Re: [ccp4bb] Strange spots - In book of John Helliwell
I have seen such features myself from time to time on diffraction pictures recorded on X-ray films in the far past and was explaining them by the presence of electron diffraction as they resemble electron diffraction patterns. Source of focused electron beam during X-ray diffraction experiment is a different story. I have no explanation for that. BTW1 when we started to use area detectors, these features disappeared. However features David Goldstone showing us is something else. We never have seen such things before. I would try to take a similar picture on X-ray film. But maybe why to bother?. Unfortunately to be a genius of X-ray diffraction physics in the present MX world will fast convert a person to homeless. My question to John Goldstone: Is it intermittent, of for certain crystal/station/detector you always get that? BTW2 I feel in contemptuous reaction of the community deep discontent of all of us.What? even diffraction physics we do not understand in depth? Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Oct 29, 2010, at 23:23 , Charles W. Carter, Jr wrote: Hi Gérard, I actually bought John's book some time ago and can provide page 321 (attached). I think perhaps John is exaggerating his case, although as I have frequently made similarly inflated claims, I am sympathetic. Here is the pdf file. If it does not go through the CCP4 filters, I would be happy to send it to the first few who request it. Charlie p321.PDF On Oct 29, 2010, at 11:03 PM, Gerard Bricogne wrote: Dear Liz, You will be disappointed. I went immediately to that link, but page 321 is not available as part of the Googlebook sample, which jumps directly from page 320 to page 325. With best wishes, Gerard. -- On Fri, Oct 29, 2010 at 09:13:27PM +0100, elizabeth.d...@diamond.ac.uk wrote: There is always hope!!! Seriously though, I have never seen anything like this before! I am watching this thread with interest to see what others suggest. THanks Also thanks should go specifically to Julian Nomme who took the trouble to send us all the Helliwell book link. Liz From: CCP4 bulletin board on behalf of Sanishvili, Ruslan Sent: Fri 29/10/2010 21:08 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange spots C'mon now! Everybody knows that frogs in real space become handsome princes in the reciprocal one... N. Ruslan Sanishvili (Nukri), Ph.D. GM/CA-CAT Biosciences Division, ANL 9700 S. Cass Ave. Argonne, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jacob Keller Sent: Friday, October 29, 2010 3:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Strange spots Yes, but the question is what in real space gives rise to reciprocal-space frog spawn? (Frogs, I guess?) - Original Message - From: Marcus Winter mailto:marcus.win...@agilent.com To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, October 29, 2010 2:56 PM Subject: Re: [ccp4bb] Strange spots Dear David, Further to the previous learned responses, surely, this is just frog spawn ? My apologies: it is a Friday evening, after all... Marcus Winter. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of David Goldstone Sent: 29 October 2010 17:08 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Strange spots Dear All, Does anyone have any insight into what the circles around the spots might be? cheers Dave -- David Goldstone, PhD National Institute for Medical Research Molecular Structure The Ridgeway Mill Hill London NW7 1AA *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Strange spots
Fig from John (I have a copy of his book in the office and will see it only tomorrow) due to better quality makes it clear (at least for me) that we see the similar effect shown by David Goldstone. Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Oct 30, 2010, at 10:16 , John R Helliwell wrote: Dear Colleagues, Here is scan of a portion of Fig 8.1b, including a zoom in, as per my earlier email. This was recorded on photographic film, which hopefully removes the worry about whether Dave's detector was malfunctioning, and secondly, being Laue, a simple X-ray optic set up (I have to check if it included a focussing X-ray mirror or not). Even if there is no sure explanation of such halo features, although I did declare 'defects in the crystal' as a possibility, we know in this case that these features were radiation sensitive. Best wishes, John On Fri, Oct 29, 2010 at 5:08 PM, David Goldstone david.goldst...@nimr.mrc.ac.uk wrote: Dear All, Does anyone have any insight into what the circles around the spots might be? cheers Dave -- David Goldstone, PhD National Institute for Medical Research Molecular Structure The Ridgeway Mill Hill London NW7 1AA -- Professor John R Helliwell DSc diffuse spots 3.jpgdiffuse spots 2 .jpg
