it on the web - is there such a thing? Thanks (even lmgtfy answers happily received), Paul. - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPiCp4UxlJ7aRr7hoRAvHYAKCjMHptUN8rAm+81cQVhbufG762twCeJFCr mtWehE4IFdFstz+NLSu8Gvo= =br0j -END PGP SIGNATURE-
Re: [ccp4bb] wwPDB and CCDC
On 13/04/12 14:30, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Paul, you are not referring to Gerard Bricogne's announcement for the grade-server, are you? http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg25770.html :) No. If not - what type of agreement do you have in mind? IIRC, I remember a picture of Gerard (K), Helen, Haruki and a smiling Colin Groom sitting along a table signing something. I was wondering if more details are available on the web. Paul.
Re: [ccp4bb] Crystal behave funny
Hi Prem, The first thing I would do is to make certain that you really have prot+DNA crystals, and not DNA alone. If you can isolate enough crystals (you may need 15 or 30, depending on how large they are), SDS page would be informative. Run protein and DNA alone and together in the same gel as controls. This could save you a lot of time/effort since you don't want to optimize DNA-only crystals. In any case, if it is the complex and still doesn't diffract well, I'd just start changing the DNA, both in length and sequence (the bases that don't matter for protein binding). Good luck, Greg On Apr 13, 2012, at 6:51 AM, Prem kumar wrote: Hi all, I got some Protein + DNA complex crystals (image attached) recently. They are needle shape some times splitted chromosome type crystals. When we pick long needles they bend so much than normal crystal but they dont break. The small needle dissolve very fast as try to open the drop's film. we try to diffract the long needle crystals and they diffract up to 20 A resolution. Any suggestion how to improve those crystal packing. Thanks in advance! -Prem IMG_1438.JPG -- Department of Biophysics Johns Hopkins University 302 Jenkins Hall 3400 N. Charles St. Baltimore, MD 21218 Phone: (410) 516-7850 (office) Phone: (410) 516-3476 (lab) gdbow...@jhu.edu http://www.jhu.edu/bowmanlab
Re: [ccp4bb] wwPDB and CCDC
Hi Paul, What you might be remembering is the following paragraph from our Validation Task Force report: The Cambridge Crystallographic Data Centre (CCDC) has recently entered into collaboration with the wwPDB partners. As part of the agreement, wwPDB will have access to Mogul, which will be integrated into the wwPDB validation pipeline, and to the experimental coordinates of ligands that are or have been deposited in the PDB and that also occur in the CSD. Taken together, this means that high-resolution reference coordinates will become available for many ligands in the PDB and that high-quality geometry-validation reports can be generated for all ligands that will be deposited in the future. Regards, Randy On 13 Apr 2012, at 15:29, Paul Emsley wrote: On 13/04/12 14:30, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Paul, you are not referring to Gerard Bricogne's announcement for the grade-server, are you? http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg25770.html :) No. If not - what type of agreement do you have in mind? IIRC, I remember a picture of Gerard (K), Helen, Haruki and a smiling Colin Groom sitting along a table signing something. I was wondering if more details are available on the web. Paul. -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] wwPDB and CCDC
Hi Paul, You saw the wwPDB/CCDC JPG in my PPT at GSK :-) Yes, wwPDB and CCDC have signed an MoU. In pounds and pennies it means, amongst a number of other things, that wwPDB will be allowed to use Mogul in its validation pipeline and that wwPDB will be allowed to incorporate and redistribute CSD coordinates of small molecules that occur in both PDB and CSD. --Gerard (K) On Fri, 13 Apr 2012, Paul Emsley wrote: On 13/04/12 14:30, Tim Gruene wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Paul, you are not referring to Gerard Bricogne's announcement for the grade-server, are you? http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg25770.html :) No. If not - what type of agreement do you have in mind? IIRC, I remember a picture of Gerard (K), Helen, Haruki and a smiling Colin Groom sitting along a table signing something. I was wondering if more details are available on the web. Paul. Best wishes, --Gerard ** Gerard J. Kleywegt http://xray.bmc.uu.se/gerard mailto:ger...@xray.bmc.uu.se ** The opinions in this message are fictional. Any similarity to actual opinions, living or dead, is purely coincidental. ** Little known gastromathematical curiosity: let z be the radius and a the thickness of a pizza. Then the volume of that pizza is equal to pi*z*z*a ! **
Re: [ccp4bb] Crystal behave funny
I'd suggest: - Find a dinosaur from my generation who can suck one into a capillary and check diffraction at room T. - Try using those loops that look like miniature tennis paddles to give the crystal a little more support - To minimize strain on the crystal when pulling it out of the drop, try to get its long dimension perpendicular to the air-water interface (usually easier said than done). - Try to find conditions where the crystals don't start to redissolve while you mount them Original message Date: Fri, 13 Apr 2012 18:51:58 +0800 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Prem kumar pk_ai...@yahoo.com) Subject: [ccp4bb] Crystal behave funny To: CCP4BB@JISCMAIL.AC.UK Hi all, I got some Protein + DNA complex crystals (image attached) recently. They are needle shape some times splitted chromosome type crystals. When we pick long needles they bend so much than normal crystal but they dont break. The small needle dissolve very fast as try to open the drop's film. we try to diffract the long needle crystals and they diffract up to 20 A resolution. Any suggestion how to improve those crystal packing. Thanks in advance! -Prem IMG_1438.JPG (2343k bytes)
Re: [ccp4bb] COOT Real Space Refinement keyboard shortcut??
