Re: [ccp4bb] difference between polar angle and eulerian angle
Dear Ed, It looks like the problem is not yet closed, so I'll add my remarks. In addition to very detailed comments by Ian, Tim and others, it might be useful to look our article in J.Appl.Cryst (1997), 30, 402-410, which addresses exactly these confusing points: what is rotated, what is fixed, in which direction rotated, polar angles vs Euler angles, etc... Maybe not so detailed as you wish but may help. Some tables may be also useful (unfortunately, there is one mistyping in Table 2, if I remember). With best wishes, Sacha Urzhumtsev De : CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Edward A. Berry [ber...@upstate.edu] Envoyé : mardi 1 avril 2014 01:12 À : CCP4BB@JISCMAIL.AC.UK Objet : Re: [ccp4bb] difference between polar angle and eulerian angle Dear Tim, Ian, Tim Gruene wrote: [...] Actually it does not depend - the rotation matrices are a representation
[ccp4bb] Post doc position at the University of Copenhagen
Post doctoral position - Structural studies of full-length membrane-bound receptors and transporters The Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences at the University of Copenhagen is offering a two-year post doctoral position within Structural studies of full-length membrane-bound receptors and transporters commencing 1st September 2014 or soon thereafter. The postdoctoral position is part of the interdisciplinary research program Fertilizing the ground and harvesting the full potential of the new neutron and X‐ray research infrastructures close to Copenhagen University (Co-NEXT) funded by the UCPH Excellence Program for Interdisciplinary Research. Job description The position will be anchored at the Biostructural Research section, Department of Drug Design and Pharmacology, University of Copenhagen. The Biostructural Research section provides the foundation for structural and functional studies of a wide range of different proteins involved in health and disease. The aim of the research project is to express and purify different mammalian membrane proteins from insect cells following already existing protocols and establish nano disc reconstitution. This project will be part of research collaboration with the Niels Bohr Institute, University of Copenhagen. The project aims at employing SANS and SAXS to capture functionally important structural states of the target proteins. Such information will facilitate structure based drug design. The successful applicant is expected to teach up to 10% of her/his time in areas relevant to the research project. Qualifications required The successful applicant would be an experimentally skilled scientist who can document original scientific production in the areas membrane protein expression using eukaryotic cells and subsequent purification for structural studies. Experience with protein reconstitution in to nano discs is an advantage. Further information For further information, please contact Professor Jette S. Kastrup (j...@sund.ku.dkmailto:j...@sund.ku.dk, tel. +45 3533 6486 or Associate professor Osman Mirza (o...@sund.ku.dkmailto:o...@sund.ku.dk, tel. +45 3533 6175). Terms of employment The terms of employment and payment are according to the agreement between the Ministry of Finance and The Danish Confederation of Professional Associations on Academics in the State. You can read more about the Faculty´s appointment procedures at: http://pharmaschool.ku.dk/ As an equal opportunities employer, the University of Copenhagen encourages all qualified applicants to apply, regardless of gender, age, religion and ethnic origin. Application Applications should include the following parts: 1) Application cover letter indicating motivation 2) Curriculum vitae 3) Copies of relevant diplomas 4) A complete list of publications indicating articles relevant to the position, including copies of these articles (max. 5) 5) Recommendation letters if relevant The parts must be clearly labeled with the above-mentioned numbers. Applications will not be taken into consideration, if the requirements listed above are not met. The application will be assessed according to the Ministerial Order no. 242 of 13 March 2012 on the Appointment of Academic Staff at Universities. The University of Copenhagen encourages all interested in this position to apply. Please submit the application with the required attachments by clicking on apply online below. Only online applications will be accepted. The closing date for applications is 1st June 2014. Applications received after the deadline will not be considered. The Faculty of Health Sciences comprises app. 7500 students, app. 1500 PhD students and app. 3200 employees. The Faculty creates new knowledge and recognition through its core activities: research, teaching, knowledge sharing and communication. With basic research fields ranging from molecular studies to studies of society, the Faculty contributes to a healthy future through its graduates, research findings and inventions for the benefit of patients and the community. Apply onlinehttps://ssl1.peoplexs.com/Peoplexs22/CandidatesPortalNoLogin/ApplicationForm.cfm?PortalID=3789VacatureID=645105 The University of Copenhagen actively influences current and future generations through excellent research, education and co-operation. UCPH is one of the highest ranked universities in Europe and is Denmark´s oldest university, founded in 1479. Today, the University has 37,000 students and 9,000 employees affiliated across six faculties: humanities, law, natural sciences, social sciences, health sciences and theology. www.ku.dk/english/. Professor Jette Sandholm Kastrup Head of GluTarget (www.glutarget.ku.dk) Biostructural Research (www.farma.ku.dk/BR) Department of Drug Design and Pharmacology Faculty of Health and Medical Sciences University of Copenhagen Universitetsparken 2 DK-2100 Copenhagen
Re: [ccp4bb] Space group problem?