Re: [ccp4bb] Help with model bias in merihedral twin + Refmac5
Peter Yes, by 2Fo-Fc type maps I was including the weighted maps you mention (which should be better). The main issue is whether 3mF(obs)-2DF(calc) and higher coefficients would be better for the twinned reflections Regards Colin From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Peter Chan Sent: 29 October 2010 18:59 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help with model bias in merihedral twin + Refmac5 Dear Colin, Thank you for the email. To be honest, I don't know exactly which map they are. From my understanding (and according to http://www.ccp4.ac.uk/schools/India-2010/tutorials/refmac/Murshudov30th. pdf), Refmac5 outputs 'normal map' 2mF(obs)-DF(calc) and 'difference map' 2mF(obs)-DF(calc). I believe this is better than looking at the typical 2F(obs)-F(calc), although there still appears to be a fair amount of model bias. Best, Peter Subject: RE: [ccp4bb] Help with model bias in merihedral twin Date: Wed, 27 Oct 2010 17:38:41 +0100 From: colin.n...@diamond.ac.uk To: pc...@hotmail.com Peter Regarding the question Although the electron density map looks good, I am not sure if I should have too much confidence in it because I was not able to obtain 'strong electron densities' from omitted sections of the model in a refinement. I don't know if this is an indicator for bias introduced somewhere. I would like to ask what may be some procedures I can try for checking and removing these biases Can you state what type of maps these are? The reason is the following. Taking PHENIX twinning maps as an example we have, in the manual - By default, data is detwinned using algebraic techniques, unless the twin fraction is above 45%, in which case detwinning is performed using proportionality of twin related Icalc values. Detwinning using the proportionality option, results in maps that are more biased towards the model, resulting in seemingly cleaner, but in the end less informative maps. The 2mFo-dFc map coefficients can be chosen to have sigmaA weighting (two_m_dtfo_d_fc) or not (two_dtfo_fc). IN both cases, the map coefficients correspond to the 'untwinned' data. A difference map can be constructed using either sigmaA weighted detwinned data (m_dtfo_d_fc), a sigmaA weighted gradient map (m_gradient) or a plain gradient map (gradient). The default is m_dtfo_d_fc but can be changed to gradient or m_gradient if desired. For the high twin fraction which you have (near 50%). my view is that 2Fo-Fc type maps are not optimal for this as they don't take account of the increased bias resulting from the extra degrees of freedom (i.e. splitting the intensity between the two reflections based on Icalc). For the reflections related by twinning higher order coefficients (e.g. 3Fo-2Fc or perhaps 4Fo-3Fc) would give noiser but less biased maps. Fibre diffraction folk do similar things when their Bessel function terms overlap. Cheers Colin From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Peter Chan Sent: 27 October 2010 02:00 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Help with model bias in merihedral twin Hello All, Not long ago I posted for some help with my twinned dataset at 1.95 A, and have confirmed the twinning of P6(5) into P6(5)22. Molecular replacement was successful and the twin refinement in Refmac yieled R/Rfree of 21%/26%, with a twin fraction of 0.46. Although the electron density map looks good, I am not sure if I should have too much confidence in it because I was not able to obtain 'strong electron densities' from omitted sections of the model in a refinement. I don't know if this is an indicator for bias introduced somewhere. I would like to ask what may be some procedures I can try for checking and removing these biases, and a few additional related questions. As suggested to me previously, I have generated a total omit map with sfcheck in ccp4i, using the refined pdb and unrefined data in P6(5). The .map file look a little worse in quality (is this because of the twinning?) but is still reasonable, with a few breaks in the main chain and side chains. Interestingly, when I do a real space refinement against the total omit map, I get slightly better Rfree at the earlier rounds of Refmac which diverges into the numbers above. Why is this the case?
