[ccp4bb] Micromatrix seeding using the mosquito
Dear all, Anyone who have had success using the mosquito for micromatrix seeding? Which way is the optimal way of adding seed solution to sitting drops on an MRC plate? Is it better to add 100 nl protein + 10 nl seed solution + 100 nl reservoir solution or to add concentrated seed solution (1 microliter) directly to the reservoir solution and then pipett 100 nl protein + 100 nl reservoir solution. Best regards and thanks in advance, Maria Håkansson __ Maria Håkansson, Ph.D. Senior Scientist, Max-lab, Lund University Phone: +46 (0) 76 8585 706 Fax: +46 (0) 46 222 47 10 Ole Römers väg 1 (P.O. Box 188) SE-221 00 Lund, Sweden Web address: www.maxlab.lu.se Email: maria.hakans...@maxlab.lu.se __
Re: [ccp4bb] Effect of NCS on estimate of data:parameter ratio
Hi Ian, Am 19.09.10 15:25, schrieb Ian Tickle: Hi Florian, Tight NCS restraints or NCS constraints (they are essentially the same thing in effect if not in implementation) both reduce the effective parameter count on a 1-for-1 basis. Restraints should not be considered as being added to the pool of X-ray observations in the calculation of the obs/param ratio, simply because restraints and X-ray observations can in no way be regarded as interchangeable (increasing the no of restraints by N is not equivalent to increasing the no of reflections by N). This becomes apparent when you try to compute the expected Rfree: the effective contribution of the restraints has to be subtracted from the parameter count, not added to the observation count. I always understood the difference between constraints and restraints such, that a constraint reduces the number of parameters by fixing certain parameters, whereas restraints are target values for parameters and as such can be counted as observations, similarly to the Fobs, which are target values for the Fcalc (although with different weights). I don't see what is wrong with this view. Do I misunderstand something? Best regards, Dirk. The complication is that a 'weak' restraint is equivalent to less than 1 parameter (I call it the 'effective no of restraints': it can be calculated from the chi-squared for the restraint). Obviously no restraint is equivalent no parameter, so you can think of it as a continuous sliding scale from no restraint (effective contribution to be subtracted from parameter count = 0) through weak restraint (0 contribution 1) through tight restraint (count ~=1) to constraint (count = 1). Cheers -- Ian On Sat, Sep 18, 2010 at 9:23 PM, Florian Schmitzberger schmitzber...@crystal.harvard.edu wrote: Dear All, I would have a question regarding the effect of non-crystallographic symmetry (NCS) on the data:parameter ratio in refinement. I am working with X-ray data to a maximum resolution of 4.1-4.4 Angstroem, 79 % solvent content, in P6222 space group; with 22 300 unique reflections and expected 1132 amino acid residues in the asymmetric unit, proper 2-fold rotational NCS (SAD phased and no high-resolution molecular replacement or homology model available). Assuming refinement of x,y,z, B and a polyalanine model (i.e. ca. 5700 atoms), this would equal an observation:parameter ratio of roughly 1:1. This I think would be equivalent to a normal protein with 50 % solvent content, diffracting to better than 3 Angstroem resolution (from the statistics I could find, at that resolution a mean data:parameter ratio of ca. 0.9:1 can be expected for refinement of x,y,z, and individual isotropic B; ignoring bond angle/length geometrical restraints at the moment). My question is how I could factor in the 2-fold rotational NCS for the estimate of the observations, assuming tight NCS restraints (or even constraint). It is normally assumed NCS reduces the noise by a factor of the square root of the NCS order, but I would be more interested how much it adds on the observation side (used as a restraint) or reduction of the parameters (used as a constraint). I don't suppose it would be correct to assume that the 2-fold NCS would half the number of parameters to refine (assuming an NCS constraint)? Regards, Florian --- Florian Schmitzberger Biological Chemistry and Molecular Pharmacology Harvard Medical School 250 Longwood Avenue, SGM 130 Boston, MA 02115, US Tel: 001 617 432 5602 -- *** Dirk Kostrewa Gene Center Munich, A5.07 Department of Biochemistry Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 25 D-81377 Munich Germany Phone: +49-89-2180-76845 Fax:+49-89-2180-76999 E-mail: kostr...@genzentrum.lmu.de WWW:www.genzentrum.lmu.de ***
Re: [ccp4bb] Micromatrix seeding using the mosquito
