Re: [ccp4bb] Density peaks to build
Dear Chris and Leo, Thank you for your suggestions. I am refining the 1.08 A structure in SHELXL and I have Hs added; I think refinement is at a final stage. I did start with the 50%, 25+25% occupancy but then I changed to 0.33 each ... I need to refine this further. For the 1.35A data, model is complete (except for some C- and N-terminal residues) and I've refined alternative conformations too. The crystallization condition is Sodium acetate and PEG3350 with ethylene glycol as cryo. I have some molecules of cryo and Na in the model. I’ll try other cat/anions and refine. Best, Fátima
Re: [ccp4bb] Density peaks to build
Dear Fatima, I have used Buster to refine alternate occupanies at 1.2 A or 1.4 A resolution, and it greatly improved the electron density as well as the output statistics. Best, Marie-Hélène Dr Marie-Hélène LeDu CEA/DSV/IBiTec-S Laboratoire de Biologie Structurale et Radiobiologie bat 144, CE Saclay 91191 Gif-sur-Yvette email : marie-helene.l...@cea.fr tel:01 69 08 71 35 Le 21/07/10 09:53, « Fatima Fonseca » ffons...@ua.pt a écrit : Dear Chris and Leo, Thank you for your suggestions. I am refining the 1.08 A structure in SHELXL and I have Hs added; I think refinement is at a final stage. I did start with the 50%, 25+25% occupancy but then I changed to 0.33 each ... I need to refine this further. For the 1.35A data, model is complete (except for some C- and N-terminal residues) and I've refined alternative conformations too. The crystallization condition is Sodium acetate and PEG3350 with ethylene glycol as cryo. I have some molecules of cryo and Na in the model. I¹ll try other cat/anions and refine. Best, Fátima
Re: [ccp4bb] Density peaks to build
Hi, Can't rotate the picture so that I can't see the distance between the nitrogen and the green blob. The green blob is elongated. Sometimes what happens is that you can have 2 waters (partial occupancies), in some unit cells in your crystals the water occupies site 1, in the other unit cells the water occupies site 2. Obviously in the model you build, care should be taken during refinement that these waters do not push each other away (VdW repulsion). Anyway blobology is a difficult part of our trade. Fred. Fatima Fonseca wrote: Dear CCP4BB users, I have 2 questions: I'm refining a very high-resolution structure (1.08 A) and I need help to solve a blob of positive density close to a modified Arg (N-omega-hydroxy-L-Arginine) - picture1. I tried to model a third conformation there but it didn't work - picture2. In another protein model (1.35 Aº) I have a lot of positive peaks in the difference map that I model as water molecules and after Refmac these waters have very high B factors (100 - 200) and appear as blobs of positive density. They make H bonds with 2-4 water molecules/protein residues within 2.66 - 3.0 A. Any suggestions would be precious. Fátima
[ccp4bb] Postdoctoral position in malaria structural biology in Oxford
Dear All, There is a position available for a post-doctoral researcher to work with Dr Matt Higgins in the Department of Biochemistry, University of Oxford, on the structural biology and biochemistry of cell surface proteins from the malaria parasite. This will involve the production and characterisation of proteins, the development of interaction assays and structural analysis by protein crystallography and nuclear magnetic resonance spectroscopy. Experience in NMR and/or crystallography would be of great benefit. Applicants should possess a doctorate in Structural Biology or Biophysics and should ideally have experience in protein production and the analysis of protein-protein interactions through biophysical and structural methods. The post is funded by a Medical Research Council project grant and is available for up to 36 months in the first instance. This position is graded on the University' Grade 7 pay scale, for which the salary range is 28,983 - 35,646 per annum. The actual starting salary offered will be based on qualifications and relevant skills acquired and will also be determined by the funding available. For further general information, an application cover sheet and other application requirements please visit the Departmental website http://www.bioch.ox.ac.uk/jobs/instructions.html or phone (01865) 613204, quoting the reference number below Applications, including three copies of the completed application cover sheet and recruitment monitoring form, three copies of a detailed CV, the names and addresses of two referees, (one of whom must be the current or most recent employer), and three copies of a letter setting out, with examples, how you meet the selection criteria for the post, should be sent to: The Deputy Administrator Department of Biochemistry University of Oxford South Parks Road Oxford OX1 3QU Please quote reference number: BR/441 The closing date for applications is Tuesday, 24th August 2010 Please send any informal enquiries to Matt at mk...@cam.ac.uk.