A works as described but t is better! In coot 0.6.2 I added the line (add-key-binding Triple Refine t (lambda () (manual-refine-residues 1))) to the file ~/.coot-preferences/coot-preferences.scm Many thanks Petra From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Paul Emsley [paul.ems...@bioch.ox.ac.uk] Sent: 13 April 2012 13:20 To: ccp4bb Subject: Re: [ccp4bb] COOT Real Space Refinement keyboard shortcut?? Are you trying to use the standard key binding a refine with auto-zone? Mine does not work whilst others such as m and n for zooming out and in do. For a I get the following message in the terminal: (graphics-general-key-press-hook 97) Key 97 not found in (scheme) key bindings Any ideas on how to fix this, anyone? The activation order is: 1) click Real Space Refine icon 2) click atom at in the residue at the centre of range (and centre of screen, typically) 3) press a (i.e. the A key) This was built into Coot pre-user-defined-key-bindings. I rarely, if ever, use it now that there is the t short-cut. In fact, as I write this, I think that I might well take it away so that A can be used for something else instead. Paul.
[ccp4bb] Rigaku R-axis IIC X-ray diffraction system
Dear all, Do you know is anyone still using Rigaku R-axis IIC X-ray diffraction system with support services? It seems Rigaku no longer support this systems. Thank you in advance. Best Regards, Leong
Re: [ccp4bb] Crystal behave funny
- Find a dinosaur from my generation who can suck one into a capillary and check diffraction at room T. - Try to find conditions where the crystals don't start to redissolve while you mount them As a matter of fact, people begin to forget that capillaries are good not only for checking the diffraction, but also for the data collection. However, the laws of Nature do not change with time, and the old dinosaur ways may still work. We just collected at SSRL a room T. data set from crystals that consisted by 75% v/v of water and 25% of a 2500 aa protein complex. The crystals did not like cryoprotectants (including Paratone), but were happy in the capillaries. On Apr 13, 2012, at 8:32 AM, Phoebe Rice wrote: I'd suggest: - Find a dinosaur from my generation who can suck one into a capillary and check diffraction at room T. - Try using those loops that look like miniature tennis paddles to give the crystal a little more support - To minimize strain on the crystal when pulling it out of the drop, try to get its long dimension perpendicular to the air-water interface (usually easier said than done). - Try to find conditions where the crystals don't start to redissolve while you mount them Original message Date: Fri, 13 Apr 2012 18:51:58 +0800 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Prem kumar pk_ai...@yahoo.com) Subject: [ccp4bb] Crystal behave funny To: CCP4BB@JISCMAIL.AC.UK Hi all, I got some Protein + DNA complex crystals (image attached) recently. They are needle shape some times splitted chromosome type crystals. When we pick long needles they bend so much than normal crystal but they dont break. The small needle dissolve very fast as try to open the drop's film. we try to diffract the long needle crystals and they diffract up to 20 A resolution. Any suggestion how to improve those crystal packing. Thanks in advance! -Prem IMG_1438.JPG (2343k bytes)
Re: [ccp4bb] Crystal behave funny
Hi Prem, In addition to other remarks made: - You could dissolve one or more crystals in water and have mass spec done to verify that your crystals are a complex. It takes many crystals (20-30) to make sure on an SDS-PAGE. You will probably need to silver stain the gel to enhance the sensitivity. And optimize for visualizing DNA (of course protein and DNA would each be control lanes). - Apart from optimizing your DNA length and overhang as suggested, you could also try to see what a detergent does for you. My experience is that they can dramatically improve the crystal quality for protein-DNA complexes. But you first need to know if the crystal consists of both protein and DNA. I am optimistic about the probability. Mark (who apparently is also a dinosaur because he practices room temperature crystallography) -Original Message- From: Prem kumar pk_ai...@yahoo.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Fri, Apr 13, 2012 5:10 am Subject: [ccp4bb] Crystal behave funny Hi all, I got some Protein + DNA complex crystals (image attached) recently. They are needle shape some times splitted chromosome type crystals. When we pick long needles they bend so much than normal crystal but they dont break. The small needle dissolve very fast as try to open the drop's film. we try to diffract the long needle crystals and they diffract up to 20 A resolution. Any suggestion how to improve those crystal packing. Thanks in advance! -Prem
Re: [ccp4bb] Crystal behave funny