Dear Chen, how sure are you that your crystals contain the protein of interest? Repeatedly washing harversted crystals in crystal stabilization solution followed by SDS-PAGE coupled to appropriate staining protocols (e.g. Coomassie, Silver staining) or western blotting can give a pretty conclusive answer. In our group, SDS-PAGE followed by Silver-staining is done routinely and in most cases leads to conclusive results. Here is a summary of the protocol: - select a drop containing substantial crystalline material. The crystals can be many and small (crystal shower) or few and large. - prepare a PCR-tube with crystal stabilization buffer (e.g. 50 uL of motherliquor containing a 10% higher concentration of precipitant). - transfer all the crystalline material from the drop into the PCR-tube using a pipet (You can use stabilization buffer from the PCR tube to collect all crystals). One can also use a cryo-loop to harvest the crystals if they are large enough to allow efficient harvesting. - centrifuge the PCR-tube at low speed for 30-60 sec and observe the crystals under the microscope to make sure they are at the bottom of the PCR-tube. - remove as much of the supernatant as you can using a pipet making sure not to remove the crystals. Then add crystal stabilization buffer to wash the crystals, and centrifuge again. - repeat this washing procedure a few times (typicaly 3-4 times). - after the final washing step, centrifugation and removal of the supernatant, add Laemmli-buffer to the crystals and use this sample for loading onto the SDS-PAGE gel. - include a positive control (e.g. solubilize another drop directly in Laemmli-buffer) and a negative control (final washing buffer). Including a pre-crystallization sample of your protein as a control is also recommended, to control for the integrity of the protein under crystallization conditions. - use silver staining to visualize the protein. --- best regards Savvas On 31 Mar 2014, at 23:48, Chen Zhao c.z...@yale.edu wrote: BTW, I forgot to mention that phenix.autosol also gave similar result. On Mon, Mar 31, 2014 at 5:46 PM, Chen Zhao c.z...@yale.edu wrote: Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen
Re: [ccp4bb] Refinement with new ligands to PDB
Dear Meisam, In the past I had similar problems, because of jligand and other cif generators can make errors when generate cif files, as it has been written before. I have solved these problems by using sketcher (ccp4) and PRODRG server (http://davapc1.bioch.dundee.ac.uk/cgi-bin/prodrg). I use the first one to create the pdb file (it is very easy to use) and the second to generate the restrains file both for refmac and for phenix.refine. I hope I've helped you. Danilo Il 2014-04-01 01:21 Meisam Nosrati ha scritto: Dear CCP4ers I have crystallized a protein with a series of ligands that are not in the PDB. I have made the pdb and the torsion libraries in the jligand and given them the new three letter codes, and then I try the refinement in the PHENIX or CCP4, but since these ligands are not in the library the refinement aborts. Even when I open the ligand files in COOT and I import the cif dictionary, still it does not recognize it, and does not let me rotate around the bonds. I need to know how to fix this problem, and how can I choose the three letter codes for my ligands that are not already chosen by other people. Thanks in advance for your help Meisam
[ccp4bb] Density averaging in Dm
Dear Friends I have a protein-peptide complex crystals, which is diffracted at 3.8 resolution with a P41212 space group. We solved the structure using molecular replacement method, which shows that there are thre molecules in asymmetric unit. Unfortunately one of the chain in this asymmetric unit is poorly defined and then I attempted density averaging using Density modification program from CCP4 suite. It is fained with error massage: *dm:(RCARDS) SOLC:content missing or out of range* The solvent content is around 75 percent and I have submitted a recent refmac mtz file for the job with rotation and translation matrix ( to have an idea anout NCS). I have also checked 'Input starting map coefficients'. Can any one help in finding what is the problem and how to sort it out? Thanking you in advance. DCB -- Mr.Dilip C. Badgujar, Senior Research Fellow, ACTREC, Tata Memorial Center, Sector-22, Kharghar, Navi Mumbai. Pin-410210
Re: [ccp4bb] difference between polar angle and eulerian angle
On 1 April 2014 00:12, Edward A. Berry ber...@upstate.edu wrote: I need to work on something else now, but when i get time I will go through Ian's derivation and see how it is in fact tractable. I should point out that it's not my derivation. This proof was given to me by Tilman Shirmer. For the original go to: http://www.biozentrum.unibas.ch/research/groups-platforms/eigene-seiten/unit/schirmer/?tx_x4epersdb_pi5%5BshowContentPid%5D=12113cHash=788e24eee4b7f8b88c842bc94fbd152b Cheers -- Ian
Re: [ccp4bb] Space group problem?