Re: [ccp4bb] Residual densities!
Could any of those strong spots at 2.1 be ice? Original message Date: Tue, 20 Jul 2010 16:13:31 -0700 From: Lijun Liu lijun@ucsf.edu Subject: Re: [ccp4bb] Residual densities! To: CCP4BB@JISCMAIL.AC.UK Pavel, Thanks for the quick response. I just listed out the Fo-Fc column of the reflection files, and it looks the strongest densities are mostly along c* and many located in the 2.1A region. I think you may be right. Lijun On Jul 20, 2010, at 4:05 PM, Pavel Afonine wrote: Hi, It looks like the data is highly anisotropic - I've seen it many times. You can either apply anisotropy correction (http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/) or use PHENIX tools to compute the maps - anyone of both should help. The major difference between the two is that PHENIX will not modify your original data in any way. Let me if using PHENIX map calculation tools does not help. Cheers, Pavel. On 7/20/10 3:19 PM, Lijun Liu wrote: Hi all, I solved a structure and the refinement is close to complete. However, some residual density puzzles me. The SG is P212121, and the c is 84.8 Å. Along c, in several small but not all regions, there are some layered, alternately appeared positive and negative residual density pieces (c is the norm of these plane-like pieces; 3-sigma contour for pos and neg difference densities). The distance between the neighboring positive pieces is measured to be ~2.1 Å, corresponding to ~1/40 *c. In one of such region, there is an indole ring of Trp that is approximately parallel to the plane-like residual density pieces (attached figure). In two other regions, there are Leu or else but not ring structure. The data is complete to 1.9 Å. Reflection check did not show any special imcompleteness (ice ring loss, wedge loss, other resolution bin loss, etc). It looks like to me that there is a systematic loss of information which made it is specially sensitive to ~2.1 Å reflections (especially those along c*). It does not look like to me to be radiation damage. Crystal was needle and showed somehow polarized (but never serious) diffraction pattern along c*. Attached is a piece of map with the above-mentioned Trp included. I would like to know if some one also met something like this before. Thanks for any comments. Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836 Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836
[ccp4bb] Crystal growth time lapse movies.
Hi all I'm doing a quick talk on crystals/crystallography for a lay audience in a couple of weeks time, and I'm looking for some time lapse movies / animated gifs of crystal growth. The one I was thinking of using was here: http://microgravity.msfc.nasa.gov/snell/vibration.html But that link is broken, and I can't find it via googling. Does anybody know of any / have any good ones that I can beg/steal/borrow? Cheers in advance, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB
Re: [ccp4bb] Crystal growth time lapse movies.
There is a good one of lysozyme crystal growth on Bernhard Rupp's website: http://www.ruppweb.org/level1/movies_list.htm http://www.ruppweb.org/level1/movies_list.htmThe Microcrystallization movie taken with the Cryscam is the one I speak of. On Wed, Jul 21, 2010 at 11:40 AM, David Briggs drdavidcbri...@gmail.comwrote: Hi all I'm doing a quick talk on crystals/crystallography for a lay audience in a couple of weeks time, and I'm looking for some time lapse movies / animated gifs of crystal growth. The one I was thinking of using was here: http://microgravity.msfc.nasa.gov/snell/vibration.html But that link is broken, and I can't find it via googling. Does anybody know of any / have any good ones that I can beg/steal/borrow? Cheers in advance, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB -- Jim Fairman, Ph D. Post-Doctoral Fellow National Institutes of Health - NIDDK Cell: 1-865-748-8672 Lab: 1-301-594-9229 E-mail: fairman@gmail.com james.fair...@nih.gov
[ccp4bb] offtopic: adjustable vertical sequencing gels.