Considering myself a meso-dinosaur, I would suggest to simply grow your crystals in capillaries and then wick off the liquid and shoot them like this without much handling. A very efficient and simple trick is to stick a capillary in an Eppendorf tube with the lid on using the Eppendorf tube as a reservoir. I admit this method was used before I was born but works well, not quite high throughput though and need some training in handling etc. As a tip use a syringe to poke a hole into the Eppendorf tube and then wiggle until the capillary fits. Jürgen On Apr 13, 2012, at 1:49 PM, mjvdwo...@netscape.netmailto:mjvdwo...@netscape.net wrote: Hi Prem, In addition to other remarks made: - You could dissolve one or more crystals in water and have mass spec done to verify that your crystals are a complex. It takes many crystals (20-30) to make sure on an SDS-PAGE. You will probably need to silver stain the gel to enhance the sensitivity. And optimize for visualizing DNA (of course protein and DNA would each be control lanes). - Apart from optimizing your DNA length and overhang as suggested, you could also try to see what a detergent does for you. My experience is that they can dramatically improve the crystal quality for protein-DNA complexes. But you first need to know if the crystal consists of both protein and DNA. I am optimistic about the probability. Mark (who apparently is also a dinosaur because he practices room temperature crystallography) -Original Message- From: Prem kumar pk_ai...@yahoo.commailto:pk_ai...@yahoo.com To: CCP4BB CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Sent: Fri, Apr 13, 2012 5:10 am Subject: [ccp4bb] Crystal behave funny Hi all, I got some Protein + DNA complex crystals (image attached) recently. They are needle shape some times splitted chromosome type crystals. When we pick long needles they bend so much than normal crystal but they dont break. The small needle dissolve very fast as try to open the drop's film. we try to diffract the long needle crystals and they diffract up to 20 A resolution. Any suggestion how to improve those crystal packing. Thanks in advance! -Prem .. 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] Postdoctoral position in structural and chemical immunology
The laboratory of Dr. Yoonsang Cho in the Department of Biochemistry and Molecular Biology at Saint Louis University School of Medicine invites a highly motivated and creative postdoctoral fellow who is enthusiastic to study ligand-receptor systems involved in inflammatory diseases and cancer. The successful candidate is expected to have strong cell biology background and will be encouraged to mainly work on a NIH-funded project to elucidate the molecular mechanisms of the interaction between a chemokine-like protein and chemokine receptors and its regulation by small molecules. The project will be highly collaborative with medicinal chemists and immunologists for designing and synthesizing new small molecules based on selected hits from high throughput screening of small molecule libraries and evaluating their therapeutic effects in animal models of human diseases. Depending on the candidate’s experience and interest, X-ray crystallography, NMR, enzyme kinetics, and high throughput screening studies will be conducted concomitantly. The laboratory is located in the state of the art Doisy Research Center (DRC) in the medical school. We have an innovative and powerful label-free biosensor-based cellular assay system for the quantitative measurement of cellular response in real time, an exclusive tissue culture room, liquid chromatography purification system, and Macintosh and Linux workstations. The department has three R-Axis IV++ imaging plate systems for high-resolution X-ray diffraction data collection, a crystallization robot, molecular graphics facility, differential scanning calorimeter, isothermal titration calorimeter, dynamic light scattering, metabolomics core facility, and bioinformatics facility. In the same building, are animal care facility, flow cytometry research core, animal imaging core, microarray core, research microscopy and confocal core, and the Center for World Health and Medicine, which provides a full range of support for drug discovery programs including design and synthesis of small molecules and their preclinical characterization. Candidates must hold a Ph.D. in immunology, cell biology, biochemistry or a related field. Solid knowledge and hands-on experience in signaling studies with immune cells expressing cytokine/chemokine receptors is essential. Structural and chemical biology experience is a plus. He or she must have excellent skills in oral and written English as well as inherent ability to organize experimental data for efficient communication and publication. Applicants should send a cover letter with their brief scientific background and motivation for the position along with a complete CV, including research experience and the names and contact information of three references to Yoonsang Cho via email. Please feel free to make informal inquiries via email. Yoonsang Cho, Ph.D. Assistant Professor of Biochemistry and Molecular Biology Saint Louis University School of Medicine 1100 South Grand Avenue St. Louis, MO 63104 Email: yc...@slu.edu URL: http://biochemweb.slu.edu/people/faculty/cho.shtml