Dear Maria In our hands Mosquito MMS worked by adding 2x protein solution + 1x seed solution + 1x reservoir. The reason for doing this was two-fold: I) ratio protein/reservoir is similar to the condition that generated the seed stock II) a deliberate bias toward the initial condition is given I) and II) might not always be helpful and possibly other ratios should be explored. Best wishes Roberto On 20 Sep 2010, at 08:55, Maria Håkansson maria.hakans...@maxlab.lu.semailto:maria.hakans...@maxlab.lu.se wrote: Dear all, Anyone who have had success using the mosquito for micromatrix seeding? Which way is the optimal way of adding seed solution to sitting drops on an MRC plate? Is it better to add 100 nl protein + 10 nl seed solution + 100 nl reservoir solution or to add concentrated seed solution (1 microliter) directly to the reservoir solution and then pipett 100 nl protein + 100 nl reservoir solution. Best regards and thanks in advance, Maria Håkansson __ Maria Håkansson, Ph.D. Senior Scientist, Max-lab, Lund University Phone: +46 (0) 76 8585 706 Fax: +46 (0) 46 222 47 10 Ole Römers väg 1 (P.O. Box 188) SE-221 00 Lund, Sweden Web address: http://www.maxlab.lu.se www.maxlab.lu.sehttp://www.maxlab.lu.se Email: mailto:maria.hakans...@maxlab.lu.se maria.hakans...@maxlab.lu.semailto:maria.hakans...@maxlab.lu.se __
[ccp4bb] 300 projects = maximum in CCP4i?
Dear all, I wanted to generate the 301'st project in the CCP4i interface Directories+Projects. The problem is that when I click on the button for the Project of this session, there is apparently no space for the 13th column (of 25 entries). I do not want to delete any old projects, so is there a workaround (I am using CCP4i 1.4.4.2)? Many thanks, Joerg Dr. Joerg Kallen Novartis Pharma AG PH222426, CPC/SBP:LAB KALLEN CHBS, WSJ-088.9.08B Novartis Pharma AG Forum 1 Novartis Campus CH-4056 Basel Switzerland Phone: +41 61 3245579 Fax: +41 61 3242686 Email : joerg.kal...@novartis.com _ CONFIDENTIALITY NOTICE The information contained in this e-mail message is intended only for the exclusive use of the individual or entity named above and may contain information that is privileged, confidential or exempt from disclosure under applicable law. If the reader of this message is not the intended recipient, or the employee or agent responsible for delivery of the message to the intended recipient, you are hereby notified that any dissemination, distribution or copying of this communication is strictly prohibited. If you have received this communication in error, please notify the sender immediately by e-mail and delete the material from any computer. Thank you.
Re: [ccp4bb] Micromatrix seeding using the mosquito
dear Maria, we routinely use Mosquito for MMS, the ratio works the best: 100nl protein + multi-aspirate mode x (15nl seeds + 85nl reservoir solution). best regards, alexey Alexey RAK Structural Biology SDI/LGCR Sanofi-aventis RD Centre de Recherche Paris 13, Quai Jules Guesde - BP 14 94403 Vitry sur Seine Cedex France Phone : +33 (0)1 58 93 86 93 Fax : +33 (0)1 58 93 80 63 Mobile : +33 (0)6 26 71 28 60 Email : alexey@sanofi-aventis.com From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Steiner, Roberto Sent: Monday, September 20, 2010 10:21 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Micromatrix seeding using the mosquito Dear Maria In our hands Mosquito MMS worked by adding 2x protein solution + 1x seed solution + 1x reservoir. The reason for doing this was two-fold: I) ratio protein/reservoir is similar to the condition that generated the seed stock II) a deliberate bias toward the initial condition is given I) and II) might not always be helpful and possibly other ratios should be explored. Best wishes Roberto On 20 Sep 2010, at 08:55, Maria Håkansson maria.hakans...@maxlab.lu.se wrote: Dear all, Anyone who have had success using the mosquito for micromatrix seeding? Which way is the optimal way of adding seed solution to sitting drops on an MRC plate? Is it better to add 100 nl protein + 10 nl seed solution + 100 nl reservoir solution or to add concentrated seed solution (1 microliter) directly to the reservoir solution and then pipett 100 nl protein + 100 nl reservoir solution. Best regards and thanks in advance, Maria Håkansson __ Maria Håkansson, Ph.D. Senior Scientist, Max-lab, Lund University Phone: +46 (0) 76 8585 706 Fax: +46 (0) 46 222 47 10 Ole Römers väg 1 (P.O. Box 188) SE-221 00 Lund, Sweden Web address: http://www.maxlab.lu.se www.maxlab.lu.se Email: mailto:maria.hakans...@maxlab.lu.se maria.hakans...@maxlab.lu.se __ attfd7c.jpg
Re: [ccp4bb] Effect of NCS on estimate of data:parameter ratio