All, Sorry for the off topic/product solicitation, but someone may be able to help. Looking for an adjustable sequencing gel that can replace the ADJ3 from Thermo Scientific. (http://www.owlsci.com/verticals/vertical.aspx?id=ADJ3 ) . Unfortunately, it's been discontinued. Needs to be able to accommodate 35 cm (wide) by 26-30cm (height) plates. Please reply privately. F - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] Residual densities!
No. No ice ring! Sorry I meant the strongest amplitudes of (Fo-Fc) at the 2.1 A around region in the reciprocal space, not the strongest densities on real space images. As Pavel suggested, this is a good case of anisotropicity. In my original email, I used polarized pattern to address this---I used the wrong nomenclature to address the same thing, I think. Lijun Could any of those strong spots at 2.1 be ice? Original message Date: Tue, 20 Jul 2010 16:13:31 -0700 From: Lijun Liu lijun@ucsf.edu Subject: Re: [ccp4bb] Residual densities! To: CCP4BB@JISCMAIL.AC.UK Pavel, Thanks for the quick response. I just listed out the Fo-Fc column of the reflection files, and it looks the strongest densities are mostly along c* and many located in the 2.1A region. I think you may be right. Lijun On Jul 20, 2010, at 4:05 PM, Pavel Afonine wrote: Hi, It looks like the data is highly anisotropic - I've seen it many times. You can either apply anisotropy correction (http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/) or use PHENIX tools to compute the maps - anyone of both should help. The major difference between the two is that PHENIX will not modify your original data in any way. Let me if using PHENIX map calculation tools does not help. Cheers, Pavel. On 7/20/10 3:19 PM, Lijun Liu wrote: Hi all, I solved a structure and the refinement is close to complete. However, some residual density puzzles me. The SG is P212121, and the c is 84.8 Å. Along c, in several small but not all regions, there are some layered, alternately appeared positive and negative residual density pieces (c is the norm of these plane-like pieces; 3-sigma contour for pos and neg difference densities). The distance between the neighboring positive pieces is measured to be ~2.1 Å, corresponding to ~1/40 *c. In one of such region, there is an indole ring of Trp that is approximately parallel to the plane-like residual density pieces (attached figure). In two other regions, there are Leu or else but not ring structure. The data is complete to 1.9 Å. Reflection check did not show any special imcompleteness (ice ring loss, wedge loss, other resolution bin loss, etc). It looks like to me that there is a systematic loss of information which made it is specially sensitive to ~2.1 Å reflections (especially those along c*). It does not look like to me to be radiation damage. Crystal was needle and showed somehow polarized (but never serious) diffraction pattern along c*. Attached is a piece of map with the above-mentioned Trp included. I would like to know if some one also met something like this before. Thanks for any comments. Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836 Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836 Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836
[ccp4bb] postdoctoral position
A postdoctoral position is available at the University of Texas Medical Branch (UTMB) to study the structure and active conformations of membrane-associated blood coagulation protein and complexes: http://www.utmb.edu/ncb/faculty/Stoilova-McPhieSvetla.asp. Highly motivated candidates with experience in macromolecular structure analysis, molecular modeling and biophysics are encouraged to apply. Experience with linux computing systems and programming will be an advantage. The Sealy Center for Structural Biology and Molecular Biophysics at UTMB, as well as the PI’s lab are fully equipped for state of the art structure determination of macromolecular complexes: http://www.scsb.utmb.edu/symposium/index.htm. UTMB is also part of the Gulf Coast Consortium: http://cohesion.rice.edu/centersandinst/gcc/ and the Texas Medical Center. Interested candidates should send a CV including areas of expertise and interest, publications list, and names and contact information for 3 references to svmcp...@utmb.edumailto:svmcp...@utmb.edu. UTMB is an equal opportunity employer. Svetla Stoilova-McPhie, PhD Assistant Professor, Department of Neuroscience and Cell Biology Scientist, Sealy Centre for Structural Biology and Molecular Biophysics University of Texas Medical Branch at Galveston 301 University Boulevard, Galveston, Texas 77555-0620 Email: svmcp...@utmb.edu http://www.utmb.edu/ncb/faculty/Stoilova-McPhieSvetla.asp
Re: [ccp4bb] Crystal growth time lapse movies.