Re: [ccp4bb] Free R with doubled cell edge
Hi, I'm not sure why you want to carry the free R reflections from the small cell to the new cell. If it's the model bias vis-a vis the reflections participating the refinement that you want to get rid of you can take another route, I think. 1) Select R-free set for the new cell (paying attention to the new NCS); 2) Take the model you obtained after phaser (4 chains) and shake it to death (either in CNS by annealing or in the ccp4 shake routine or the USF equivalent). By then, your model should have got rid of the bias and you can start refinement. Gurus, have I cut any corner by suggesting this route? Would there be any objection from referees (...)? Good luck, Boaz - Original Message - From: Kay Diederichs kay.diederi...@uni-konstanz.de Date: Saturday, October 30, 2010 14:23 Subject: Re: [ccp4bb] Free R with doubled cell edge To: CCP4BB@JISCMAIL.AC.UK Hi Ed, in the new cell (long a axis), the reflections H K L are related by H=2*h K=k L=l to those of the old (short a) cell. I would expect that the R-factor of those H K L reflections with even H from the new crystal form is low (at least at low resolution) against the h k l reflections of the old crystal form. (I'd also expect that they are stronger than the odd-H reflections.) You can obtain the R-free flag for these reflections by creating a file with h k l R-free-flag from your old dataset, multiplying all h by 2 (it should be possible to do this with the CCP4 program reindex, using reindex HKL h+h, k, l as the only input line), and using that for the new data. This procedure gives you R-free flags for half of the reflections of your new dataset (those with even H). Those reflections with odd H are of course new; they are not (directly) related to any reflections of the old crystal form. You may want to randomly assign R-free flags to them; there is (I think) a task in ccp4i which takes care of partially missing R-free flags. HTH, Kay Am 20:59, schrieb Thomas Edwards: Dear BB Sages, I have a problem where I think I could very easily do the wrong thing. And I don't really want to do that... We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU - Shelx, RESOLVE, ARPwARP. Cool.). In p21 30 109 65 90 105 90 at 2.5A However, we have now collected 1.9A data. In p21... 60 109 65 90 107 90 4 chains per AU instead of 2 with a doubling of a. Self rotations with the new data suggest 2 two-folds, one quite near crystallographic. It seems that the doubling of the a edge is adding an NCS two- fold that is almost crystallographic. Now, having refined against the 2.5A data to R/Rfree of about 25/30 we would like to use that model to do MR against the new high res data (We didn't collect Zn peak data for the new crystal - didn't think we'd need it.). I have done that and found 4 mols with Phaser in about 60 seconds. Still cool. So, we would like to transfer Free R flags to the new data to avoid refining against what had been labelled as Free R. My problem is - how do I do that properly? I am worried that some of the working data in the bigger cell will be correlated with Free data via the near crystallographic NCS. I clearly don't want to just copy them from the old mtz file with a0 I recall some discussion about this from years ago on the BB but can't find the right threads. Can anybody point me to the correct way to do this please - I presumably want to label with Free R flags symmetry related Free R labelled reflections from the old data that are related by the new NCS 2-fold (that is close to crystallographic) in the new data. Right?? If I have worded that correctly... I am hoping that will make sense to somebody. I think that the solutions that were recently suggested for lower vs higher symmetry in the same unit cell do not apply here. One suggestion has been to do the MolRep, choose new free Rs, give it all a good hard shake with high temp simulated annealing and hope that any bias is gone. I'm not sure that I am comfortable with the word hope here... But, if the consensus of opinion of the wise folk at the BB is that this will pass muster at the point where the charming and delightful referees are commenting on the extremely high impact (obviously :-) manuscript, then I will quote you all! I await your wise words. Free R. Again. Sorry. Cheers Ed __ T.Edwards Ph.D. Garstang 8.53d Astbury Centre for Structural Molecular Biology University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- A new scientific truth does not triumph by convincing opponents and making them see the light, but rather because its opponents eventually die, and a new generation grows up that is familiar with it. ~Max Planck Boaz Shaanan, Ph.D.