Hi Dirk First, constraints are just a special case of restraints in the limit of infinite weights, in fact one way of getting constraints is simply to use restraints with very large weights (though not too large that you get rounding problems). These 'pseudo-constraints' will be indistinguishable in effect from the 'real thing'. So why treat restraints and constraints differently as far as the statistics are concerned: the difference is purely one of implementation. Second, restraints are not interchangeable 1-for-1 with X-ray data as far as the statistics are concerned: N restraints cannot be considered as equivalent to N X-ray data, which would be the implication of adding together the number of restraints and the number of X-ray data. This can be seen in the estimation of the expected values of the residuals (chi-squared) for the working test sets, which are used to estimate the expected Rfree. If you take a look at our 1998 AC paper (D54, 547-557), Table 2 (p.551), the last row of the table (labelled 'RGfree/RG') shows the expected residuals for the working set (denominator) and test set (numerator) for the cases of no restraints, restrained and constrained refinement: No restraints (or constraints): Dwork = f - m Dfree = f + m Restrained: Dwork = f - (m - r + Drest) Dfree = f + (m - r + Drest) Constrained: Dwork = f - (m - r) Dfree = f + (m - r) where: Dwork = expected working set residual (chi-squared), Dfree = expected test set residual (chi-squared), f = no of reflections in working set, m = no of parameters, r = no of restraints and/or constraints, Drest = restraint residual (chi-squared). The constrained case is obviously just a special case of the restrained case with Drest = 0, i.e. in the constrained case the difference between the refined and target values is zero, and the 'no restraints' case is a special case of this with r = 0. We can generalise all of this by writing simply: Dwork = f - m' Dfree = f + m' where m' is the effective no of parameters corrected for restraints and/or constraints (m' = m - r + Drest); the effective no of parameters is reduced whether you're using restraints or constraints. In the case where you had both restraints and constraints r would be the total no of restraints + constraints, however constraints contribute nothing to Drest. The 'effectiveness' of a restraint depends on its contribution to Drest (Z^2), a smaller value means it's more effective. A contribution of Z^2 = 1 to Drest completely cancels the effect of increasing r by 1 by adding the restraint (i.e. the restraint has no effect). This incidentally shows that the effect of over-fitting (adding redundant effective parameters) is to reduce the working set and increase the test set residuals. If you consider the working set residual in the general case: Dwork = f - (m - r + Drest) = f + r - m - Drest it certainly appears from this that the number of X-ray data (f) and the number of restraints (r) are being added. However if you consider the test set residual: Dfree = f + (m - r + Drest) = f - r + m + Drest this is clearly not the case. All you can say is that the effective number of parameters is reduced by the number of restraints + constraints. Cheers -- Ian On Mon, Sep 20, 2010 at 9:20 AM, Dirk Kostrewa kostr...@genzentrum.lmu.de wrote: Hi Ian, Am 19.09.10 15:25, schrieb Ian Tickle: Hi Florian, Tight NCS restraints or NCS constraints (they are essentially the same thing in effect if not in implementation) both reduce the effective parameter count on a 1-for-1 basis. Restraints should not be considered as being added to the pool of X-ray observations in the calculation of the obs/param ratio, simply because restraints and X-ray observations can in no way be regarded as interchangeable (increasing the no of restraints by N is not equivalent to increasing the no of reflections by N). This becomes apparent when you try to compute the expected Rfree: the effective contribution of the restraints has to be subtracted from the parameter count, not added to the observation count. I always understood the difference between constraints and restraints such, that a constraint reduces the number of parameters by fixing certain parameters, whereas restraints are target values for parameters and as such can be counted as observations, similarly to the Fobs, which are target values for the Fcalc (although with different weights). I don't see what is wrong with this view. Do I misunderstand something? Best regards, Dirk. The complication is that a 'weak' restraint is equivalent to less than 1 parameter (I call it the 'effective no of restraints': it can be calculated from the chi-squared for the restraint). Obviously no restraint is equivalent no parameter, so you can think of it as a continuous sliding scale from no restraint (effective contribution to be subtracted from parameter count = 0) through weak restraint (0 contribution 1)