Dear David, You are welcome to use this movie from our homepage: http://shelx.uni-ac.gwdg.de/xtal/xtal.htm#xtalgrowth 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 Wed, 21 Jul 2010, David Briggs wrote: Hi all I'm doing a quick talk on crystals/crystallography for a lay audience in a couple of weeks time, and I'm looking for some time lapse movies / animated gifs of crystal growth. The one I was thinking of using was here: http://microgravity.msfc.nasa.gov/snell/vibration.html But that link is broken, and I can't find it via googling. Does anybody know of any / have any good ones that I can beg/steal/borrow? Cheers in advance, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB
[ccp4bb] AKTA Explorer 100 needs new home [Off-topic]
Hi all, We have an AKTA Explorer 100 from a closing lab that we would like to find a new home for. Please respond off-line for price and configuration info, and apologies for off-topic post. Thanks, Erin Curry ress...@gmail.com (510) 344-6633
[ccp4bb] How to get the program OUTLIAR?
Hi there, I read the paper Detecting outliers in non-redundant diffraction data (Read, Acta Cryst D55,1759), which described a program called OUTLIAR. Can anbody tell me how to get this program? Seems hard to find it on the web. Best Regards, Hailiang
Re: [ccp4bb] Residual densities!
Another possible cause of this layering might be a single strong rogue reflection at about 2.1A along the C* axis. Since hex ice has a lattice spacing around here, a single crystal of ice (a snowflake captured from the LN2 or growing on the crystal in the cold stream) could result in such spots. If one of them was close enough to the predicted spot of your crystal, it could get integrated and show up in your data list. Not an ice ring, you understand, but an ice diffraction spot appearing to be a protein diffraction spot. I think this is what Phoebe was referring to. If the anisotropy correction doesn't give a good map, try making a map with resolution limited to 2.3 A. If that looks ok, extend the resolution in small steps through the 2.1A range. If you layers suddenly returns with one of these steps, you could eliminate a thin shell, or narrow it down to the particular spot and eliminate that. -ed- Lijun Liu wrote: No. No ice ring! Sorry I meant the strongest amplitudes of (Fo-Fc) at the 2.1 A around region in the reciprocal space, not the strongest densities on real space images. As Pavel suggested, this is a good case of anisotropicity. In my original email, I used polarized pattern to address this---I used the wrong nomenclature to address the same thing, I think. Lijun Could any of those strong spots at 2.1 be ice? Original message Date: Tue, 20 Jul 2010 16:13:31 -0700 From: Lijun Liu lijun@ucsf.edu mailto:lijun@ucsf.edu Subject: Re: [ccp4bb] Residual densities! To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Pavel, Thanks for the quick response. I just listed out the Fo-Fc column of the reflection files, and it looks the strongest densities are mostly along c* and many located in the 2.1A region. I think you may be right. Lijun On Jul 20, 2010, at 4:05 PM, Pavel Afonine wrote: Hi, It looks like the data is highly anisotropic - I've seen it many times. You can either apply anisotropy correction (http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/) or use PHENIX tools to compute the maps - anyone of both should help. The major difference between the two is that PHENIX will not modify your original data in any way. Let me if using PHENIX map calculation tools does not help. Cheers, Pavel. On 7/20/10 3:19 PM, Lijun Liu wrote: Hi all, I solved a structure and the refinement is close to complete. However, some residual density puzzles me. The SG is P212121, and the c is 84.8 Å. Along c, in several small but not all regions, there are some layered, alternately appeared positive and negative residual density pieces (c is the norm of these plane-like pieces; 3-sigma contour for pos and neg difference densities). The distance between the neighboring positive pieces is measured to be ~2.1 Å, corresponding to ~1/40 *c. In one of such region, there is an indole ring of Trp that is approximately parallel to the plane-like residual density pieces (attached figure). In two other regions, there are Leu or else but not ring structure. The data is complete to 1.9 Å. Reflection check did not show any special imcompleteness (ice ring loss, wedge loss, other resolution bin loss, etc). It looks like to me that there is a systematic loss of information which made it is specially sensitive to ~2.1 Å reflections (especially those along c*). It does not look like to me to be radiation damage. Crystal was needle and showed somehow polarized (but never serious) diffraction pattern along c*. Attached is a piece of map with the above-mentioned Trp included. I would like to know if some one also met something like this before. Thanks for any comments. Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836 Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836 Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836
Re: [ccp4bb] How to get the program OUTLIAR?