Re: [ccp4bb] Free R with doubled cell edge
On Saturday, October 30, 2010, Boaz Shaanan wrote: Hi, I'm not sure why you want to carry the free R reflections from the small cell to the new cell. If it's the model bias vis-a vis the reflections participating the refinement that you want to get rid of you can take another route, I think. 1) Select R-free set for the new cell (paying attention to the new NCS); 2) Take the model you obtained after phaser (4 chains) and shake it to death (either in CNS by annealing or in the ccp4 shake routine or the USF equivalent). By then, your model should have got rid of the bias and you can start refinement. Gurus, have I cut any corner by suggesting this route? I think it is better to do exactly what was requested - carry forward the old Rfree to the new data set. Since the a axis is double, the Rfree of smallcell [h k l] is transferred to bigcell [2h k l] One problem with shaking things up as you describe is that if the original model was refined against higher resolution data than your new data set, you will probably never get back to the same model quality as you started with (see the ongoing discussion in another thread). And if the new data set is higher resolution, then you face the same problem in reverse. If you want to take your eventual new, higher resolution model back into the old cell you want subsequent refinement to have unbiased Rfree on that end also. Ethan Would there be any objection from referees (...)? Good luck, Boaz - Original Message - From: Kay Diederichs kay.diederi...@uni-konstanz.de Date: Saturday, October 30, 2010 14:23 Subject: Re: [ccp4bb] Free R with doubled cell edge To: CCP4BB@JISCMAIL.AC.UK Hi Ed, in the new cell (long a axis), the reflections H K L are related by H=2*h K=k L=l to those of the old (short a) cell. I would expect that the R-factor of those H K L reflections with even H from the new crystal form is low (at least at low resolution) against the h k l reflections of the old crystal form. (I'd also expect that they are stronger than the odd-H reflections.) You can obtain the R-free flag for these reflections by creating a file with h k l R-free-flag from your old dataset, multiplying all h by 2 (it should be possible to do this with the CCP4 program reindex, using reindex HKL h+h, k, l as the only input line), and using that for the new data. This procedure gives you R-free flags for half of the reflections of your new dataset (those with even H). Those reflections with odd H are of course new; they are not (directly) related to any reflections of the old crystal form. You may want to randomly assign R-free flags to them; there is (I think) a task in ccp4i which takes care of partially missing R-free flags. HTH, Kay Am 20:59, schrieb Thomas Edwards: Dear BB Sages, I have a problem where I think I could very easily do the wrong thing. And I don't really want to do that... We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU - Shelx, RESOLVE, ARPwARP. Cool.). In p21 30 109 65 90 105 90 at 2.5A However, we have now collected 1.9A data. In p21... 60 109 65 90 107 90 4 chains per AU instead of 2 with a doubling of a. Self rotations with the new data suggest 2 two-folds, one quite near crystallographic. It seems that the doubling of the a edge is adding an NCS two- fold that is almost crystallographic. Now, having refined against the 2.5A data to R/Rfree of about 25/30 we would like to use that model to do MR against the new high res data (We didn't collect Zn peak data for the new crystal - didn't think we'd need it.). I have done that and found 4 mols with Phaser in about 60 seconds. Still cool. So, we would like to transfer Free R flags to the new data to avoid refining against what had been labelled as Free R. My problem is - how do I do that properly? I am worried that some of the working data in the bigger cell will be correlated with Free data via the near crystallographic NCS. I clearly don't want to just copy them from the old mtz file with a0 I recall some discussion about this from years ago on the BB but can't find the right threads. Can anybody point me to the correct way to do this please - I presumably want to label with Free R flags symmetry related Free R labelled reflections from the old data that are related by the new NCS 2-fold (that is close to crystallographic) in the new data. Right?? If I have worded that correctly... I am hoping that will make sense to somebody. I think that the solutions that were recently suggested for lower vs higher symmetry in the same unit cell do not apply here. One suggestion has been to do the MolRep, choose new free Rs, give it all a good hard shake with high
Re: [ccp4bb] DLS