Re: [ccp4bb] Graphics for notebook
Eric, if you want to use stereo on a notebook you have very limited options. I have stereo running using a Lenovo T61p with a Nvidia Quadro FX570M under XP, not tried Linux yet. Officially this card is not even supported by Nvidia for 3D application, but works anyway in conjunction with a docking station, which has a DVI adaptor. Don't go with a Quadro NV series, they did not work in our hands. HTH Carsten -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Eric Karg Sent: Sunday, September 19, 2010 11:46 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Graphics for notebook Dear all, I wanted to know which type of graphics card is more suitable for a notebook which is going to be used for structural biology. Integrated or dedicated? ATI or NVIDIA? At the moment I have to choose between an integrated Intel HD Graphics or a dedicated NVIDIA NVS 3100M Graphics. Any suggestions are highly appreciated. Thanks! Eric
Re: [ccp4bb] Graphics for notebook
The Quadro FX 3700M in my Dell Precision 6400M notebook works great with stereo in Windows, no support for Linux yet though. On Mon, Sep 20, 2010 at 8:57 AM, Schubert, Carsten [PRDUS] cschu...@its.jnj.com wrote: Eric, if you want to use stereo on a notebook you have very limited options. I have stereo running using a Lenovo T61p with a Nvidia Quadro FX570M under XP, not tried Linux yet. Officially this card is not even supported by Nvidia for 3D application, but works anyway in conjunction with a docking station, which has a DVI adaptor. Don't go with a Quadro NV series, they did not work in our hands. HTH Carsten -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Eric Karg Sent: Sunday, September 19, 2010 11:46 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Graphics for notebook Dear all, I wanted to know which type of graphics card is more suitable for a notebook which is going to be used for structural biology. Integrated or dedicated? ATI or NVIDIA? At the moment I have to choose between an integrated Intel HD Graphics or a dedicated NVIDIA NVS 3100M Graphics. Any suggestions are highly appreciated. Thanks! Eric -- Jim Fairman, Ph D. Post-Doctoral Fellow National Institutes of Health - NIDDK Lab: 1-301-594-9229 E-mail: fairman@gmail.com james.fair...@nih.gov
Re: [ccp4bb] Deposition of riding H: R-factor is overrated
Hi Nicholas, Thank you for your reply. snip it seems that we are trying to deposit one model to satisfy two different purposes - one for model validation and the other for model interpretation (use in docking etc), and what's good for one purpose might not be necessarily good for the other. /snip This has been discussed before on this list, but allow me to repeat it: You would have expected that the crystallographers' aim would be to deposit the model that maximises the product (likelihood * prior). Clearly, this is not what we do, mainly because (a) the calculation of likelihood is only based on a subset of the 'data' that are obtained from an X-ray diffraction experiment (for example, we ignore diffuse scattering as Ian pointed-out), (b) we consciously avoid 'prior' because this would make the models 'subjective', meaning that better informed people would deposit (for the same data) different models than the less well informed, (c) the format of the PDB does not offer much room for 'creative interpretations' of the electron density maps [for example, you can't have discrete disorder on the backbone (or has this changed ?)]. I sense that what is being deposited is not the 'best model' in any conceivable way, but the model that 'best' accounts for the final 2mFo-DFc map within the limitations of the program used for the final refinement. I don't quite understand your point. We currently deposit electron densities and movies, I don't see how depositing an energy minimized structure is so difficult. It doesn't need to be on the same pdb file as the model used in refinement nor does it need to be deposited into the PDB server, but even if it does, is it not possible to have it as a new Chain or new atom type in the current pdb file format? ps. May I say parenthetically that making the deposited models dependant on their intended usage, would possibly qualify as 'fraud' ;-) I don't quite understand this either. When I prepare a protein model for simulation, I would remove all alternative conformations, add hydrogens, and then minimize the structure. If I make such a minimized structure available for others to use with full