[ccp4bb] off-topic: Compound search by chemical similarity
Dear All, Sorry for the off-topic question. Is there an easy way to search chemically close molecules and know if they are commercially available ? Many Thanks. -- .. Dr. Cedric Bauvois Cristallographie des protéines Institut de Recherches Microbiologiques J.-M. Wiame -IRMW Campus CERIA - Av. E. Gryson, 1 B-1070 Bruxelles, BELGIUM e-mail: cedric.bauv...@ulb.ac.be tél: +32 (0)2 5273634 fax: +32 (0)2 5267273 ..
[ccp4bb] Gb3 glycolipid coordinate
Dear All, Sorry for this off-topic question. I am looking for the coordinates of the full (not only the carbohydrate part) Gb3 (Globotriaosylceramide) and GM1 glycolipids. I am pretty sure that the Gb3 glycolipid has been solved by NMR (Nunez, 1982). Any help by providing me the coordinates will be greatly appreciated. thanks a lot, Julie = = = = Julie Ménétrey Institut Curie - UMR144 Laboratoire de motilité structurale 26 rue d'Ulm 75248 Paris cedex 05 Tel : 33-(0)1-56-24-68-11 !!! New phone number Fax : 33-(0)1-56-24-63-19 email : julie.menet...@curie.fr http://www.curie.fr/equipe/27 = = = =
Re: [ccp4bb] off-topic: Compound search by chemical similarity
Sigma-Aldrich has such tool under the advanced search option. Anthony On Fri, 20 Mar 2009 09:13:38 +0100, cedric bauvois wrote Dear All, Sorry for the off-topic question. Is there an easy way to search chemically close molecules and know if they are commercially available ? Many Thanks. -- .. Dr. Cedric Bauvois Cristallographie des protéines Institut de Recherches Microbiologiques J.-M. Wiame -IRMW Campus CERIA - Av. E. Gryson, 1 B-1070 Bruxelles, BELGIUM e-mail: cedric.bauv...@ulb.ac.be tél: +32 (0)2 5273634 fax: +32 (0)2 5267273 .. - Anthony Addlagatta, Ph.D. Ramanujan Fellow and Senior Scientist Center for Chemical Biology Indian Institute of Chemical Technology [IICT] Tarnaka, Hyderabad- 57, INDIA Tel:+91-40-27191583 Url: http://www.iictindia.org/zacb/Dr.%20Anthony.aspx
[ccp4bb] fake images
Yes, Harry, indeed there is a program for simulating diffraction patterns. You can get a development snapshot of it here: http://bl831.als.lbl.gov/~jamesh/mlfsom/development_snapshot.tar.gz MLFSOM (mosflm in reverse) is not the only program of its kind in existence and I don't think it is a good idea to keep the fact that they exist a secret in any way. In fact, MLFSOM has been extremely instructive in establishing how important different sources of error are to the structure determination process, and where the threshold of solvability is in real-world units. I am writing this up now and I have given several talks about it recently but it has always puzzled me that noone has EVER asked me if there is some way to identify as fake the images that come from MLFSOM. The answer to this question is: Yes, there are several. Moving on... When it comes to Garib's original question of do we need the images, I am definitely of the opinion that the answer to this question is yes. In fact, I would like to ask Garib and everyone else if they can answer the question: Why is Rcryst/Rfree from almost every protein crystal structure on the order of 20% when the intensities are generally measured to better than 5%? What are we missing? Small molecule structures are unpublishable with Rcryst Rsym because this means that the presented model does not explain the observations to within experimental error. I will tell you for nothing that if you feed fake data from MLFSOM into Elves and ARP/wARP, you will get back an Rcryst/Rfree that is roughly equal to Rsym (~5-6%), so this means that none of the sources of error I have included in MLFSOM: photon-counting noise, shutter jitter, beam flicker, sample vibration, diffuse scatter, absorption effects, funny spot shapes, radiation damage, detector read-out and calibration noise. None of these sources of error are big enough to explain the R-factor gap in macromolecular crystallography. So, if you don't know what it is we are missing, how can you be sure it is not in the image data? -James Holton MAD Scientist harry powell wrote: Hi I've heard of a tool from the Golden State which could (potentially) be used for forging diffraction images... I believe it's called mlfsom. On 18 Mar 2009, at 17:50, Felix Frolow wrote: One convincing argument I have: We will be able to catch fraud ultimately. Fraud is a devastation for structural biology. ...Unless they will be smart enough to forge diffraction data images, not a big deal. The second one - in the case of a controversy of the deposited results (possible thing) we can try to re-interpret the space group and Bravais lattice And one more, when we have time we can show that we know better to process and to refine ;-) Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972 3640 8723 Fax: ++972 3640 9407 Cellular: ++972 547 459 608 On Mar 18, 2009, at 6:41 PM, Garib Murshudov wrote: Dear all Before going into and trying to find a technical solution to the problem it would be good if decide if we need images. As far as I know if we face with a problem to solve and we know that it is necessary to solve then we find technical solution to the problem (either from other fields or we find our own solution with some elements of reinvention of new MX wheels). Do we need images to store? What kind of information we can extract from images that we cannot from amplitudes, intensities (even unmerged)? Does anybody have a convincing argument for favour of images? regards Garib On 18 Mar 2009, at 16:32, Herbert J. Bernstein wrote: Actually the radiologists who manage CT and PET scans of brains do have a solution, called DICOM, see http://medical.nema.org/. If we work together as a community we should be able to do as well as the rocket scientists and the brain surgeons' radiologists, perhaps even better. -- Herbert = Herbert J. Bernstein, Professor of Computer Science Dowling College, Kramer Science Center, KSC 121 Idle Hour Blvd, Oakdale, NY, 11769 +1-631-244-3035 y...@dowling.edu = On Wed, 18 Mar 2009, Jacob Keller wrote: Apparently it DOES take a rocket scientist to solve this problem. Maybe the brain surgeons also have a solution? JPK *** 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 *** - Original Message - From: Klaas Decanniere klaas.decanni...@vub.ac.be To:
Re: [ccp4bb] fake images
Hi James I think the answer to your question about the 'R-factor gap' is easy to answer (but not so easy to solve!). It's obviously due to the inadequacy of our models, particularly with regards to thermal motion (anisotropy, anharmonicity etc), disorder motion correlation/diffuse scatter, also bonding effects, none of which you have included in your simulation (yes I know you said you included diffuse scatter but I guess it's only the basic isotropic 'Einstein model', not the more realistic models of acoustic/optic scattering taking into account motion correlation between different atom densities). Granted most of these (with the obvious exception of anisotropy and possibly bonding effects) are not included in small molecule models either, which would lead one to conclude that the remaining effects of anharmonicity, motion correlation and disorder are much more severe in a protein crystal. Having said all that, I think it's a great idea to have the capability to generate fake data, and the more realistic it is the better! It's so much easier debugging programs when you know the right answer! Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of James Holton Sent: 20 March 2009 12:29 To: CCP4BB@jiscmail.ac.uk Subject: fake images Yes, Harry, indeed there is a program for simulating diffraction patterns. You can get a development snapshot of it here: http://bl831.als.lbl.gov/~jamesh/mlfsom/development_snapshot.tar.gz MLFSOM (mosflm in reverse) is not the only program of its kind in existence and I don't think it is a good idea to keep the fact that they exist a secret in any way. In fact, MLFSOM has been extremely instructive in establishing how important different sources of error are to the structure determination process, and where the threshold of solvability is in real-world units. I am writing this up now and I have given several talks about it recently but it has always puzzled me that noone has EVER asked me if there is some way to identify as fake the images that come from MLFSOM. The answer to this question is: Yes, there are several. Moving on... When it comes to Garib's original question of do we need the images, I am definitely of the opinion that the answer to this question is yes. In fact, I would like to ask Garib and everyone else if they can answer the question: Why is Rcryst/Rfree from almost every protein crystal structure on the order of 20% when the intensities are generally measured to better than 5%? What are we missing? Small molecule structures are unpublishable with Rcryst Rsym because this means that the presented model does not explain the observations to within experimental error. I will tell you for nothing that if you feed fake data from MLFSOM into Elves and ARP/wARP, you will get back an Rcryst/Rfree that is roughly equal to Rsym (~5-6%), so this means that none of the sources of error I have included in MLFSOM: photon-counting noise, shutter jitter, beam flicker, sample vibration, diffuse scatter, absorption effects, funny spot shapes, radiation damage, detector read-out and calibration noise. None of these sources of error are big enough