Dear Savvas, Thank you for your reply and your nice protocol. I am also worried of this problem so I have already run my crystals on the gel for several times. The result is so clean that you can hardly draw any other conclusions except that the crystals are made up of the full-length molecule. But possibility remains regarding this concern if my molecule of interest is not in crystalline state but other molecules that cannot be stained are. Best, Chen On Tue, Apr 1, 2014 at 3:30 AM, Savvas Savvides savvas.savvi...@ugent.bewrote: Dear Chen, how sure are you that your crystals contain the protein of interest? Repeatedly washing harversted crystals in crystal stabilization solution followed by SDS-PAGE coupled to appropriate staining protocols (e.g. Coomassie, Silver staining) or western blotting can give a pretty conclusive answer. In our group, SDS-PAGE followed by Silver-staining is done routinely and in most cases leads to conclusive results. Here is a summary of the protocol: - select a drop containing substantial crystalline material. The crystals can be many and small (crystal shower) or few and large. - prepare a PCR-tube with crystal stabilization buffer (e.g. 50 uL of motherliquor containing a 10% higher concentration of precipitant). - transfer all the crystalline material from the drop into the PCR-tube using a pipet (You can use stabilization buffer from the PCR tube to collect all crystals). One can also use a cryo-loop to harvest the crystals if they are large enough to allow efficient harvesting. - centrifuge the PCR-tube at low speed for 30-60 sec and observe the crystals under the microscope to make sure they are at the bottom of the PCR-tube. - remove as much of the supernatant as you can using a pipet making sure not to remove the crystals. Then add crystal stabilization buffer to wash the crystals, and centrifuge again. - repeat this washing procedure a few times (typicaly 3-4 times). - after the final washing step, centrifugation and removal of the supernatant, add Laemmli-buffer to the crystals and use this sample for loading onto the SDS-PAGE gel. - include a positive control (e.g. solubilize another drop directly in Laemmli-buffer) and a negative control (final washing buffer). Including a pre-crystallization sample of your protein as a control is also recommended, to control for the integrity of the protein under crystallization conditions. - use silver staining to visualize the protein. --- best regards Savvas On 31 Mar 2014, at 23:48, Chen Zhao c.z...@yale.edu wrote: BTW, I forgot to mention that phenix.autosol also gave similar result. On Mon, Mar 31, 2014 at 5:46 PM, Chen Zhao c.z...@yale.edu wrote: Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen
[ccp4bb] AW: [ccp4bb] Space group problem?