instead of DLS you could also look into buying a fancy PCR machine ~30K where you can perform a) regular PCR b) quantitative PCR c) thermal stability tests for your protein Ericsson et al. Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem (2006) vol. 357 (2) pp. 289-98 Crowther et al. Buffer Optimization of Thermal Melt Assays of Plasmodium Proteins for Detection of Small-Molecule Ligands. Journal of biomolecular screening : the official journal of the Society for Biomolecular Screening (2009) pp. Sure you are missing the quantification for how homogeneous your sample is and an estimation of the molecular weight of the particles in solution but the correlation between crystallizability and DLS is not so good compared to thermal shifts as pointed out in Ericsson 2006. You will have a high end purification system so you could get a sense of your sample homogeneity via SEC instead Just a thought, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Oct 30, 2010, at 12:02 AM, Mohd Shukuri Mohamad Ali wrote: Hi there Sorry for the off ccp4 topics. I am planning to get a DLS to set up my crystallography lab. I got a tight budget. Around USD 8 perhaps. I'm quite new to this field. Thanks in advance for your suggestions Regards Shukuri
Re: [ccp4bb] DLS
I second the question of need: a decent PCR machine capable of 'thermofluor'-like experiments should cost around $34-$38K, which leaves about the same amount of $$ for a decent purification machine. One of the questions you have to figure out is: are you setting up a high-throughput lab or a regular kind of lab? If you're doing HT work then perhaps a plate-based DLS is a good idea, although you will undoubtedly need more $$ for the rest of the stuff. On the other hand if you're setting up to do 'regular' i.e. low-throughput (I prefer the term 'hand crafted', by the way) purification then an investment in a Thermofluor machine (be it PCR or a multiwell spectrofluorimeter with temperature control) and another useful device might be more wise. Having written this, I would have to add for the sake of honesty that my lab has both - but we are in fact straddling the boundary between HT and hand-crafted work and we have use both instruments daily. Artem On Fri, Oct 29, 2010 at 11:02 PM, Mohd Shukuri Mohamad Ali shuk...@biotech.upm.edu.my wrote: Hi there Sorry for the off ccp4 topics. I am planning to get a DLS to set up my crystallography lab. I got a tight budget. Around USD 8 perhaps. I'm quite new to this field. Thanks in advance for your suggestions Regards Shukuri
Re: [ccp4bb] DLS
Hi Shukuri, If you're on a tight budget and you only want to use DLS to verify sample homogeneity I would save my money or use it for something else (a crystallization robot, for example). You definitely do NOT need DLS to solve crystal structures, including those of membrane proteins. Many monodisperse samples do not crystallize and I can tell you from experience that polydisperse samples can crystallize perfectly well. Gel filtration chromatography is a good and much cheaper alternative to DLS, and you'll have the columns already anyway. Admittedly, DLS will give you somewhat more info than gel filtration, but again, if money is an issue I would not get a DLS instrument. For me its comparable to a UV microscope for verification of crystals; nice for sure, but not worth the $$$. Cheers, Bert On 10/30/10 12:02 AM, Mohd Shukuri Mohamad Ali shuk...@biotech.upm.edu.my wrote: Hi there Sorry for the off ccp4 topics. I am planning to get a DLS to set up my crystallography lab. I got a tight budget. Around USD 8 perhaps. I'm quite new to this field. Thanks in advance for your suggestions Regards Shukuri
Re: [ccp4bb] DLS
hear-hear wise words. may I add that if you are buying a DLS anyway, maybe consider getting a model which can also be connected online to an FPLC to do Static LS measurements, ... after all a DLS signal is basically an autocorrelation of the static signal over time ... which will tell you about dispersion, but is incapable of measuring MW, and can only get Rg, unlike static LS (MALLS) that will give you absolute MW over your FPLC - which is handy. A. On Oct 30, 2010, at 20:10, Artem Evdokimov wrote: I second the question of need: a decent PCR machine capable of 'thermofluor'-like experiments should cost around $34-$38K, which leaves about the same amount of $$ for a decent purification machine. One of the questions you have to figure out is: are you setting up a high-throughput lab or a regular kind of lab? If you're doing HT work then perhaps a plate-based DLS is a good idea, although you will undoubtedly need more $$ for the rest of the stuff. On the other hand if you're setting up to do 'regular' i.e. low-throughput (I prefer the term 'hand crafted', by the way) purification then an investment in a Thermofluor machine (be it PCR or a multiwell spectrofluorimeter with temperature control) and another useful device might be more wise. Having written this, I would have to add for the sake of honesty that my lab has both - but we are in fact straddling the boundary between HT and hand-crafted work and we have use both instruments daily. Artem On Fri, Oct 29, 2010 at 11:02 PM, Mohd Shukuri Mohamad Ali shuk...@biotech.upm.edu.my wrote: Hi there Sorry for the off ccp4 topics. I am planning to get a DLS to set up my crystallography lab. I got a tight budget. Around USD 8 perhaps. I'm quite new to this field. Thanks in advance for your suggestions Regards Shukuri P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