to explain the R-factor gap in macromolecular crystallography. So, if you don't know what it is we are missing, how can you be sure it is not in the image data? -James Holton MAD Scientist harry powell wrote: Hi I've heard of a tool from the Golden State which could (potentially) be used for forging diffraction images... I believe it's called mlfsom. On 18 Mar 2009, at 17:50, Felix Frolow wrote: One convincing argument I have: We will be able to catch fraud ultimately. Fraud is a devastation for structural biology. ...Unless they will be smart enough to forge diffraction data images, not a big deal. The second one - in the case of a controversy of the deposited results (possible thing) we can try to re-interpret the space group and Bravais lattice And one more, when we have time we can show that we know better to process and to refine ;-) Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972 3640 8723 Fax: ++972 3640 9407 Cellular: ++972 547 459 608 On Mar 18, 2009, at 6:41 PM, Garib Murshudov wrote: Dear all Before going into and trying to find a technical solution to the problem it would be good if decide if we need images. As far as I know if we face with a problem to solve and we know that it is necessary to solve then we find technical solution to the problem (either from other fields or we find our own solution with some elements of reinvention of new MX wheels). Do we need images to store? What kind
Re: [ccp4bb] fake images
Dear James, About 30 years ago I wrote a clone of the program ORTEP; the clone was called XP, but unfortunately Microsoft later stole the name and brought it into disrepute. So that I would always be able to see at a glance which plots were 'genuine ORTEP' and which were my clone, I built in a sort of watermark (a slightly different way of shading the ellipsoids). I hope that you have done something similar in mlfsom! But please don't reveal what it is to ccp4bb, I suggest that you deposit the information in encripted form with the PDB. Otherwise you may suffer the same fate as I did: the 'ORTEP' plot on the cover of the February 2009 Acta C appears to have been made by a clone of my clone! The information that data produced by mlfsom give Rcryst ~ Rsym is very interesting and makes it even more likely that the typical R-values of 19.9% for proteins have something to do with the ca. 50% of the structure that is rather poorly modeled - the 'solvent' region. Best wishes, 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 Fri, 20 Mar 2009, James Holton wrote: Yes, Harry, indeed there is a program for simulating diffraction patterns. You can get a development snapshot of it here: http://bl831.als.lbl.gov/~jamesh/mlfsom/development_snapshot.tar.gz MLFSOM (mosflm in reverse) is not the only program of its kind in existence and I don't think it is a good idea to keep the fact that they exist a secret in any way. In fact, MLFSOM has been extremely instructive in establishing how important different sources of error are to the structure determination process, and where the threshold of solvability is in real-world units. I am writing this up now and I have given several talks about it recently but it has always puzzled me that noone has EVER asked me if there is some way to identify as fake the images that come from MLFSOM. The answer to this question is: Yes, there are several. Moving on... When it comes to Garib's original question of do we need the images, I am definitely of the opinion that the answer to this question is yes. In fact, I would like to ask Garib and everyone else if they can answer the question: Why is Rcryst/Rfree from almost every protein crystal structure on the order of 20% when the intensities are generally measured to better than 5%? What are we missing? Small molecule structures are unpublishable with Rcryst Rsym because this means that the presented model does not explain the observations to within experimental error. I will tell you for nothing that if you feed fake data from MLFSOM into Elves and ARP/wARP, you will get back an Rcryst/Rfree that is roughly equal to Rsym (~5-6%), so this means that none of the sources of error I have included in MLFSOM: photon-counting noise, shutter jitter, beam flicker, sample vibration, diffuse scatter, absorption effects, funny spot shapes, radiation damage, detector read-out and calibration noise. None of these sources of error are big enough to explain the R-factor gap in macromolecular crystallography. So, if you don't know what it is we are missing, how can you be sure it is not in the image data? -James Holton MAD Scientist harry powell wrote: Hi I've heard of a tool from the Golden State which could (potentially) be used for forging diffraction images... I believe it's called mlfsom. On 18 Mar 2009, at 17:50, Felix Frolow wrote: One convincing argument I have: We will be able to catch fraud ultimately. Fraud is a devastation for structural biology. ...Unless they will be smart enough to forge diffraction data images, not a big deal. The second one - in the case of a controversy of the deposited results (possible thing) we can try to re-interpret the space group and Bravais lattice And one more, when we have time we can show that we know better to process and to refine ;-) Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972 3640 8723 Fax: ++972 3640 9407 Cellular: ++972 547 459 608 On Mar 18, 2009, at 6:41 PM, Garib Murshudov wrote: Dear all Before going into and trying to find a technical solution to the problem it would be good if decide if we need images. As far as I know if we face with a problem to solve and we know that it is necessary to solve then we find technical solution to the problem (either from other fields or we find our own solution with some elements of reinvention of new MX wheels). Do we need images to store? What kind of information we can extract from images that we cannot from
Re: [ccp4bb] purification
Hi Kien- Are you basing extensive proteolysis (degradation) on an SDS-PAGE result alone? Are you monitoring the elution profile from Ni/NTA? Do you see numerous A280 peaks for your elution? Sample prep of membrane proteins for SDS-PAGE is very trial-and-error. Heating the samples may cause weird aggregation to occur. You often see a ladder effect and anomalous mobilities of membrane proteins on SDS-PAGE. This is clarified (normally) with a native gel and/or with analytical SEC. Now, I admit, this is normally for oligomeric membrane proteins- I'm not sure if yours is oligomeric or not. And this may very well not be your main problem because I would expect you would see some signal on your blot from your Ni/NTA flow through. Hope this helps- Brad On Thu, Mar 19, 2009 at 7:53 PM, Kn Ly kn...@auckland.ac.nz wrote: Hello everyone, I am expressing a 100 KDa eukaryotic membrane protein in E coli. The protein is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa. However, the protein get severely degraded so after putting through a Ni-NTA column, the protein came out with a lot of contaminant bands. I did a western blot using antibody against his tag. The total cell lysate gave signals in many bands. The flow through did not give any signal and the eluted fraction again gave many band signals, indicating the protein got degraded copiously even before purification. I used Roche protease inhibitor tablet and still got a lot of degradation. Can anyone suggest a way to avoid the problem or a purification method so that I can purify the intact protein while keeping away the unwanted degraded fractions. Thanks heaps in advance. Kien
Re: [ccp4bb] fake images and R-Rfree values
I must add something... ID14-2 beam line in our hands produced during first decade of 2000's data sets that for structures at about 1.8 - 1.6 Angstrom constantly leaded to a very good R - Rfree (in the range of 12 %-18%) As an example see PDB entry 1Y9A. If will be needed I will supply diffraction data for several structures. Such quality produces problems as a referee accused us by over-fitting Rfree whatever it means. He also new what is a theoretical difference of R-Rfree in any resolution? During our discussions recently with Dr Alexander Popov, responsible for the ESRF ID23-1 beam line on this matter Dr Popov suggest that it is because the X-ray beam on this station is very large. I also think that its is largely parallel, old quality of the synchrotron lines. Maybe other people who noticed similar thing may add more. BTW final data set scaling statistics (SCALEPAK) is shown: Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I errorstat. Chi**2 R- facR-fac 20.00 3.79 7081.7 258.9 136.6 1.172 0.029 0.031 3.79 3.01 3894.0 157.2 95.2 1.2780.038 0.040 3.01 2.63 1514.9 97.1 64.8 0.9560.056 0.140 2.63 2.39 938.8 75.4 57.5 0.9760.074 0.080 2.39 2.22 717.1 74.5 59.5 0.9250.093 0.100 2.22 2.09 534.2 71.6 62.2 0.9470.122 0.116 2.09 1.99 398.6 70.6 65.1 0.9150.164 0.169 1.99 1.90 258.1 69.1 66.3 0.957 0.256 0.299 All reflections 1875.3 108.175.5 1.018 0.051 0.046 Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972 3640 8723 Fax: ++972 3640 9407 Cellular: ++972 547 459 608 On Mar 20, 2009, at 2:29 PM, James Holton wrote: Yes, Harry, indeed there is a program for simulating diffraction patterns. You can get a development snapshot of it here: http://bl831.als.lbl.gov/~jamesh/mlfsom/development_snapshot.tar.gz MLFSOM (mosflm in reverse) is not the only program of its kind in