Dear Chen, I am not an expert on SAD and MAD. However, at this stage I would not worry too much about density going through the 2-fold axis. There might be a sulfate ion or some other buffer component present at that position, or it may just be an artifact that will go away once the structure has been built and refined. The questions I would worry about is: how much too small is your unit cell? Is it just crowded, say 25-30% solvent, or would your protein molecule not fit at all? Does the amount of solvent as estimated from your SAD/MAD maps agree with the amount of solvent obtained from the calculation of the Matthews volume? How many (SeMet?) sites do you expect, 6, more, less? If everything looks ok except that the unit cell is rather crowded, I would go ahead and try to build the structure. However, if even a single protein molecule would not fit in your unit cell, or you find many more sites than you can explain, you should start worrying about twinning. Even than the structure can probably be solved, but then you need some real experts! My 2 cents, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Chen Zhao Gesendet: Montag, 31. März 2014 23:46 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Space group problem? Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen
[ccp4bb] Workshop: Chemical Probe-based Open Science: Uncovering New Human and Plant Biology - Brazil, April 28-29th
Dear all, On April 28-29th Nature conferences, SGC and University of Campinas will realize a workshop entitled Chemical Probe-based Open Science: Uncovering New Human and Plant Biology. It will be held at the University of Campinas (Unicamp), in Campinas-São Paulo, Brazil. It is a good opportunity to meet some great scientists in this field. For more information: http://www.nature.com/ natureconferences/sgc2014/ index.html http://www.nature.com/natureconferences/sgc2014/index.html -- *Izabella A. Pena Neshich* *PhD Student, Genetics and Molecular Biology* Centro de Biologia Molecular e Engenharia Genética - CBMEG Universidade Estadual de Campinas - UNICAMP
[ccp4bb] error xds
Dear CCP4 users, while I am running CORRECT within XDS, the program suddenly stops, and gives the following message: forrtl: severe (24): end-of-file during read, unit 2, file /data/almudena/160314SLS/dts2/bin1_01.tmp I actually don't know what it means. I could run it with no problems with 300 frames, but not with 600 or with 500. I get this message when I do so. Any suggestions are welcome. I'm looking forward to hearing from you. Best wishes, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] error xds
sorry, I missed this part of the error message: !!! ERROR !!! AUTOMATIC DETERMINATION OF SPOT SIZE PARAMETERS HAS FAILED. YOU MAY RESTART THIS STEP AFTER SPECIFYING VALUES IN XDS.INP FOR: REFLECTING_RANGE=REFLECTING_RANGE_E.S.D.= BEAM_DIVERGENCE= BEAM_DIVERGENCE_E.S.D.= 2014-04-01 17:27 GMT+02:00 Almudena Ponce Salvatierra maps.fa...@gmail.com : Dear CCP4 users, while I am running CORRECT within XDS, the program suddenly stops, and gives the following message: forrtl: severe (24): end-of-file during read, unit 2, file /data/almudena/160314SLS/dts2/bin1_01.tmp I actually don't know what it means. I could run it with no problems with 300 frames, but not with 600 or with 500. I get this message when I do so. Any suggestions are welcome. I'm looking forward to hearing from you. Best wishes, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
[ccp4bb] Postdoctoral position at Harvard Medical School
A *post-doctoral position* is available to study the molecular mechanism for self vs. non-self discrimination by the innate immune system. We use a combination of X-ray crystallography, biochemistry and cell biology to characterize structures and functions of key host molecules that recognize foreign molecules, in particular viral nucleic acids. Applicants should have received (or expect to receive) a PhD and have a strong background in X-ray crystallography, biochemistry, cellular immunology or virology. Please send a cover letter, CV, names, e-mail addresses and telephone numbers of three references to Sun Hur at sun@childrens.harvard.edu Please refer to http://hurlab.tch.harvard.edu/ and http://hurlab.tch.harvard.edu/publications/ to learn more about our research.
Re: [ccp4bb] error xds
Dear Almudena, You can try to run XDS with 300 frames. At the end, you have to open the file called INTEGRATE.LP and copy the refined value of REFLECTING_RANGE= REFLECTING_RANGE_E.S.D.= BEAM_DIVERGENCE=BEAM_DIVERGENCE_E.S.D.= at the end of your XDS.INP. Now, you can re-run XDS with all your frames. However, if xds fails, it means that your data are not good. This is only a strategy to overcome the error. Danilo Il 2014-04-01 17:28 Almudena Ponce Salvatierra ha scritto: sorry, I missed this part of the error message: !!! ERROR !!! AUTOMATIC DETERMINATION OF SPOT SIZE PARAMETERS HAS FAILED. YOU MAY RESTART THIS STEP AFTER SPECIFYING VALUES IN XDS.INP FOR: REFLECTING_RANGE= REFLECTING_RANGE_E.S.D.= BEAM_DIVERGENCE= BEAM_DIVERGENCE_E.S.D.= 2014-04-01 17:27 GMT+02:00 Almudena Ponce Salvatierra maps.fa...@gmail.com: Dear CCP4 users, while I am running CORRECT within XDS, the program suddenly stops, and gives the following message: forrtl: severe (24): end-of-file during read, unit 2, file /data/almudena/160314SLS/dts2/bin1_01.tmp I actually don't know what it means. I could run it with no problems with 300 frames, but not with 600 or with 500. I get this message when I do so. Any suggestions are welcome. I'm looking forward to hearing from you. Best wishes, Almudena -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany -- Almudena Ponce-Salvatierra Macromolecular crystallography and Nucleic acid chemistry Max Planck Institute for Biophysical Chemistry Am Fassberg 11 37077 Göttingen Germany
Re: [ccp4bb] AW: [ccp4bb] Space group problem?