Have you tried sending e-mail to Randy Read? Frances = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 = On Wed, 21 Jul 2010, Hailiang Zhang wrote: Hi there, I read the paper Detecting outliers in non-redundant diffraction data (Read, Acta Cryst D55,1759), which described a program called OUTLIAR. Can anbody tell me how to get this program? Seems hard to find it on the web. Best Regards, Hailiang
[ccp4bb] Job Opening in Crystallisation at the Swiss Light Source
The Paul Scherrer Institute is with 1300 employees the largest research centre for the natural and engineering sciences in Switzerland and a worldwide leading user laboratory. Its research activities are concentrated on the three main topics structure of matter, energy and environmental research as well as human health. The Swiss Light Source (SLS) is one of the most advanced synchrotron radiation sources worldwide. The SLS operates two state-of-the-art undulator beam lines for protein crystallography, and a third, highly automated beam line located at a superbend magnet (X06DA). At beam line X06DA a crystallisation facility which is integrated with the beam line in order to allow for in situ X-ray diffraction screening is being implemented. The Macromolecular Crystallography Group is involved in several aspects of protein crystallography including the design and construction of new beam line components as well as various structural biology projects, especially in collaboration with the Biomolecular Research Group under the guidance of Prof. Dr Gebhard Schertler. We are looking for a Crystallisation Facility Scientist Your tasksYou will lead our efforts to extend and automatise the high-throughput crystallisation facility and its integration with beam line X06DA at the Swiss Light Source and build up the user programme of this facility. In collaboration with scientists and engineers of the SLS MX-group you will further develop the *in situ* X-ray diffraction screening capabilities of the facility and coordinate its integration with the sample data base. Further tasks are the implementation of crystallisation in lipidic cubic phase as well as the establishment of protein quality control protocols in collaboration with the PSI structural biology unit. In addition, there are excellent opportunities to pursue your own research projects in structural biology. Your profileYou hold a *Ph.D.*degree in biology or (bio-) chemistry, and have significant experience in high-throughput crystallisation methods and protein crystallography. Practical knowledge in synchrotron related research and in project management is of advantage. If you are self-motivated, flexible, and a good team player this position will offer great opportunities to establish your research career in an exciting and highly multidisciplinary environment. For further information please contact: Dr Clemens Schulze-Briese, phone +41 56 310 45 33, clemens.schu...@psi.ch or Dr Vincent Olieric, phone +41 56 310 52 33, vincent.olie...@psi.ch Please submit your application including CV quoting the ref. code by e-mail to elke.baum...@psi.ch or to Paul Scherrer Institut, Human Resources, ref. code 6112-03, Elke Baumann, 5232 Villigen PSI, Switzerland. http://www.psi.ch/pa/offenestellen/Wissenschaft/2389
Re: [ccp4bb] Residual densities!