existence and I don't think it is a good idea to keep the fact that they exist a secret in any way. In fact, MLFSOM has been extremely instructive in establishing how important different sources of error are to the structure determination process, and where the threshold of solvability is in real-world units. I am writing this up now and I have given several talks about it recently but it has always puzzled me that noone has EVER asked me if there is some way to identify as fake the images that come from MLFSOM. The answer to this question is: Yes, there are several. Moving on... When it comes to Garib's original question of do we need the images, I am definitely of the opinion that the answer to this question is yes. In fact, I would like to ask Garib and everyone else if they can answer the question: Why is Rcryst/Rfree from almost every protein crystal structure on the order of 20% when the intensities are generally measured to better than 5%? What are we missing? Small molecule structures are unpublishable with Rcryst Rsym because this means that the presented model does not explain the observations to within experimental error. I will tell you for nothing that if you feed fake data from MLFSOM into Elves and ARP/ wARP, you will get back an Rcryst/Rfree that is roughly equal to Rsym (~5-6%), so this means that none of the sources of error I have included in MLFSOM: photon-counting noise, shutter jitter, beam flicker, sample vibration, diffuse scatter, absorption effects, funny spot shapes, radiation damage, detector read-out and calibration noise. None of these sources of error are big enough to explain the R-factor gap in macromolecular crystallography. So, if you don't know what it is we are missing, how can you be sure it is not in the image data? -James Holton MAD Scientist harry powell wrote: Hi I've heard of a tool from the Golden State which could (potentially) be used for
Re: [ccp4bb] fake images and R-Rfree values
Felix I would be very surprised if anyone could calculate the expected Rfree in this case with any degree of reliability, since it seems to have been refined with a combination of TLS and rigid-bond/sphericity anisotropic ADP restraints. Taking account of restraints, particularly thermal ones which have to be assigned ad hoc weights in the absence of experimentally observed ones (as is the case with geometrical restraints) in the calculation of Rfree is non-trivial! Cheers -- Ian -Original Message- From: owner-ccp...@jiscmail.ac.uk [mailto:owner-ccp...@jiscmail.ac.uk] On Behalf Of Felix Frolow Sent: 20 March 2009 14:31 To: James Holton Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] fake images and R-Rfree values I must add something... ID14-2 beam line in our hands produced during first decade of 2000's data sets that for structures at about 1.8 - 1.6 Angstrom constantly leaded to a very good R - Rfree (in the range of 12 %-18%) As an example see PDB entry 1Y9A. If will be needed I will supply diffraction data for several structures. Such quality produces problems as a referee accused us by over-fitting Rfree whatever it means. He also new what is a theoretical difference of R-Rfree in any resolution? During our discussions recently with Dr Alexander Popov, responsible for the ESRF ID23-1 beam line on this matter Dr Popov suggest that it is because the X-ray beam on this station is very large. I also think that its is largely parallel, old quality of the synchrotron lines. Maybe other people who noticed similar thing may add more. BTW final data set scaling statistics (SCALEPAK) is shown: Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I errorstat. Chi**2 R- facR-fac 20.00 3.797081.7 258.9 136.6 1.172 0.029 0.031 3.79 3.013894.0 157.2 95.2 1.2780.038 0.040 3.01 2.631514.9 97.1 64.8 0.9560.056 0.140 2.63 2.39 938.8 75.4 57.5 0.9760.074 0.080 2.39 2.22 717.1 74.5 59.5 0.9250.093 0.100 2.22 2.09 534.2 71.6 62.2 0.9470.122 0.116 2.09 1.99 398.6 70.6 65.1 0.9150.164 0.169 1.99 1.90 258.1 69.1 66.3 0.957 0.256 0.299 All reflections 1875.3 108.175.5 1.018 0.051 0.046 Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica D, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972 3640 8723 Fax: ++972 3640 9407 Cellular: ++972 547 459 608 On Mar 20, 2009, at 2:29 PM, James Holton wrote: Yes, Harry, indeed there is a program for simulating diffraction patterns. You can get a development snapshot of it here: http://bl831.als.lbl.gov/~jamesh/mlfsom/development_snapshot.tar.gz MLFSOM (mosflm in reverse) is not the only program of its kind in existence and I don't think it is a good idea to keep the fact that they exist a secret in any way. In fact, MLFSOM has been extremely instructive in establishing how important different sources of error are to the structure determination process, and where the threshold of solvability is in real-world units. I am writing this up now and I have given several talks about it recently but it has always puzzled me that noone has EVER asked me if there is some way to identify as fake the images that come from MLFSOM. The answer to this question is: Yes, there are several. Moving on... When it comes to Garib's original question of do we need the images, I am definitely of the opinion that the answer to this question is yes. In fact, I would like to ask Garib and everyone else if they can answer the question: Why is Rcryst/Rfree from almost every protein crystal structure on the order of 20% when the intensities are generally measured to better than 5%? What are we missing? Small molecule structures are unpublishable with Rcryst Rsym because this means that the presented model does not explain the observations to within experimental error. I will tell you for nothing that if you feed fake data from MLFSOM into Elves and ARP/
Re: [ccp4bb] purification
Try running the Ni column as fast as possible and putting concentrated EDTA in the fraction collector tubes before you start, to minimize opportunities for metal-dependent proteases. It may not be a magic bullet but it can't hurt. Phoebe Original message Date: Thu, 19 Mar 2009 23:53:14 + From: Kn Ly kn...@auckland.ac.nz Subject: [ccp4bb] purification To: CCP4BB@JISCMAIL.AC.UK Hello everyone, I am expressing a 100 KDa eukaryotic membrane protein in E coli. The protein is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa. However, the protein get severely degraded so after putting through a Ni-NTA column, the protein came out with a lot of contaminant bands. I did a western blot using antibody against his tag. The total cell lysate gave signals in many bands. The flow through did not give any signal and the eluted fraction again gave many band signals, indicating the protein got degraded copiously even before purification. I used Roche protease inhibitor tablet and still got a lot of degradation. Can anyone suggest a way to avoid the problem or a purification method so that I can purify the intact protein while keeping away the unwanted degraded fractions. Thanks heaps in advance. Kien Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] purification
Dear Kien, you might also try a different resin/ metal ion. If I remember correctly, the technician where I did my PhD had much better results with a Talon resin using Co instead of Ni: you can use much less Imidazole, only 5-10mM for washing and 50mM for elution. If you can go back to cloning, try a C-terminal fusion protein. That should prevent you from purifying shorter product caused by truncation of translation: if the His-tag is at the C-terminus, everything before (your target protein) would be there, too! Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Fri, 20 Mar 2009, Phoebe Rice wrote: Try running the Ni column as fast as possible and putting concentrated EDTA in the fraction collector tubes before you start, to minimize opportunities for metal-dependent proteases. It may not be a magic bullet but it can't hurt. Phoebe Original message Date: Thu, 19 Mar 2009 23:53:14 + From: Kn Ly kn...@auckland.ac.nz Subject: [ccp4bb] purification To: CCP4BB@JISCMAIL.AC.UK Hello everyone, I am expressing a 100 KDa eukaryotic membrane protein in E coli. The protein is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa. However, the protein get severely degraded so after putting through a Ni-NTA column, the protein came out with a lot of contaminant bands. I did a western blot using antibody against his tag. The total cell lysate gave signals in many bands. The flow through did not give any signal and the eluted fraction again gave many band signals, indicating the protein got degraded copiously even before purification. I used Roche protease inhibitor tablet and still got a lot of degradation. Can anyone suggest a way to avoid the problem or a purification method so that I can purify the intact protein while keeping away the unwanted degraded fractions. Thanks heaps in advance. Kien Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] fake images