Dear all, I am just taking advantage of this particular thread to add my query also. I hope you don't mind Chen. We haven't solved any structure in our lab using SAD phasing, so pardon me for sounding naive. I have a 6.5 A data of anomalous scattering with a 3.5 A data using the Cu anode.My crystals dont grow any better. I have around 10 Met distributed fairly evenly through out the sequence. So is it possible to get any anomalous signals form this data? Moreover, I am not able to index my 3.5A data at all. The spots don't fit well. What could be the problem? Hope this thread can be of benefit for both me and Chen. Thanks On Tue, Apr 1, 2014 at 8:21 PM, Chen Zhao c.z...@yale.edu wrote: Dear Herman, Thank you so much for your suggestions. The density that passes through the rotational axis is so strong and extended that can be considered as a significant portion of the molecule. However, some density in the middle might show some features. I have no experience and this could be only artifact. The unit cell seems to be able to fit 1-2 molecules. But if there is one molecule/ASU, the solvent content is about 89%, which is possible but unlikely. Although the resolution of the crystal is not high, it is rather rigid and easy to handle, which might indicate a not-too-high solvent content. I soaked the native crystal with heavy atom compounds and I have no clear idea of the relationship between metal binding and sequence, so I don't know how many sites to expect. Best, Chen On Tue, Apr 1, 2014 at 9:42 AM, herman.schreu...@sanofi.com wrote: Dear Chen, I am not an expert on SAD and MAD. However, at this stage I would not worry too much about density going through the 2-fold axis. There might be a sulfate ion or some other buffer component present at that position, or it may just be an artifact that will go away once the structure has been built and refined. The questions I would worry about is: how much too small is your unit cell? Is it just crowded, say 25-30% solvent, or would your protein molecule not fit at all? Does the amount of solvent as estimated from your SAD/MAD maps agree with the amount of solvent obtained from the calculation of the Matthews volume? How many (SeMet?) sites do you expect, 6, more, less? If everything looks ok except that the unit cell is rather crowded, I would go ahead and try to build the structure. However, if even a single protein molecule would not fit in your unit cell, or you find many more sites than you can explain, you should start worrying about twinning. Even than the structure can probably be solved, but then you need some real experts! My 2 cents, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Chen Zhao *Gesendet:* Montag, 31. März 2014 23:46 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] Space group problem? Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
Re: [ccp4bb] AW: [ccp4bb] Space group problem?