Re: [ccp4bb] Disorder or poor phases?
Francis, I think in the cases you describe the region in question is disordered. Time and time again I have users coming to my beamline wanting to clean up a questionable region by getting experimental phases. Ahh! If only I had a nickle for each one. Oh wait, I suppose I kind of do? I take that back! Go MAD everyone! Much as I hate to discourage people from using my favorite technique, Tim is right: phases are not region-specific in electron density maps. Dale does make a good point that there is such a thing as model bias and one can argue that experimental phases don't have it. But, this is only true if you have not yet applied solvent flattening. How long has it been since you looked at a raw experimentally-phased map (before solvent flattening)? I'm willing to bet a while. With very few exceptions, raw experimental phases are lousy. We have actually become quite dependent on density modification to clean them up. In fact, solvent flattening is the only reason why SAD works at all. However, you CAN use anomalous differences to clear up disordered regions in a different way. Something I started calling SeMet scanning a number of years ago. A few of my users have done this, and a good example of it is Figure 3 of Huang et al. 2004 (doi:10.1038/nsmb826). Basically, you mutate residues in the disordered region one at a time to SeMet, and look at phased anomalous difference Fourier (PADF) maps. These maps are surprisingly clear, even when the anomalous difference signal is so weak as to make experimental phasing hopeless. Yes, the best phases to use for PADF maps are model phases, but, as always, it is prudent to refine the model after omitting the thing you are looking for before calculating such phases. Another way to get residue-specific labeling for low-resolution chain tracing is radiation damage. If you expose for the right amount of time, Asp and Glu side chains will be specifically burnt off, but not Asn and Gln. You will also see Met loosing its head, etc. So, as long as you have read Burmeister (2000), an Fo-Fo map of damaged vs undamaged can be used to guide sequence assignment, even at 4.5 A and worse. Anyway, when it comes to the question of is it disordered or is it model bias?, I think it is usually the former. It is very difficult to make model bias suppress a region that is actually well-ordered. Try it! After all, this is the whole reason why we bother looking at fo-fc maps. Then again, it is always possible to have a model so bad that the phase error is enough to squash anything. An excellent example of this can be found in the Book of Fourier. Taking amplitudes from the image of a cat, you can see what happens when you use the phases of a duck: http://www.ysbl.york.ac.uk/~cowtan/fourier/picduckcatfft.gif as opposed to what happens if you use the phases of a manx: http://www.ysbl.york.ac.uk/~cowtan/fourier/piccatmanx2.gif A manx is a species of cat that doesn't have a tail, so no animals were harmed in obtaining these phases. My point here is that the cat's tail can be seen quite readily in the 2fo-fc map if most of the structure is already right, but if your model is completely unrelated to the true structure (fitting a duck into a cat-shaped hole), then everything is in the noise. Real structures are usually somewhere between these two extremes, and I think an important shortcoming in modern crystallography is that we don't have a good quantitative description of this middle-ground. We all like to think we know what model bias is, but we don't exactly have units for it. Should we be using a scale of 0 to 1? Or perhaps duck to cat? Yes, I know we have figure of merit, but FOM is not region-specific. In my experience, as long as you have ~50% of the electrons in the right place (and none of them in the wrong place), then you can generally trust that the biggest difference feature in the fo-fc map is real, and build from there. As the model becomes more complete, the phases should continue to get better, not worse. Eventually, this does break down, although I'm not really sure why. With small molecules, the maximum fo-fc peak keeps getting bigger (on a sigma scale) as you add more and more atoms, and the biggest one you will ever see is the last one. For macromolecules, the difference features keep getting smaller and smaller as you build. Perhaps small errors (like non-Gaussian atomic displacement distributions being modeled as Gaussians) slowly accumulate? Perhaps there are other sources of systematic error that we don't yet fully understand? Eventually, for whatever reason, you stop building. Having electrons in the wrong place is about twice as bad as not having them at all, which I think is why we trim models so aggressively for molecular replacement, and also why we are so reticent to model in things that we are not sure about. Disordered regions, of course, will
Re: [ccp4bb] Disorder or poor phases?