After consultation with Ton Spek, I should correct my last email. It turns out that my 'watermark' was not clever enough, because PLATON - his program used to make the picture that I had randomly picked as an example - can emulate the XP watermark (the way of shading the ellipsoids which I intended to be different from the 'genuine ORTEP') rather well, and had indeed done so in the picture in question. However it is technically an 'ORTEP clone' (like XP), not an 'XP clone'. I am using 'clone' to mean that one has intentionally created the 'same look and feel' as an existing program, not to imply that the same code is used. This shows how difficult it will be for James to include an unforgeable 'watermark' in his calculated frames, though I would still encourage him to try. Being able to emulate the experiment so well is wonderful for debugging the programs used to process the frames, but it will surely make fraud more difficult to detect! 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 Fri, 20 Mar 2009, George M. Sheldrick wrote: Dear James, About 30 years ago I wrote a clone of the program ORTEP; the clone was called XP, but unfortunately Microsoft later stole the name and brought it into disrepute. So that I would always be able to see at a glance which plots were 'genuine ORTEP' and which were my clone, I built in a sort of watermark (a slightly different way of shading the ellipsoids). I hope that you have done something similar in mlfsom! But please don't reveal what it is to ccp4bb, I suggest that you deposit the information in encripted form with the PDB. Otherwise you may suffer the same fate as I did: the 'ORTEP' plot on the cover of the February 2009 Acta C appears to have been made by a clone of my clone! The information that data produced by mlfsom give Rcryst ~ Rsym is very interesting and makes it even more likely that the typical R-values of 19.9% for proteins have something to do with the ca. 50% of the structure that is rather poorly modeled - the 'solvent' region. Best wishes, 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 Fri, 20 Mar 2009, James Holton wrote: Yes, Harry, indeed there is a program for simulating diffraction patterns. You can get a development snapshot of it here: http://bl831.als.lbl.gov/~jamesh/mlfsom/development_snapshot.tar.gz MLFSOM (mosflm in reverse) is not the only program of its kind in existence and I don't think it is a good idea to keep the fact that they exist a secret in any way. In fact, MLFSOM has been extremely instructive in establishing how important different sources of error are to the structure determination process, and where the threshold of solvability is in real-world units. I am writing this up now and I have given several talks about it recently but it has always puzzled me that noone has EVER asked me if there is some way to identify as fake the images that come from MLFSOM. The answer to this question is: Yes, there are several. Moving on... When it comes to Garib's original question of do we need the images, I am definitely of the opinion that the answer to this question is yes. In fact, I would like to ask Garib and everyone else if they can answer the question: Why is Rcryst/Rfree from almost every protein crystal structure on the order of 20% when the intensities are generally measured to better than 5%? What are we missing? Small molecule structures are unpublishable with Rcryst Rsym because this means that the presented model does not explain the observations to within experimental error. I will tell you for nothing that if you feed fake data from MLFSOM into Elves and ARP/wARP, you will get back an Rcryst/Rfree that is roughly equal to Rsym (~5-6%), so this means that none of the sources of error I have included in MLFSOM: photon-counting noise, shutter jitter, beam flicker, sample vibration, diffuse scatter, absorption effects, funny spot shapes, radiation damage, detector read-out and calibration noise. None of these sources of error are big enough to explain the R-factor gap in macromolecular crystallography. So, if you don't know what it is we are missing, how can you be sure it is not in the image data? -James Holton MAD Scientist harry powell wrote: Hi I've heard of a tool from the Golden State which could (potentially) be used for forging diffraction images... I believe it's called mlfsom. On 18 Mar 2009, at 17:50, Felix Frolow wrote: One convincing argument I have: We will be able to catch fraud ultimately. Fraud is a devastation for structural
[ccp4bb] Refmac failed at the end of run
Hi all, I am using refmac in ccp4i 6.1.1 (installed in windows). It runs well when using automatic weight, but failed when using user-specified weight (0.12 in this case). I attach the error message as follow. Your help is very much appreciated. .. 18 0.2064 0.2348 0.846407628. 21897.0 0.0075 0.318 1.170 0.502 0.076 19 0.2065 0.2343 0.846407625. 21896.4 0.0075 0.318 1.169 0.501 0.076 20 0.2065 0.2347 0.846407629. 21897.3 0.0075 0.318 1.167 0.501 0.076 $$ $TEXT:Warning: $$ comment $$ WARNING: CCPOPN Logical name C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac has no asso $$ Open failed: Unit: 9, File: C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac (logical: C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac) Refmac_5.5.0072: Open failed: File: C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac Times: User: 0.0s System:0.0s Elapsed:27:38 /pre /html *** * Information from CCP4Interface script *** The program run with command: refmac5 XYZIN C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/472cr8c10_v1-coot-4_refmac1.pdb XYZOUT C:/Ccp4Temp/47crc10_3_2_pdb_1.tmp HKLIN C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/472cr8nsls_refmac1.mtz HKLOUT C:/Ccp4Temp/47crc10_3_3_mtz_1.tmp LIBIN C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/libcheck_C10.cif TLSIN C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/472cr8c10_v1-coot-4_edit_tls1_refmac1.tls TLSOUT C:/Ccp4Temp/47crc10_3_4_tls_1.tmp LIBOUT C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/47crc10_3_lib.cif has failed with error message Last system error message: Unknown error Refmac_5.5.0072: Open failed: File: C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac *** #CCP4I TERMINATION STATUS 0 Last system error message: Unknown error Refmac_5.5.0072: Open failed: File: C:/Chen/Xtal/cvir/472cr8_c10/work2/refmac/DepositFiles/472cr8nsls/unknown211008:17:49:34.refmac #CCP4I TERMINATION TIME 20 Mar 2009 13:23:28 #CCP4I MESSAGE Task failed -- Best regards, Joe
Re: [ccp4bb] fake images
Steganographic encoding of a PGP-encoded graphical segment should not be difficult to incorporate... The problem of course is that the delicate signal of such a 'hidden' message would be subject to deliberate erasure unless the encoding is actually done on the level of reflections themselves (i.e. something clever like arranging background pixels differing by 1 bit in a specific pattern around specific reflections). The alternative is to ask for ubiquitous incorporation of such watermarks into all detector-generated images. The absence of watermarks would indicate either 1) an older detector which cannot be software-updated to produce the codes or 2) fake image. Returning to real world - even if images are faked and fake structures are produced, and all sorts of N-S-C papers are written - experience shows that fraud does not survive for too long, especially when the subject matter is 'hot' and interesting, and doubly so if the results are unexpected or remarkable in some other way. I doubt that people would go to the trouble of faking routine structures, since those generally do not make their authors any money/fame/recognition. Artem After consultation with Ton Spek, I should correct my last email. It turns out that my 'watermark' was not clever enough, because PLATON - his program used to make the picture that I had randomly picked as an example - can emulate the XP watermark (the way of shading the ellipsoids which I intended to be different from the 'genuine ORTEP') rather well, and had indeed done so in the picture in question. However it is technically an 'ORTEP clone' (like XP), not an 'XP clone'. I am using 'clone' to mean that one has intentionally created the 'same look and feel' as an existing program, not to imply that the same code is used. This shows how difficult it will be for James to include an unforgeable 'watermark' in his calculated frames, though I would still encourage him to try. Being able to emulate the experiment so well is wonderful for debugging the programs used to process the frames, but it will surely make fraud more difficult to detect! 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 Fri, 20 Mar 2009, George M. Sheldrick wrote: Dear James, About 30 years ago I wrote a clone of the program ORTEP; the clone was called XP, but unfortunately Microsoft later stole the name and brought it into disrepute. So that I would always be able to see at a glance which plots were 'genuine ORTEP' and which were my clone, I built in a sort of watermark (a slightly different way of shading the ellipsoids). I hope that you have done something similar in mlfsom! But please don't reveal what it is to ccp4bb, I suggest that you deposit the information in encripted form with the PDB. Otherwise you may suffer the same fate as I did: the 'ORTEP' plot on the cover of the February 2009 Acta C appears to have been made by a clone of my clone! The information that data produced by mlfsom give Rcryst ~ Rsym is very interesting and makes it even more likely that the typical R-values of 19.9% for proteins have something to do with the ca. 50% of the structure that is rather poorly modeled - the 'solvent' region. Best wishes, 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 Fri, 20 Mar 2009, James Holton wrote: Yes, Harry, indeed there is a program for simulating diffraction patterns. You can get a development snapshot of it here: http://bl831.als.lbl.gov/~jamesh/mlfsom/development_snapshot.tar.gz MLFSOM (mosflm in reverse) is not the only program of its kind in existence and I don't think it is a good idea to keep the fact that they exist a secret in any way. In fact, MLFSOM has been extremely instructive in establishing how important different sources of error are to the structure determination process, and where the threshold of solvability is in real-world units. I am writing this up now and I have given several talks about it recently but it has always puzzled me that noone has EVER asked me if there is some way to identify as fake the images that come from MLFSOM. The answer to this question is: Yes, there are several. Moving on... When it comes to Garib's original question of do we need the images, I am definitely of the opinion that the answer to this question is yes. In fact, I would like to ask Garib and everyone else if they can answer the question: Why is Rcryst/Rfree from almost every protein crystal structure on the order of 20% when the intensities are generally measured to better than 5%? What are we missing? Small molecule structures are unpublishable with Rcryst Rsym because this