Ed, Thanks for the comment! Anisotropic correction hits the problem, and makes good improvement! You suggestion about one strong reflection around 2.1 Å is very interesting. However, one single reflection's (along c*) ripples passes along c and give sin/cos pattern along c, which means along c everywhere should give such a residual map pattern throughout the unit cell, which is not observed. As I mentioned, only several small regions showed such residual maxima (please do not argue with me I also had lost 1 or 2 big low ordered reflections, :). Also, this reflection needs to be very strong and should not be overload-ignored during processing. Anyway, reflections (0 0 39-41) are pretty weak or normal. Cheers, the great FT!!! Lijun On Jul 21, 2010, at 2:28 PM, Edward A. Berry wrote: Another possible cause of this layering might be a single strong rogue reflection at about 2.1A along the C* axis. Since hex ice has a lattice spacing around here, a single crystal of ice (a snowflake captured from the LN2 or growing on the crystal in the cold stream) could result in such spots. If one of them was close enough to the predicted spot of your crystal, it could get integrated and show up in your data list. Not an ice ring, you understand, but an ice diffraction spot appearing to be a protein diffraction spot. I think this is what Phoebe was referring to. If the anisotropy correction doesn't give a good map, try making a map with resolution limited to 2.3 A. If that looks ok, extend the resolution in small steps through the 2.1A range. If you layers suddenly returns with one of these steps, you could eliminate a thin shell, or narrow it down to the particular spot and eliminate that. -ed- Lijun Liu wrote: No. No ice ring! Sorry I meant the strongest amplitudes of (Fo-Fc) at the 2.1 A around region in the reciprocal space, not the strongest densities on real space images. As Pavel suggested, this is a good case of anisotropicity. In my original email, I used polarized pattern to address this---I used the wrong nomenclature to address the same thing, I think. Lijun Could any of those strong spots at 2.1 be ice? Original message Date: Tue, 20 Jul 2010 16:13:31 -0700 From: Lijun Liu lijun@ucsf.edu mailto:lijun@ucsf.edu Subject: Re: [ccp4bb] Residual densities! To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Pavel, Thanks for the quick response. I just listed out the Fo-Fc column of the reflection files, and it looks the strongest densities are mostly along c* and many located in the 2.1A region. I think you may be right. Lijun On Jul 20, 2010, at 4:05 PM, Pavel Afonine wrote: Hi, It looks like the data is highly anisotropic - I've seen it many times. You can either apply anisotropy correction (http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/) or use PHENIX tools to compute the maps - anyone of both should help. The major difference between the two is that PHENIX will not modify your original data in any way. Let me if using PHENIX map calculation tools does not help. Cheers, Pavel. On 7/20/10 3:19 PM, Lijun Liu wrote: Hi all, I solved a structure and the refinement is close to complete. However, some residual density puzzles me. The SG is P212121, and the c is 84.8 Å. Along c, in several small but not all regions, there are some layered, alternately appeared positive and negative residual density pieces (c is the norm of these plane-like pieces; 3-sigma contour for pos and neg difference densities). The distance between the neighboring positive pieces is measured to be ~2.1 Å, corresponding to ~1/40 *c. In one of such region, there is an indole ring of Trp that is approximately parallel to the plane-like residual density pieces (attached figure). In two other regions, there are Leu or else but not ring structure. The data is complete to 1.9 Å. Reflection check did not show any special imcompleteness (ice ring loss, wedge loss, other resolution bin loss, etc). It looks like to me that there is a systematic loss of information which made it is specially sensitive to ~2.1 Å reflections (especially those along c*). It does not look like to me to be radiation damage. Crystal was needle and showed somehow polarized (but never serious) diffraction pattern along c*. Attached is a piece of map with the above-mentioned Trp included. I would like to know if some one also met something like this before. Thanks for any comments. Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836 Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836 Lijun Liu Cardiovascular Research Institute University of California, San Francisco 1700 4th Street, Box 2532 San Francisco, CA 94158 Phone: (415)514-2836 Lijun Liu Cardiovascular Research Institute University of
Re: [ccp4bb] Residual densities!