a recent experience in our lab with molecular replacement (wt and disordered point mutant; same space group and unit cell) was solved with a combination of two methods. 1. We made omit maps in the disordered region at several lower resolutions. The region became interpretable after suffereing through these maps, building residue by residue and refinement. 2. Then we had the bright idea to make Fwt-Fmutant maps to confirm our interpretation. Happily this map did confirm the unexpected large structural changed caused by a point mutant. On Fri, Apr 13, 2012 at 1:31 PM, James Holton jmhol...@lbl.gov wrote: Francis, I think in the cases you describe the region in question is disordered. Time and time again I have users coming to my beamline wanting to clean up a questionable region by getting experimental phases. Ahh! If only I had a nickle for each one. Oh wait, I suppose I kind of do? I take that back! Go MAD everyone! Much as I hate to discourage people from using my favorite technique, Tim is right: phases are not region-specific in electron density maps. Dale does make a good point that there is such a thing as model bias and one can argue that experimental phases don't have it. But, this is only true if you have not yet applied solvent flattening. How long has it been since you looked at a raw experimentally-phased map (before solvent flattening)? I'm willing to bet a while. With very few exceptions, raw experimental phases are lousy. We have actually become quite dependent on density modification to clean them up. In fact, solvent flattening is the only reason why SAD works at all. However, you CAN use anomalous differences to clear up disordered regions in a different way. Something I started calling SeMet scanning a number of years ago. A few of my users have done this, and a good example of it is Figure 3 of Huang et al. 2004 (doi:10.1038/nsmb826). Basically, you mutate residues in the disordered region one at a time to SeMet, and look at phased anomalous difference Fourier (PADF) maps. These maps are surprisingly clear, even when the anomalous difference signal is so weak as to make experimental phasing hopeless. Yes, the best phases to use for PADF maps are model phases, but, as always, it is prudent to refine the model after omitting the thing you are looking for before calculating such phases. Another way to get residue-specific labeling for low-resolution chain tracing is radiation damage. If you expose for the right amount of time, Asp and Glu side chains will be specifically burnt off, but not Asn and Gln. You will also see Met loosing its head, etc. So, as long as you have read Burmeister (2000), an Fo-Fo map of damaged vs undamaged can be used to guide sequence assignment, even at 4.5 A and worse. Anyway, when it comes to the question of is it disordered or is it model bias?, I think it is usually the former. It is very difficult to make model bias suppress a region that is actually well-ordered. Try it! After all, this is the whole reason why we bother looking at fo-fc maps. Then again, it is always possible to have a model so bad that the phase error is enough to squash anything. An excellent example of this can be found in the Book of Fourier. Taking amplitudes from the image of a cat, you can see what happens when you use the phases of a duck: http://www.ysbl.york.ac.uk/~cowtan/fourier/picduckcatfft.gif as opposed to what happens if you use the phases of a manx: http://www.ysbl.york.ac.uk/~cowtan/fourier/piccatmanx2.gif A manx is a species of cat that doesn't have a tail, so no animals were harmed in obtaining these phases. My point here is that the cat's tail can be seen quite readily in the 2fo-fc map if most of the structure is already right, but if your model is completely unrelated to the true structure (fitting a duck into a cat-shaped hole), then everything is in the noise. Real structures are usually somewhere between these two extremes, and I think an important shortcoming in modern crystallography is that we don't have a good quantitative description of this middle-ground. We all like to think we know what model bias is, but we don't exactly have units for it. Should we be using a scale of 0 to 1? Or perhaps duck to cat? Yes, I know we have figure of merit, but FOM is not region-specific. In my experience, as long as you have ~50% of the electrons in the right place (and none of them in the wrong place), then you can generally trust that the biggest difference feature in the fo-fc map is real, and build from there. As the model becomes more complete, the phases should continue to get better, not worse. Eventually, this does break down, although I'm not really sure why. With small molecules, the maximum fo-fc peak keeps getting bigger (on a sigma scale) as you add more and more atoms, and the biggest one you will ever see is the last one. For macromolecules, the
Re: [ccp4bb] Who is using 64-bit Linux?