Re: [ccp4bb] purification
Hi Kien, As Artem pointed out earlier. Are you sure that your protein folded correctly. You want to make sure that the protein is expressed in lipid membrane not in inclusion body. If so, considering changing the host might be a good idea. Also, did you use any kind of detergent when you extract the protein? Choice of detergent is also very important in purifying a membrane protein. Try different detergents if you havent used one and it may work. Talon resin works fine with me when I was working with ion channel. Hope that helps, Puey On Fri, Mar 20, 2009 at 9:32 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Kien, you might also try a different resin/ metal ion. If I remember correctly, the technician where I did my PhD had much better results with a Talon resin using Co instead of Ni: you can use much less Imidazole, only 5-10mM for washing and 50mM for elution. If you can go back to cloning, try a C-terminal fusion protein. That should prevent you from purifying shorter product caused by truncation of translation: if the His-tag is at the C-terminus, everything before (your target protein) would be there, too! Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Fri, 20 Mar 2009, Phoebe Rice wrote: Try running the Ni column as fast as possible and putting concentrated EDTA in the fraction collector tubes before you start, to minimize opportunities for metal-dependent proteases. It may not be a magic bullet but it can't hurt. Phoebe Original message Date: Thu, 19 Mar 2009 23:53:14 + From: Kn Ly kn...@auckland.ac.nz Subject: [ccp4bb] purification To: CCP4BB@JISCMAIL.AC.UK Hello everyone, I am expressing a 100 KDa eukaryotic membrane protein in E coli. The protein is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KDa. However, the protein get severely degraded so after putting through a Ni-NTA column, the protein came out with a lot of contaminant bands. I did a western blot using antibody against his tag. The total cell lysate gave signals in many bands. The flow through did not give any signal and the eluted fraction again gave many band signals, indicating the protein got degraded copiously even before purification. I used Roche protease inhibitor tablet and still got a lot of degradation. Can anyone suggest a way to avoid the problem or a purification method so that I can purify the intact protein while keeping away the unwanted degraded fractions. Thanks heaps in advance. Kien Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] fake images
I just want to emphasize the usefulness of MLFSOM as a educational tool. I made numerous figures showing principles and problems of data collection using James' program. I can also assure you that in order to use it properly, you need to know something about image data. I only scratched the surface and I think it would be hard work to fake the images in a way that later expert forensics would not readily provide evidence. Also, there are 'watermarks' available from cryptographic methods that are even 'post-processing' resistant. Regrettably, as long as demonstrated forgeries and irresponsibly ignorant mistakes and their cover-ups have no consequences whatsoever, the temptation will remain. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of George M. Sheldrick Sent: Friday, March 20, 2009 10:27 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] fake images After consultation with Ton Spek, I should correct my last email. It turns out that my 'watermark' was not clever enough, because PLATON - his program used to make the picture that I had randomly picked as an example - can emulate the XP watermark (the way of shading the ellipsoids which I intended to be different from the 'genuine ORTEP') rather well, and had indeed done so in the picture in question. However it is technically an 'ORTEP clone' (like XP), not an 'XP clone'. I am using 'clone' to mean that one has intentionally created the 'same look and feel' as an existing program, not to imply that the same code is used. This shows how difficult it will be for James to include an unforgeable 'watermark' in his calculated frames, though I would still encourage him to try. Being able to emulate the experiment so well is wonderful for debugging the programs used to process the frames, but it will surely make fraud more difficult to detect!
[ccp4bb] surface area calcualtion
Hello all can any one suggest any server or tool which can calculate the burried surface area between the domains of the same monomer? PISA calculates the interfacial surace area between the monomer or oligomer. Can areamol calculate it? Thanks in advance Thomas
Re: [ccp4bb] surface area calcualtion
Thomas, The Areaimol program within the CCP4 suite should do what you're looking for: http://www.ccp4.ac.uk/html/areaimol.html Although its a calculation of surface accessability, you can delete sections of your protein to get at the buried area between two sections of your protein. Cheers, Jim On Fri, Mar 20, 2009 at 2:48 PM, Jhon Thomas jhon1.tho...@gmail.com wrote: Hello all can any one suggest any server or tool which can calculate the burried surface area between the domains of the same monomer? PISA calculates the interfacial surace area between the monomer or oligomer. Can areamol calculate it? Thanks in advance Thomas -- 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
[ccp4bb] Problems with phasing a protein (1300aa)
Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. 'Se' labeled crystals diffracts maximally up to 3.5 to 4 A and dies very quickly on most of the beamlines. We have scanned at Se wavelength and it gives very strong signal as it contain ~45 Se in AU (1300 aa). It is difficult to collect a complete dataset (maximally we get 50-60 % completion with Rmerge ~15) out of one crystal on regular beamline. At microfocus beamline (APS), we were able to collect data in 3-4 batches and merge them to get a complete dataset (Rmerge ~18-20) out of one crystal. We used data collected on microfocus beamline (at peak wavelength) for locating heavy atom position using SHELXD, Solve and Phenix.hyss. SOlve and Phenix.hyss find very few heavy atom sites 1-5 whereas SHELX-CDE lists many but shows no difference in original and inverted (contrast and connectivity). Our phasing attempts with datasets obtained after merging two incomplete dataset from two different crystal has also been disappointing. My another worry is absolute value of average intensity, which seems to be quite low in most of the datasets. Below I have pasted last table of scale.log (HKL2000). Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 7.5345.4 1.6 1.3 1.295 0.055 0.047 7.53 5.9811.4 1.3 1.3 0.672 0.135 0.114 5.98 5.2311.2 1.6 1.6 0.643 0.171 0.152 5.23 4.7516.8 2.0 1.9 0.736 0.148 0.118 4.75 4.4118.8 2.2 2.2 0.739 0.143 0.132 4.41 4.1514.6 2.4 2.4 0.653 0.190 0.175 4.15 3.9411.3 2.5 2.5 0.582 0.247 0.226 3.94 3.7710.1 2.8 2.8 0.511 0.280 0.191 3.77 3.63 8.0 3.1 3.1 0.450 0.315 0.285 3.63 3.50 7.6 3.3 3.2 0.483 0.311 0.270 All reflections 15.5 2.3 2.2 0.694 0.153 0.106 Now, I want you to help me by answering some of my queries: 1. Is it possible to get MAD/SAD phasing done from a dataset having more than 15% Rmerge and resolution in the range of 4 - 4.5 Ang? 2. Will a complete data set obtained from merging various batches(30-40 frames each) from one or more than one crystal will have proper anomalous signal for phasing? I am worried as weak anomalous signal may get lost while merging. 3. Will such a low value of average Intensities (as shown above from HKL scale log file) will be good enough for MAD/SAD phasing or I really need to improve crystal quality for stronger diffraction. 4. For MAD/SAD phasing, till what resolution we need to have anomalous signal ? Many of my datasets shows anomalous signal maximally up to 6-8 A (calculated using Phenix.xtriage). 5. Since I have low resolution (3.5 to 4 A)data, relatively high Rmerge (14-15%), lower value of average intensity, anomalous signal up to 6 A or so. which programs will be more useful for heavy atom location and to prevent false positives from being selected? We have been also trying our luck with heavy atom soak but that also has not been very encouraging. I would appreciate any suggestions in this regard. Thanks in advance and sorry for such a long mail. Kumar