Dear Lijun, Glad to hear the anisotropy correction is helping. As for the layering being visible in only certain areas, I agree this would be incompatible with a single strong reflection if it were vastly stronger than the good reflections. But if not, you can imagine it going in and out of phase with valid reflections of slightly higher and lower resolution, and being most noticeable where in phase (or maybe not- if the valid reflections add up to the true structure, then the rogue reflection would add a wave right across the crystal). It's more complicated than that, because the phases of the neighboring reflections will be messed up trying to compensate for the rogue reflection. Or maybe we have to propose two rogue reflections of slightly different frequency and same direction, beating against each other, going in and out of phase? Anyway, good luck. Ed Lijun Liu wrote: Ed, Thanks for the comment! Anisotropic correction hits the problem, and makes good improvement! You suggestion about one strong reflection around 2.1 Å is very interesting. However, one single reflection's (along c*) ripples passes along c and give sin/cos pattern along c, which means along c everywhere should give such a residual map pattern throughout the unit cell, which is not observed. As I mentioned, only several small regions showed such residual maxima (please do not argue with me I also had lost 1 or 2 big low ordered reflections, :). Also, this reflection needs to be very strong and should not be overload-ignored during processing. Anyway, reflections (0 0 39-41) are pretty weak or normal. Cheers, the great FT!!! Lijun On Jul 21, 2010, at 2:28 PM, Edward A. Berry wrote: Another possible cause of this layering might be a single strong rogue reflection at about 2.1A along the C* axis. Since hex ice has a lattice spacing around here, a single crystal of ice (a snowflake captured from the LN2 or growing on the crystal in the cold stream) could result in such spots. If one of them was close enough to the predicted spot of your crystal, it could get integrated and show up in your data list. Not an ice ring, you understand, but an ice diffraction spot appearing to be a protein diffraction spot. I think this is what Phoebe was referring to. If the anisotropy correction doesn't give a good map, try making a map with resolution limited to 2.3 A. If that looks ok, extend the resolution in small steps through the 2.1A range. If you layers suddenly returns with one of these steps, you could eliminate a thin shell, or narrow it down to the particular spot and eliminate that. -ed- Lijun Liu wrote: No. No ice ring! Sorry I meant the strongest amplitudes of (Fo-Fc) at the 2.1 A around region in the reciprocal space, not the strongest densities on real space images. As Pavel suggested, this is a good case of anisotropicity. In my original email, I used polarized pattern to address this---I used the wrong nomenclature to address the same thing, I think. Lijun Could any of those strong spots at 2.1 be ice? Original message Date: Tue, 20 Jul 2010 16:13:31 -0700 From: Lijun Liu lijun@ucsf.edu mailto:lijun@ucsf.edu Subject: Re: [ccp4bb] Residual densities! To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Pavel, Thanks for the quick response. I just listed out the Fo-Fc column of the reflection files, and it looks the strongest densities are mostly along c* and many located in the 2.1A region. I think you may be right. Lijun On Jul 20, 2010, at 4:05 PM, Pavel Afonine wrote: Hi, It looks like the data is highly anisotropic - I've seen it many times. You can either apply anisotropy correction (http://www.doe-mbi.ucla.edu/~sawaya/anisoscale/) or use PHENIX tools to compute the maps - anyone of both should help. The major difference between the two is that PHENIX will not modify your original data in any way. Let me if using PHENIX map calculation tools does not help. Cheers, Pavel. On 7/20/10 3:19 PM, Lijun Liu wrote: Hi all, I solved a structure and the refinement is close to complete. However, some residual density puzzles me. The SG is P212121, and the c is 84.8 Å. Along c, in several small but not all regions, there are some layered, alternately appeared positive and negative residual density pieces (c is the norm of these plane-like pieces; 3-sigma contour for pos and neg difference densities). The distance between the neighboring positive pieces is measured to be ~2.1 Å, corresponding to ~1/40 *c. In one of such region, there is an indole ring of Trp that is approximately parallel to the plane-like residual density pieces (attached figure). In two other regions, there are Leu or else but not ring structure. The data is complete to 1.9 Å. Reflection check did not show any special imcompleteness (ice ring loss, wedge loss, other resolution bin loss, etc). It looks like to me that there is a systematic loss of information which made it is specially sensitive to ~2.1 Å reflections (especially those