I myself recently had the misfortune of trying to get a java program relying on the (apparently 32-bit only) JMF package to run on 64-bit linux. This wasted almost an entire week of my life! I tried downgrading the operating system to 32-bit, but that reduced the number of CPUs available in the system from 24 to 8. Still don't know why that is (I'm not all that familiar with Ubuntu, and don't want to be), but I imagine one could call that a performance hit. On the whole, however, I have not seen any significant performance advantage of 64 over 32 bit running crystallography programs side-by-side on equivalent hardware. I have also been unimpressed with the supposed memory access advantages of 64 bit. I had to do a LOT of recompiling programs in order to create maps or MTZ files bigger than 2 GB, and I also still have certain programs running out of virtual memory at 4GB as well. Despite the fact that the relevant machine has 48 GB of RAM and 80 GB of swap. I tell you. Technology just doesn't work. -James Holton MAD Scientist On 4/4/2012 2:21 AM, Takanori Nakane wrote: Dear Tim, 64-bit is about memory addressing - why would you expect a performance boost? I have wondered where this notion originated from. The x86_64 architecture has more registers than 32bit (x86) architecture. Register access is faster than memory access so the more data programs can put on registers, the faster it runs. Best regards, Takanori Nakane
Re: [ccp4bb] Who is using 64-bit Linux?
I tell you. Technology just doesn't work. developers and user's don't, technology is usually ok, but I feel your pain.
[ccp4bb] Good way to check ion sites on Coot
Dear all. can someone tell me what is the best way to check for ion binding sites on my structure? I mean, a text with coordination examples, or maybe a tool on coot for it ... best wishes Andre
Re: [ccp4bb] Who is using 64-bit Linux?
On Apr 13, 2012, at 1:24 PM, James Holton wrote: I tried downgrading the operating system to 32-bit, but that reduced the number of CPUs available in the system from 24 to 8. Still don't know why that is I'm probably wrong, but I'll guess that a 32 bit operating system can only spare 3 of those bits to address CPUs ;-) James
Re: [ccp4bb] Who is using 64-bit Linux?
No, that's a limit set by the ubuntu 32 bit kernel maintainers when they configured and compiled the kernel (again, see my comment about the problem being with developers and users). I think the limit is 256 for x86, 4096 for ia64 (itanium), even old versions of RHEL supported 16 and 32 logical CPUs for x86 : http://support.bull.com/ols/product/system/linux/redhat/help/kbf/g/inst/PrKB11417 http://cateee.net/lkddb/web-lkddb/NR_CPUS.html On Fri, Apr 13, 2012 at 3:08 PM, James Stroud xtald...@gmail.com wrote: On Apr 13, 2012, at 1:24 PM, James Holton wrote: I tried downgrading the operating system to 32-bit, but that reduced the number of CPUs available in the system from 24 to 8. Still don't know why that is I'm probably wrong, but I'll guess that a 32 bit operating system can only spare 3 of those bits to address CPUs ;-) James
Re: [ccp4bb] Good way to check ion sites on Coot
Hi This is what I do: ValidateHighly coordinated waters From: Andre Godoy Sent: Friday, April 13, 2012 3:57 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Good way to check ion sites on Coot Dear all. can someone tell me what is the best way to check for ion binding sites on my structure? I mean, a text with coordination examples, or maybe a tool on coot for it ... best wishes Andre
Re: [ccp4bb] Who is using 64-bit Linux?