Re: [ccp4bb] Problems with phasing a protein (1300aa)
Radiation Damage? Why don't you try scaling back on the time of each frame, get better completeness and redundancy, while taking a hit on resolution? That could get you your heavy atoms, and then you could apply those phases to a roasted crystal. The heavy atoms are the first to go, it seems. Jacob *** 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 *** - Original Message - From: Kumar To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, March 20, 2009 2:26 PM Subject: [ccp4bb] Problems with phasing a protein (1300aa) Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. 'Se' labeled crystals diffracts maximally up to 3.5 to 4 A and dies very quickly on most of the beamlines. We have scanned at Se wavelength and it gives very strong signal as it contain ~45 Se in AU (1300 aa). It is difficult to collect a complete dataset (maximally we get 50-60 % completion with Rmerge ~15) out of one crystal on regular beamline. At microfocus beamline (APS), we were able to collect data in 3-4 batches and merge them to get a complete dataset (Rmerge ~18-20) out of one crystal. We used data collected on microfocus beamline (at peak wavelength) for locating heavy atom position using SHELXD, Solve and Phenix.hyss. SOlve and Phenix.hyss find very few heavy atom sites 1-5 whereas SHELX-CDE lists many but shows no difference in original and inverted (contrast and connectivity). Our phasing attempts with datasets obtained after merging two incomplete dataset from two different crystal has also been disappointing. My another worry is absolute value of average intensity, which seems to be quite low in most of the datasets. Below I have pasted last table of scale.log (HKL2000). Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 7.5345.4 1.6 1.3 1.295 0.055 0.047 7.53 5.9811.4 1.3 1.3 0.672 0.135 0.114 5.98 5.2311.2 1.6 1.6 0.643 0.171 0.152 5.23 4.7516.8 2.0 1.9 0.736 0.148 0.118 4.75 4.4118.8 2.2 2.2 0.739 0.143 0.132 4.41 4.1514.6 2.4 2.4 0.653 0.190 0.175 4.15 3.9411.3 2.5 2.5 0.582 0.247 0.226 3.94 3.7710.1 2.8 2.8 0.511 0.280 0.191 3.77 3.63 8.0 3.1 3.1 0.450 0.315 0.285 3.63 3.50 7.6 3.3 3.2 0.483 0.311 0.270 All reflections 15.5 2.3 2.2 0.694 0.153 0.106 Now, I want you to help me by answering some of my queries: 1. Is it possible to get MAD/SAD phasing done from a dataset having more than 15% Rmerge and resolution in the range of 4 - 4.5 Ang? 2. Will a complete data set obtained from merging various batches(30-40 frames each) from one or more than one crystal will have proper anomalous signal for phasing? I am worried as weak anomalous signal may get lost while merging. 3. Will such a low value of average Intensities (as shown above from HKL scale log file) will be good enough for MAD/SAD phasing or I really need to improve crystal quality for stronger diffraction. 4. For MAD/SAD phasing, till what resolution we need to have anomalous signal ? Many of my datasets shows anomalous signal maximally up to 6-8 A (calculated using Phenix.xtriage). 5. Since I have low resolution (3.5 to 4 A)data, relatively high Rmerge (14-15%), lower value of average intensity, anomalous signal up to 6 A or so. which programs will be more useful for heavy atom location and to prevent false positives from being selected? We have been also trying our luck with heavy atom soak but that also has not been very encouraging. I would appreciate any suggestions in this regard. Thanks in advance and sorry for such a long mail. Kumar
Re: [ccp4bb] Problems with phasing a protein (1300aa)
Well, what do you expect the anomalous signal contributed from your 45 seleniums in a perfect world to be when the asymmetric unit contains 1300 aa? Do you think a dataset with Rmerge of ~15% is good enough to detect a signal of even 2%? (Note: I did not do the calculation, so I just made up the number of 2%.) Jim On Fri, 20 Mar 2009, Kumar wrote: Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. ...
Re: [ccp4bb] Problems with phasing a protein (1300aa)
One can estimate this from http://www.ruppweb.org/new_comp/anomalous_scattering.htm and the answer as Jim says is no. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Pflugrath Sent: Friday, March 20, 2009 1:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems with phasing a protein (1300aa) Well, what do you expect the anomalous signal contributed from your 45 seleniums in a perfect world to be when the asymmetric unit contains 1300 aa? Do you think a dataset with Rmerge of ~15% is good enough to detect a signal of even 2%? (Note: I did not do the calculation, so I just made up the number of 2%.) Jim On Fri, 20 Mar 2009, Kumar wrote: Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. ...
Re: [ccp4bb] Purification
You can try using affinity tags on both the N- and C-termini of the protein, eg. MBP on N and His on C. ho Date: Thu, 19 Mar 2009 23:53:14 + From: Kn Ly kn...@auckland.ac.nz Subject: purification Hello everyone, I am expressing a 100 KDa eukaryotic membrane protein in E coli. The prot= ein is fused to 6His-MBP in the N terminus and the resulting mass is ~ 150 KD= a. However, the protein get severely degraded so after putting through a Ni-= NTA column, the protein came out with a lot of contaminant bands. I did a western blot using antibody against his tag. The total cell lysate gave signals in many bands. The flow through did not give any signal and the eluted fraction again gave many band signals, indicating the protein got degraded copiously even before purification. I used Roche protease inhibitor tablet and still got a lot of degradation= . Can anyone suggest a way to avoid the problem or a purification method so= that I can purify the intact protein while keeping away the unwanted degraded fractions. Thanks heaps in advance. Kien
Re: [ccp4bb] Problems with phasing a protein (1300aa)
this cant be true, in the idea case (not Rmerge 15%, then again one can apply a resolution cutoff, perhaps, while this sounds like a very desperate case) the answer must be yes. didnt do the calculation right now (but it _should_ back this up) we for instance have solved a structure long time ago -- and this probably wasnt on the limit (well it was at the time but not anymore), 365 res x 8 in AU and 80 Se. at least looking at the SnB success list (very old list ) http://www.hwi.buffalo.edu/SnB/SnBSuccesses.htm there are plenty of others. -tommi On Mar 20, 2009, at 10:53 PM, Bernhard Rupp wrote: One can estimate this from http://www.ruppweb.org/new_comp/anomalous_scattering.htm and the answer as Jim says is no. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Pflugrath Sent: Friday, March 20, 2009 1:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems with phasing a protein (1300aa) Well, what do you expect the anomalous signal contributed from your 45 seleniums in a perfect world to be when the asymmetric unit contains 1300 aa? Do you think a dataset with Rmerge of ~15% is good enough to detect a signal of even 2%? (Note: I did not do the calculation, so I just made up the number of 2%.) Jim On Fri, 20 Mar 2009, Kumar wrote: Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. ... Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] Problems with phasing a protein (1300aa)
Kumar, As many here have pointed out - this is not likely to work. However if you already have crystals, why not to try phasing using tried and true heavy atom derivatives. Who knows, you might get lucky and one of them might actually improve the diffraction (this happens more often than people think). If you need specific advice - contact me directly. Alternatively, you can try to improve your crystals a bit - again, feel free to contact me directly for specifics, as the subscribers of this list may not be interested - this topic is pretty frequent around here. Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Kumar Sent: Friday, March 20, 2009 3:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Problems with phasing a protein (1300aa) Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. 'Se' labeled crystals diffracts maximally up to 3.5 to 4 A and dies very quickly on most of the beamlines. We have scanned at Se wavelength and it gives very strong signal as it contain ~45 Se in AU (1300 aa). It is difficult to collect a complete dataset (maximally we get 50-60 % completion with Rmerge ~15) out of one crystal on regular beamline. At microfocus beamline (APS), we were able to collect data in 3-4 batches and merge them to get a complete dataset (Rmerge ~18-20) out of one crystal. We used data collected on microfocus beamline (at peak wavelength) for locating heavy atom position using SHELXD, Solve and Phenix.hyss. SOlve and Phenix.hyss find very few heavy atom sites 1-5 whereas SHELX-CDE lists many but shows no difference in original and inverted (contrast and connectivity). Our phasing attempts with datasets obtained after merging two incomplete dataset from two different crystal has also been disappointing. My another worry is absolute value of average intensity, which seems to be quite low in most of the datasets. Below I have pasted last table of scale.log (HKL2000). Shell Lower Upper Average Average Norm. Linear Square limitAngstrom I error stat. Chi**2 R-fac R-fac 50.00 7.5345.4 1.6 1.3 1.295 0.055 0.047 7.53 5.9811.4 1.3 1.3 0.672 0.135 0.114 5.98 5.2311.2 1.6 1.6 0.643 0.171 0.152 5.23 4.7516.8 2.0 1.9 0.736 0.148 0.118 4.75 4.4118.8 2.2 2.2 0.739 0.143 0.132 4.41 4.1514.6 2.4 2.4 0.653 0.190 0.175 4.15 3.9411.3 2.5 2.5 0.582 0.247 0.226 3.94 3.7710.1 2.8 2.8 0.511 0.280 0.191 3.77 3.63 8.0 3.1 3.1 0.450 0.315 0.285 3.63 3.50 7.6 3.3 3.2 0.483 0.311 0.270 All reflections 15.5 2.3 2.2 0.694 0.153 0.106 Now, I want you to help me by answering some of my queries: 1. Is it possible to get MAD/SAD phasing done from a dataset having more than 15% Rmerge and resolution in the range of 4 - 4.5 Ang? 2. Will a complete data set obtained from merging various batches(30-40 frames each) from one or more than one crystal will have proper anomalous signal for phasing? I am worried as weak anomalous signal may get lost while merging. 