Dear Jurgen, The beam position is fine. we have collected many data sets before and after this data. Moreover, we the Technical scientist always checks the beam position before we mount the crystals. Thanks On Tue, Apr 1, 2014 at 11:23 PM, Jurgen Bosch jbos...@jhu.edu wrote: check your beam position .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Apr 1, 2014, at 1:26 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab nazia.nasi...@nii.ac.in wrote: Dear all, I am just taking advantage of this particular thread to add my query also. I hope you don't mind Chen. We haven't solved any structure in our lab using SAD phasing, so pardon me for sounding naive. I have a 6.5 A data of anomalous scattering with a 3.5 A data using the Cu anode.My crystals dont grow any better. I have around 10 Met distributed fairly evenly through out the sequence. So is it possible to get any anomalous signals form this data? Moreover, I am not able to index my 3.5A data at all. The spots don't fit well. What could be the problem? Hope this thread can be of benefit for both me and Chen. Thanks On Tue, Apr 1, 2014 at 8:21 PM, Chen Zhao c.z...@yale.edu wrote: Dear Herman, Thank you so much for your suggestions. The density that passes through the rotational axis is so strong and extended that can be considered as a significant portion of the molecule. However, some density in the middle might show some features. I have no experience and this could be only artifact. The unit cell seems to be able to fit 1-2 molecules. But if there is one molecule/ASU, the solvent content is about 89%, which is possible but unlikely. Although the resolution of the crystal is not high, it is rather rigid and easy to handle, which might indicate a not-too-high solvent content. I soaked the native crystal with heavy atom compounds and I have no clear idea of the relationship between metal binding and sequence, so I don't know how many sites to expect. Best, Chen On Tue, Apr 1, 2014 at 9:42 AM, herman.schreu...@sanofi.com wrote: Dear Chen, I am not an expert on SAD and MAD. However, at this stage I would not worry too much about density going through the 2-fold axis. There might be a sulfate ion or some other buffer component present at that position, or it may just be an artifact that will go away once the structure has been built and refined. The questions I would worry about is: how much too small is your unit cell? Is it just crowded, say 25-30% solvent, or would your protein molecule not fit at all? Does the amount of solvent as estimated from your SAD/MAD maps agree with the amount of solvent obtained from the calculation of the Matthews volume? How many (SeMet?) sites do you expect, 6, more, less? If everything looks ok except that the unit cell is rather crowded, I would go ahead and try to build the structure. However, if even a single protein molecule would not fit in your unit cell, or you find many more sites than you can explain, you should start worrying about twinning. Even than the structure can probably be solved, but then you need some real experts! My 2 cents, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Chen Zhao *Gesendet:* Montag, 31. März 2014 23:46 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] Space group problem? Dear all, I am now trying to phase a structure in C2 using anomalous scattering at 5-6 A. It is hard to improve the derivative resolution at the moment. Shelxd is able to locate 6 sites with a distinct CC and FOM. After density modification in shelxe, the contrast of the two enantiomers is 0.59/0.38 for SAD and 0.7/0.3 for MAD. When I looked at the electron density, the maps from SAD and MAD are similar, and the solvent boundary is quite clear. However, the problem is that the electron density blob passes through the 2-fold rotation axis, even at 3 rmsd contour level. Also, the unit cell seems to be too small for the molecule. I am afraid that the space group assignment is wrong, but I am a beginner so I nearly have no clue. I did reprocess the data in P1 and looked at the self-rotation function with a radius at 200 A. From the list it seems that there is only one 2-fold rotation axis. I am quite confused. Could anybody give me some hint of this problem? Thanks a lot in advance! Sincerely, Chen -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi
Re: [ccp4bb] Refinement with new ligands to PDB
Thank you very much everyone for your comments. I will try your ideas and try to get this thing to work. I appreciate your help Meisam On Mon, Mar 31, 2014 at 7:21 PM, Meisam Nosrati meisam.nosr...@gmail.comwrote: Dear CCP4ers I have crystallized a protein with a series of ligands that are not in the PDB. I have made the pdb and the torsion libraries in the jligand and given them the new three letter codes, and then I try the refinement in the PHENIX or CCP4, but since these ligands are not in the library the refinement aborts. Even when I open the ligand files in COOT and I import the cif dictionary, still it does not recognize it, and does not let me rotate around the bonds. I need to know how to fix this problem, and how can I choose the three letter codes for my ligands that are not already chosen by other people. Thanks in advance for your help Meisam
Re: [ccp4bb] AW: [ccp4bb] Space group problem?