disclosure, how would that constitute fraud? I was going to start offering minimized models on our future structures on our lab website, but if that constitutes fraud, then I might have to rethink. I don't know enough to argue with anyone here and that's not the intention of my posts - I am just trying to help figure out a way to resolve a significant problem that will likely to resurface down the road. It would be helpful if the more experienced people here can start a discussion of 'how to resolve' the problems exposed by this thread so far - assuming that you agree that it's a problem worth your time. Cheers, Quyen __ Quyen Hoang, Ph.D Assistant Professor Department of Biochemistry and Molecular Biology, Stark Neurosciences Research Institute Indiana University School of Medicine 635 Barnhill Drive, Room MS0013D Indianapolis, Indiana 46202-5122 Phone: 317-274-4371 Fax: 317-274-4686 email: qqho...@iupui.edu -- Dr Nicholas M. Glykos, Department of Molecular Biology and Genetics, Democritus University of Thrace, University Campus, Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office) +302551030620, Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/
[ccp4bb] EMBL-EBI Services Survey
Dear colleagues, The EBI is carrying out a user survey about its services, collecting input from current and potential users. The survey lives here: http://www.surveymonkey.com/s/EMBL-EBI - It should take 10-15 minutes. - There are no compulsory questions. - You can be entered into a prize draw where you could win an iPad. We will be collecting data until the end of September. If you have technical problems with the survey please let us know at http://www.ebi.ac.uk/support Thank you for your help, --Gerard --- Gerard J. Kleywegt, PDBe, EMBL-EBI, Hinxton, UK ger...@ebi.ac.uk . pdbe.org Secretary: Pauline Haslam pdbe_ad...@ebi.ac.uk
Re: [ccp4bb] Graphics for notebook
It's best to have dedicated NVIDIA (don't have much experience with ATI, but it is my understanding that they may be more difficult to configire sometimes). However, Intel on-board graphics has gotten much better recently (in fact, Intel releases drivers as open source (guess because they are not big players in video cards). I am not sure you'll actually notice any difference between NVIDIA and Intel integrated, since coot is not as demanding as the bleeding edge videogames for which high-speed graphics matters. I'd recommend to search various linux forums for information about the laptop you are considering - if it's a fairly new model, there are often issues you'll need to resolve to get it going. Ed. On Sun, 2010-09-19 at 16:45 +0100, wrote: Dear all, I wanted to know which type of graphics card is more suitable for a notebook which is going to be used for structural biology. Integrated or dedicated? ATI or NVIDIA? At the moment I have to choose between an integrated Intel HD Graphics or a dedicated NVIDIA NVS 3100M Graphics. Any suggestions are highly appreciated. Thanks! Eric -- I'd jump in myself, if I weren't so good at whistling. Julian, King of Lemurs
Re: [ccp4bb] Graphics for notebook
Hi Eric, I wanted to know which type of graphics card is more suitable for a notebook which is going to be used for structural biology. Integrated or dedicated? ATI or NVIDIA? At the moment I have to choose between an integrated Intel HD Graphics or a dedicated NVIDIA NVS 3100M Graphics. Any suggestions are highly appreciated. These responses are in line with what I'd recommend. First, NVidia presently has the drivers with fewest number of PyMOL bugs (then ATI, then Intel) across all platforms. Their Linux support, has for years, outstripped the competition. Next, if you're doing anything graphics related, I'd highly suggest moving away from integrated boards. Last, the integrated mobile boards are the worst offenders--I'm looking at you Intel GM945!--so if you can help it, stay away from them. Cheers, -- Jason -- Jason Vertrees, PhD PyMOL Product Manager Schrodinger, LLC (e) jason.vertr...@schrodinger.com (o) +1 (603) 374-7120
[ccp4bb] Postdoctoral Position
A postdoctoral position in protein crystallography is available in Dr. Min Lu’s lab at Public Health Research Institute Center, New Jersey Medical School, University of Medicine and Dentistry of New Jersey. Our research mainly focuses on the three-dimensional structures and biological functions of proteins involved in HIV-1 entry and maturation. Successful candidate should have a PhD degree in biochemistry, molecular biology or related field. Knowledge and experience in protein crystallography is a must. Interested applicants should send a CV and a list of two references to Dr. Lu at l...@umdnj.edu