3. Will such a low value of average Intensities (as shown above from HKL scale log file) will be good enough for MAD/SAD phasing or I really need to improve crystal quality for stronger diffraction. 4. For MAD/SAD phasing, till what resolution we need to have anomalous signal ? Many of my datasets shows anomalous signal maximally up to 6-8 A (calculated using Phenix.xtriage). 5. Since I have low resolution (3.5 to 4 A)data, relatively high Rmerge (14-15%), lower value of average intensity, anomalous signal up to 6 A or so. which programs will be more useful for heavy atom location and to prevent false positives from being selected? We have been also trying our luck with heavy atom soak but that also has not been very encouraging. I would appreciate any suggestions in this regard. Thanks in advance and sorry for such a long mail. Kumar
Re: [ccp4bb] fake images
Bernhard Rupp wrote: I only scratched the surface and I think it would be hard work to fake the images in a way that later expert forensics would not readily provide evidence. Also, there are 'watermarks' available from cryptographic methods that are even 'post-processing' resistant. A practical question is, even if it could be detected in theory, is anyone looking for it in practice? How many journal reviewers are going to audit this information? Today some structures still don't get deposited with structure factors. Not all journals enforce deposition policies well. Even if some future image repository can do automated image auditing, people who want to commit overt fraud will pick a journal that doesn't enforce deposition requirements, and decline to provide their images. A watermark or statistical analysis won't help you if you don't have access to the image. This discussion started regarding a place for people who _want_ to deposit images; unfortunately some people aren't going to do it voluntarily. -Eric --
[ccp4bb] Question about stereo on LCD monitor
Dear CCP4BB users, After one long year I'm back to crystallography again :) Actually I try to organize a crystallographic lab in new work place. Of course one of important thing what I need is a good computer machine for crystallographic calculations and molecular graphics. And now I need yours advices/opinion... - is it actually possible use a LCD monitor in stereo mode, for example in COOT or O, with shutterglasses? - what kind of graphic card, not so expensive, but with good quality of generated graphic is actually on the top? During my PhD I worked on machine with Quadro FX1300 graphic card with shutterglasses, but with CRT Iiyama monitor. Maybe after 2-3 years something changes in this subject... With best wishes Rafal Rafal Dolot, Ph.D Polish Academy of Sciences Centre of Molecular and Macromolecular Studies Department of Bioorganic Chemistry Sienkiewicza 112 90-363 Lodz, Poland
Re: [ccp4bb] Problems with phasing a protein (1300aa)
What is not true? Also in your case the applet estimates an expected 4-5% signal which is quite doable with decent data. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tommi Kajander Sent: Friday, March 20, 2009 3:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems with phasing a protein (1300aa) this cant be true, in the idea case (not Rmerge 15%, then again one can apply a resolution cutoff, perhaps, while this sounds like a very desperate case) the answer must be yes. didnt do the calculation right now (but it _should_ back this up) we for instance have solved a structure long time ago -- and this probably wasnt on the limit (well it was at the time but not anymore), 365 res x 8 in AU and 80 Se. at least looking at the SnB success list (very old list ) http://www.hwi.buffalo.edu/SnB/SnBSuccesses.htm there are plenty of others. -tommi On Mar 20, 2009, at 10:53 PM, Bernhard Rupp wrote: One can estimate this from http://www.ruppweb.org/new_comp/anomalous_scattering.htm and the answer as Jim says is no. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Pflugrath Sent: Friday, March 20, 2009 1:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems with phasing a protein (1300aa) Well, what do you expect the anomalous signal contributed from your 45 seleniums in a perfect world to be when the asymmetric unit contains 1300 aa? Do you think a dataset with Rmerge of ~15% is good enough to detect a signal of even 2%? (Note: I did not do the calculation, so I just made up the number of 2%.) Jim On Fri, 20 Mar 2009, Kumar wrote: Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. ... Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] fake images
It took from the 'official' IUCR recommendation in 2000 Guss M (2000) Guidelines for the deposition and release of macromolecular coordinate and experimental data. Acta Crystallogr. D56(1), 2. until Feb 2008 for structure factor amplitude deposition to the PDB to become mandatory. Nature started to provide coordinates and SFs to reviewers already around 2006 So, with luck, mandatory image deposition will happen in less than a decade. By then we'll have dirt cheap solid state disks which we stick into a port labeled 'solve structure' on our 0.5 lb Windux handheld which then wirelessly transmits the model and data to the repository, automatically linking to your auto-generated paper's identifier, while an artificial intelligence pre-editing tool rejects your manuscript for political incorrectness. Mark my words. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Eric Bennett Sent: Friday, March 20, 2009 4:10 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] fake images Bernhard Rupp wrote: I only scratched the surface and I think it would be hard work to fake the images in a way that later expert forensics would not readily provide evidence. Also, there are 'watermarks' available from cryptographic methods that are even 'post-processing' resistant. A practical question is, even if it could be detected in theory, is anyone looking for it in practice? How many journal reviewers are going to audit this information? Today some structures still don't get deposited with structure factors. Not all journals enforce deposition policies well. Even if some future image repository can do automated image auditing, people who want to commit overt fraud will pick a journal that doesn't enforce deposition requirements, and decline to provide their images. A watermark or statistical analysis won't help you if you don't have access to the image. This discussion started regarding a place for people who _want_ to deposit images; unfortunately some people aren't going to do it voluntarily. -Eric --
Re: [ccp4bb] fake images
And by then, the IUCR will have established the CFDU (Crystallographic Fraud Detection Unit), onto which Jack Bauer would be enlisted as a consultant (24 may still be running, who knows, so Jack won't be able to serve full-time on the job). This would be fun. Boaz - Original Message - From: Bernhard Rupp b...@ruppweb.org Date: Saturday, March 21, 2009 1:30 Subject: Re: [ccp4bb] fake images To: CCP4BB@JISCMAIL.AC.UK It took from the 'official' IUCR recommendation in 2000 Guss M (2000) Guidelines for the deposition and release of macromolecular coordinate and experimental data. Acta Crystallogr. D56(1), 2. until Feb 2008 for structure factor amplitude deposition to the PDB to become mandatory. Nature started to provide coordinates and SFs to reviewers already around 2006 So, with luck, mandatory image deposition will happen in less than a decade. By then we'll have dirt cheap solid state disks which we stick into a port labeled 'solve structure' on our 0.5 lb Windux handheld which then wirelessly transmits the model and data to the repository, automaticallylinking to your auto-generated paper's identifier, while an artificial intelligencepre-editing tool rejects your manuscript for political incorrectness. Mark my words. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Eric Bennett Sent: Friday, March 20, 2009 4:10 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] fake images Bernhard Rupp wrote: I only scratched the surface and I think it would be hard work to fake the images in a way that later expert forensics would not readily provide evidence. Also, there are 'watermarks' available from cryptographic methods that are even 'post-processing' resistant. A practical question is, even if it could be detected in theory, is anyone looking for it in practice? How many journal reviewers are going to audit this information? Today some structures still don't get deposited with structure factors. Not all journals enforce deposition policies well. Even if some future image repository can do automated image auditing, people who want to commit overt fraud will pick a journal that doesn't enforce deposition requirements, and decline to provide their images. A watermark or statistical analysis won't help you if you don't have access to the image. This discussion started regarding a place for people who _want_ to deposit images; unfortunately some people aren't going to do it voluntarily. -Eric -- Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel Phone: 972-8-647-2220 ; Fax: 646-1710 Skype: boaz.shaanan