On the whole, however, I have not seen any significant performance advantage of 64 over 32 bit running crystallography programs side-by-side on equivalent hardware. I have also been unimpressed with the supposed memory access advantages of 64 bit. I had to do a LOT of recompiling programs in order to create maps or MTZ files bigger than 2 GB, and I also still have certain programs running out of virtual memory at 4GB as well. Despite the fact that the relevant machine has 48 GB of RAM and 80 GB of swap. Eventually all of the programs using cute memory tricks to deal with the restrictions of 70s, 80s, and early 90s systems will be patched, or replaced by ones which don't use these acrobatics. But I don't think it'll be anytime soon. Pete I tell you. Technology just doesn't work. -James Holton MAD Scientist On 4/4/2012 2:21 AM, Takanori Nakane wrote: Dear Tim, 64-bit is about memory addressing - why would you expect a performance boost? I have wondered where this notion originated from. The x86_64 architecture has more registers than 32bit (x86) architecture. Register access is faster than memory access so the more data programs can put on registers, the faster it runs. Best regards, Takanori Nakane
[ccp4bb] Postdoctoral position at the University of Edinburgh
Dear all, A postdoctoral position is available in the Wellcome Trust Centre for Cell Biology, University of Edinburgh, for combined crystallographic and biochemical studies of microtubule associated proteins and molecular motors. The advert is below and applications can be made online until May 14th. Regards, Julie Welburn Postdoctoral Research Associate Applications are invited for a Postdoctoral Research Associate in the laboratory of Dr. Julie Welburn, funded by Cancer Research UK. The laboratory investigates fundamental mechanisms of chromosome segregation at the molecular and cellular level. The focus of our laboratory is the function and properties of microtubules in mitosis and how microtubule-binding proteins, motors and signaling molecules can utilize and modify the dynamics of microtubule subpopulations. Our laboratory is developing quantitative cell biology tools to define how chromosomes oscillate and how different populations of microtubules are regulated. We also use biochemical and structural approaches on mitotic molecular motor complexes, focusing on kinesin microtubule depolymerase family and other microtubule-associated proteins we have identified, to understand their contribution to the regulation of microtubule dynamics and chromosome segregation. We are now looking for an enthusiastic and talented researcher to join our team investigating the molecular mechanisms that control microtubule-based processes in mitosis using structural biology. You should have, or will shortly obtain, a PhD in a relevant subject area and will have experience in crystallography/structural biology and/or biochemistry. An outstanding academic track record is essential and at least one first author publication in a prominent journal is expected. Fixed Term: 2 Years Salary Scale: £30,122 to £35,938 per annum Please Quote Ref: 3015591 Closing Date: 14 May 2012 Further details about the position and the application process can be found at: http://www.jobs.ed.ac.uk/vacancies/index.cfm?fuseaction=vacancies.furtherdetailsvacancy_ref=3015591 The University of Edinburgh is a charitable body, registered in Scotland, with registration number SC005336.
Re: [ccp4bb] Good way to check ion sites on Coot
Hi Andre, Majorie Harding wrote a few nice papers on metal ion binding sites and their associated coordination geometry, examples are: Harding MM (2006) Acta D vol. 62 pp. 678-82 or Harding MM (2004) Acta D vol. 60 pp. 849-59 Best Marc Dr. Marc Kvansakul Laboratory Head, NHMRC CDA Fellow Dept. of Biochemistry| La Trobe University | Bundoora Rm 218, Phys Sci Bld 4, Kingsbury Drive, Melbourne, 3086, Australia T: 03 9479 2263 | F: 03 9479 2467 | E: m.kvansa...@latrobe.edu.au | From: Zhijie Li zhijie...@utoronto.camailto:zhijie...@utoronto.ca Reply-To: Zhijie Li zhijie...@utoronto.camailto:zhijie...@utoronto.ca Date: Fri, 13 Apr 2012 16:37:43 -0400 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Good way to check ion sites on Coot Hi This is what I do: ValidateHighly coordinated waters From: Andre Godoymailto:andre_go...@yahoo.com.br Sent: Friday, April 13, 2012 3:57 PM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Good way to check ion sites on Coot Dear all. can someone tell me what is the best way to check for ion binding sites on my structure? I mean, a text with coordination examples, or maybe a tool on coot for it ... best wishes Andre