By the way, I have another question related to the number of sites. I just rethought about what Herman mentioned, and I just realized that the number of sites, at least strong sites, could be guessed from an anomalous Patterson map. Therefore I looked at the anomalous Patterson of my data. The map indicates that the signal is very weak, and there are only 4 non-origin positive peaks (3 effective peaks, because 2 are symmetry equivalent), indicating there are at most 3 heavy atoms (3x2=6) if all of them contribute to the map. However, in shelxd, I can get optimal results from 6 sites out of the same data set. I am not sure whether this is because the anomalous Patterson is not sensitive enough. Now I am more confused about the criteria in choosing the parameters when running shelx and all other programs. What should be the general practice? Should I rely more on the anomalous Patterson or more on the better scores reported? When I tried to optimize the results by changing the parameters (either when locating sites or when phasing), am I more heading for the real signal or more simply tweaking the parameters? Maybe the golden standard would be whether you could solve the structure. Another question is, although from the Euler's equation I could understand negative Patterson peaks (maybe I am wrong), I am still not clear what negative Patterson peaks represent and why people generally don't talk about it. In the specific data I am talking about above, there is a negative Patterson peak at the same position as a positive Patterson peak with the same height. Is there any indication about this? Thank you so much for your attention! On Tue, Apr 1, 2014 at 2:00 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab nazia.nasi...@nii.ac.in wrote: Dear Jurgen, The beam position is fine. we have collected many data sets before and after this data. Moreover, we the Technical scientist always checks the beam position before we mount the crystals. Thanks On Tue, Apr 1, 2014 at 11:23 PM, Jurgen Bosch jbos...@jhu.edu wrote: check your beam position .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Apr 1, 2014, at 1:26 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab nazia.nasi...@nii.ac.in wrote: Dear all, I am just taking advantage of this particular thread to add my query also. I hope you don't mind Chen. We haven't solved any structure in our lab using SAD phasing, so pardon me for sounding naive. I have a 6.5 A data of anomalous scattering with a 3.5 A data using the Cu anode.My crystals dont grow any better. I have around 10 Met distributed fairly evenly through out the sequence. So is it possible to get any anomalous signals form this data? Moreover, I am not able to index my 3.5A data at all. The spots don't fit well. What could be the problem? Hope this thread can be of benefit for both me and Chen. Thanks On Tue, Apr 1, 2014 at 8:21 PM, Chen Zhao c.z...@yale.edu wrote: Dear Herman, Thank you so much for your suggestions. The density that passes through the rotational axis is so strong and extended that can be considered as a significant portion of the molecule. However, some density in the middle might show some features. I have no experience and this could be only artifact. The unit cell seems to be able to fit 1-2 molecules. But if there is one molecule/ASU, the solvent content is about 89%, which is possible but unlikely. Although the resolution of the crystal is not high, it is rather rigid and easy to handle, which might indicate a not-too-high solvent content. I soaked the native crystal with heavy atom compounds and I have no clear idea of the relationship between metal binding and sequence, so I don't know how many sites to expect. Best, Chen On Tue, Apr 1, 2014 at 9:42 AM, herman.schreu...@sanofi.com wrote: Dear Chen, I am not an expert on SAD and MAD. However, at this stage I would not worry too much about density going through the 2-fold axis. There might be a sulfate ion or some other buffer component present at that position, or it may just be an artifact that will go away once the structure has been built and refined. The questions I would worry about is: how much too small is your unit cell? Is it just crowded, say 25-30% solvent, or would your protein molecule not fit at all? Does the amount of solvent as estimated from your SAD/MAD maps agree with the amount of solvent obtained from the calculation of the Matthews volume? How many (SeMet?) sites do you expect, 6, more, less? If everything looks ok except that the unit cell is rather crowded, I would go ahead and try to build the structure. However, if even a single
[ccp4bb] Structure factor equation
Encouraged by recent help from the BB in filling in gaps in my understanding, maybe I can get help with another question: At the top of page 121 in Blundell and Johnson, it is written: The total wave scattered by a small unit of volume dv at a position r relative to the wave scattered from the origin will therefore have an amplitude proportional to Rho(r)dv and phase 2Pi i(r.S)dv (OK so far) i.e. wave scattered = Rho(r)exp(2Pi i r.S)dv How is that a wave? r and S are constant vectors. My best explanation so far is to say this is a complex coefficient that will adjust the weight and phase of the wave scattered by this point. Say the wave scattered from one electron at the origin will result in a temporal cosine wave at the surface of the detector: E = exp(2Pi i wt) = cos(2Pi wt) (not sure if 2Pi is needed when w is radians/sec) Then the wave at the same point, scattered by dv at r, would be the same multiplied by the quantity in question: E = rho(r)exp(2Pi i r.s)dv * exp(2pi i wt) = rho(r)exp(2pi i (wt - r.S)) i.e. phase-shifted by 2Pi (r.S), and multiplied by Rho(r)dv Is that more or less it? (since these quantities add up to the Structure Factor F(s), I guess I'm really asking what a structure factor is. Rupp says a structure fator is a vector representing the diffracted X-rays, which i take to be consistent with this if vector is in the complex plane)