Re: [ccp4bb] fake images
Just to dot the T's and cross the I's here - I actually don't want to *have to* deposit images routinely. I definitely hope that this is not going to be a mandatory step in PDB deposition any time soon - for a simple reason: at the moment this is too much trouble. Connections are too slow, and images are too bothersome to store beyond say 12 months. Add to this the need to also deposit 'site files' (or their equivalents for various software) and one gets to deal with an unholy mess. I certainly see the merit of depositing images for 'new and hot' structures, especially if data is questionable, and doubly so if the publication results in extraordinary or revolutionary conclusions. But please - I beg you - don't make me deposit images for yet another PTP1B structure. There - now if the movement towards having an image depot fizzles out you can blame me for that. Artem --- When the Weasel comes to give New Year's greetings to the Chickens no good intentions are in his mind. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Eric Bennett Sent: Friday, March 20, 2009 7:10 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] fake images Bernhard Rupp wrote: I only scratched the surface and I think it would be hard work to fake the images in a way that later expert forensics would not readily provide evidence. Also, there are 'watermarks' available from cryptographic methods that are even 'post-processing' resistant. A practical question is, even if it could be detected in theory, is anyone looking for it in practice? How many journal reviewers are going to audit this information? Today some structures still don't get deposited with structure factors. Not all journals enforce deposition policies well. Even if some future image repository can do automated image auditing, people who want to commit overt fraud will pick a journal that doesn't enforce deposition requirements, and decline to provide their images. A watermark or statistical analysis won't help you if you don't have access to the image. This discussion started regarding a place for people who _want_ to deposit images; unfortunately some people aren't going to do it voluntarily. -Eric --
Re: [ccp4bb] Problems with phasing a protein (1300aa)
Bijvoet amplitude ratios seem always more pessimistic then they should be. Sure, no while lines considered in the edge calc, for example. BR
Re: [ccp4bb] Problems with phasing a protein (1300aa)
|delta F|/F *c \approx |delta I|/ I , with c somewhere between 2.5 and 3. Bijvoet amplitude ratios seem always more pessimistic then they should be. The 4 to 5% |delta F|/F would translate into a 10 to 12% expected difference for individual intensities. P 2009/3/20 Bernhard Rupp b...@ruppweb.org: What is not true? Also in your case the applet estimates an expected 4-5% signal which is quite doable with decent data. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tommi Kajander Sent: Friday, March 20, 2009 3:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems with phasing a protein (1300aa) this cant be true, in the idea case (not Rmerge 15%, then again one can apply a resolution cutoff, perhaps, while this sounds like a very desperate case) the answer must be yes. didnt do the calculation right now (but it _should_ back this up) we for instance have solved a structure long time ago -- and this probably wasnt on the limit (well it was at the time but not anymore), 365 res x 8 in AU and 80 Se. at least looking at the SnB success list (very old list ) http://www.hwi.buffalo.edu/SnB/SnBSuccesses.htm there are plenty of others. -tommi On Mar 20, 2009, at 10:53 PM, Bernhard Rupp wrote: One can estimate this from http://www.ruppweb.org/new_comp/anomalous_scattering.htm and the answer as Jim says is no. BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Pflugrath Sent: Friday, March 20, 2009 1:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems with phasing a protein (1300aa) Well, what do you expect the anomalous signal contributed from your 45 seleniums in a perfect world to be when the asymmetric unit contains 1300 aa? Do you think a dataset with Rmerge of ~15% is good enough to detect a signal of even 2%? (Note: I did not do the calculation, so I just made up the number of 2%.) Jim On Fri, 20 Mar 2009, Kumar wrote: Hello CCP4bb members, I have been trying to obtain phases for a protein which contain ~1300aa. We have obtained native data to a resolution of 3.3A (Space group I222 or I212121). But we are having tough time phasing it. ... Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi -- - P.H. Zwart Beamline Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org CCTBX: http://cctbx.sf.net -
Re: [ccp4bb] Question about stereo on LCD monitor
Rafal, Actually, things only really started to change last month... Please see http://pymol.org and scroll down most of the way to the February 13th entry regarding the $600 Zalman ZM-M220W stereo-capable LCD. In my opinion, that is the best option on the market until the powers that be start supporting 120 Hz LCDs with OpenGL drivers -- which could be days or months, but hopefully not years. Once that happens, 120 Hz LCDs will likely become the preferred option. I don't know whether O, Coot, or CCP4 support this monitor yet, but they really should if they do not! (It only takes a couple dozen lines of code beyond standard QBS stereo -- developers please see the code I've posted at http://pymol.org/zalman/howto.html ) Cheers, Warren -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Rafal Dolot Sent: Friday, March 20, 2009 4:17 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Question about stereo on LCD monitor Dear CCP4BB users, After one long year I'm back to crystallography again :) Actually I try to organize a crystallographic lab in new work place. Of course one of important thing what I need is a good computer machine for crystallographic calculations and molecular graphics. And now I need yours advices/opinion... - is it actually possible use a LCD monitor in stereo mode, for example in COOT or O, with shutterglasses? - what kind of graphic card, not so expensive, but with good quality of generated graphic is actually on the top? During my PhD I worked on machine with Quadro FX1300 graphic card with shutterglasses, but with CRT Iiyama monitor. Maybe after 2-3 years something changes in this subject... With best wishes Rafal Rafal Dolot, Ph.D Polish Academy of Sciences Centre of Molecular and Macromolecular Studies Department of Bioorganic Chemistry Sienkiewicza 112 90-363 Lodz, Poland
Re: [ccp4bb] images
On Mar 19, 2009, at 3:26 AM, Andrew Purkiss-Trew wrote: On Wed, 2009-03-18 at 18:19 +, Frank von Delft wrote: Maybe, but images without experimental context (sequence? ligands? purification? crystallization format? -- PURPOSE OF EXPERIMENT!?!! relationship to the other 15 similar datasets) are as good as no images. And as far as I know, there's no good discussion on the table for that. At least, no-one on the thread mentioned it, so they're probably not thinking about it either. I suppose efforts like PIMS or are a start, and maybe they can even have enough information (my feeling is they currently don't). But that's where the discussion should start: how to index (in sense of annotate) the datasets. The technicalities are just that: technicalities. Or even closer to home: does ANY detector/beamline write even timestamps into the image header...? Never mind ring current, intensity of the beam, size of beam, size of crystal, length of direct beam path, etc etc... As far as I know, most detectors write the current time into the image header. Certainly our in house MAR image plate systems do, as do the detectors at Diamond and ESRF (for those that I've looked at this morning). FYI at my beamline (ALS 12.3.1), in addition to the usual useful metadata, we also put in the beamline ID and the serial number of the detector. In theory anything can be added if you take the time to customize the detector code. HEADER_BYTES= 512; DIM=2; BYTE_ORDER=little_endian; TYPE=unsigned_short; SIZE1=3072; SIZE2=3072; PIXEL_SIZE=0.102592; BIN=2x2; BIN_TYPE=HW; ADC=fast; CREV=1; BEAMLINE=ALS1231; DETECTOR_SN=907; DATE=Tue Feb 3 11:07:38 2009; TIME=10.00; ACC_TIME=11516; DISTANCE=649.80; TWOTHETA=0.00; PHI=191.310; OSC_START=191.310; OSC_RANGE=1.000; WAVELENGTH=1.033184; BEAM_CENTER_X=155.70; BEAM_CENTER_Y=157.40; DENZO_X_BEAM=157.76; DENZO_Y_BEAM=155.70; Scott
Re: [ccp4bb] Problems with phasing a protein (1300aa)
Sure, no while lines considered in the edge calc, for example. That and (more important) that 2*abs(a-b)/(a+b) 2*abs(sqrt(a)-sqrt(b))/(sqrt(a)+sqrt(b)) See Acta Cryst. D61, 1437–1448. (btw, while is overrated, use for and a break statement) p
