Re: [ccp4bb] fixing position in the refinement: Shelx, refmac5
For SHELX there are two ways to fix selected atoms: either put AFIX 1 in front of the fixed atoms and AFIX 0 after them, or use the BLOC instruction. e.g. BLOC 1 N_1 C_53 N_57 C61 N_62 CA_62 C_62 O_62 N_63 LAST BLOC -1 N_1 C_53 N_57 C61 N_62 CA_62 C_62 O_62 N_63 LAST would fix residues 54-56 and the side-chain of residue 62 (but you may need to consider the order of atoms). This would be easier than using AFIX if you are already using AFIX for riding hydrogens, however it is OK to fix atoms with AFIX 1 and then use HFIX in the same job to add hydrogens. I have deliberately chosen a complicated example to show how easy it is! More details on SHELXL refinements are now available from the CCP4 Wiki: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/SHELXL and for SHELXC, D and E see: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/SHELX_C/D/E Note that since this is a Wiki, users are encouraged to contribute! 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. +49-551-39-2582 On Thu, 3 Apr 2008, U Sam wrote: How one can fix position of few residues when refining in SHELX or Refmac5. Thanks. Sam. _ Use video conversation to talk face-to-face with Windows Live Messenger. http://www.windowslive.com/messenger/connect_your_way.html?ocid=TXT_TAGLM_WL_Refresh_messenger_video_042008
Re: [ccp4bb] fixing position in the refinement: Shelx, refmac5
No. Better way is restraining internal degrees of freedom. Something like elastic network model: Tirion MM (1996) Phys Rev Letters 77 pp 1905-1908 But care should be taken with weights and which distances should be restrained. Fiixing position is not good way of using prior information (In my view of course, which is debatable) Garib On 4 Apr 2008, at 06:56, Frank von Delft wrote: Is that also how you would restrain refinement at low resolution when using a high-resolution model? (e.g. a 1.8A model into 4A data) phx. Garib Murshudov wrote: In newer version of refmac you can use harmonic restraints. Take the new stable version of refmac from www.ysbl.york.ac.uk/YSBLPrograms/index.jsp and use harmonic restraints as described in: http://www.ysbl.york.ac.uk/~garib/refmac/data/refmac_news.html http://www.ysbl.york.ac.uk/%7Egarib/refmac/data/refmac_news.html Keyword is: external harmonic residues from [residue_number] [chain_name] to [residue_number] [chain_name] sigma [value] sigma 0.1 With the current ccp4i version you can use developers option and add an external keyword file that contains this keyword for residues you want to fix. I hope it helps. Garib On 4 Apr 2008, at 00:39, U Sam wrote: How one can fix position of few residues when refining in SHELX or Refmac5. Thanks. Sam. _ Use video conversation to talk face-to-face with Windows Live Messenger. http://www.windowslive.com/messenger/connect_your_way.html? ocid=TXT_TAGLM_WL_Refresh_messenger_video_042008
[ccp4bb] Opening for a X-ray facility/computing manager
The VIB Department of Molecular and Cellular Interactions at the Vrije Universiteit Brussel is looking for a highly motivated X-ray facility/computing manager. The successful candidate will provide expert support to our in house structural biology community and will conduct research in the area of structural biology through internal and/or external collaborations. The department currently contains four groups involved in structural biology totalling around 40 people. A Bio-NMR group is to be established beginning next year. Details on the research conducted in the department can be found on our websites (http://www.vib.be/ and http://ultr.vub.ac.be/). Experience with UNIX/LINUX system administration is a must and experience with the computational side of protein crystallography is highly desirable. The main service duties will include: - Maintaining and upgrading the crystallographic and NMR computing facilities and reassuring of data back-up - Maintenance of the in-house X-ray data collection facilities - Organizing and streamlining synchrotron trips Interested candidates should send their cv to Remy Loris ([EMAIL PROTECTED]) together with the names and contact details of at least two reference persons. Salary will be discussed and will depend on the level of experience and qualifications of the successful candidate. Remy Loris Vrije Universiteit Brussel Pleinlaan 2 1050 Brussel Belgium
[ccp4bb] Post doctoral postion in computational methods development, Leiden, NL
A two year post doctoral position is available at Leiden University, The Netherlands (http://www.bfsc.leidenuniv.nl/) in computational methods development. The position will be for the derivation and implementation of new algorithms for exploiting all the available information in macromolecular crystal structure solutions using multivariate statistical methods. To get a better idea of the methods we use, check out our references for some applications already done: (http://www.bfsc.leidenuniv.nl/software/crank/references.html) The methods are implemented within our Crank suite (http://www.bfsc.leidenuniv.nl/software/crank/) [*] and in Refmac in collaboration with Garib Murshudov (http://www.ysbl.york.ac.uk/~garib/refmac/latest_refmac.html#experimental). A good knowledge of mathematics/probabilistic methods and computer programming is important for the successful completion of the project. Funding for this position is from the Dutch organization for scientific research (Netherlandse organisatie voor wetenschappelijk onderzoek: http://www.nwo.nl). For more information, please feel free to contact me! * An official (and *much* improved) version of Crank will be in the upcoming CCP4 6.1 release and from our web site in Leiden - I'll send another email when it is available for download.
[ccp4bb] Off topic: McDonalds Happy Meal and Crystallography
I took my 5 year old niece to a McDonald's (in England) for a happy meal today - Spiderwick Chronicles promotion. The toy's light effect reminded me of a visual aid I saw to help introduce X-ray diffraction and the crystal lattice to students. My niece was more interested in using the visual effect to see if her toy was a goodie or baddie. Cheers! _ Going green? See the top 12 foods to eat organic. http://green.msn.com/galleries/photos/photos.aspx?gid=164ocid=T003MSN51N1653A
[ccp4bb] off topic - C-ter cleavable his tag
Hi, I am looking for a vector with a cleavable C-terminal his tag for prokaryotic protein expression. I found pET-52b but it has also got a N-terminal cleavable strep-tag which I cannot avoid. Thanks Vijay
Re: [ccp4bb] off topic - C-ter cleavable his tag
Hi Vijay, You may consider having a protease site in your 3' PCR primer, since you probably have to remove the stop codon at the same time. It's probably easier to do than finding a vector with C-terminal cleavable tag and C-His will be in-frame without any extra sequence. Many pET vectors with N-His also have C-His. You may also consider HRV3C or TEV. Not because Accelagen sells those proteases, but they are more specific than proteases like thrombin and easier to remove. Cheers, Chun Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 mailto:[EMAIL PROTECTED] [EMAIL PROTECTED] www.accelagen.com http://www.accelagen.com/ _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Vijay Kumar Sent: Friday, April 04, 2008 8:40 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic - C-ter cleavable his tag Hi, I am looking for a vector with a cleavable C-terminal his tag for prokaryotic protein expression. I found pET-52b but it has also got a N-terminal cleavable strep-tag which I cannot avoid. Thanks Vijay
Re: [ccp4bb] Off topic: McDonalds Happy Meal and Crystallography
I saw that toy - Personally, I do not like the idea of encouraging children to aim a light (low powered led) directly at one's eye... Ezra P Hubbard wrote: I took my 5 year old niece to a McDonald's (in England) for a happy meal today - Spiderwick Chronicles promotion. The toy's light effect reminded me of a visual aid I saw to help introduce X-ray diffraction and the crystal lattice to students. My niece was more interested in using the visual effect to see if her toy was a goodie or baddie. Cheers! Going green? See the top 12 foods to eat organic. http://green.msn.com/galleries/photos/photos.aspx?gid=164ocid=T003MSN51N1653A
[ccp4bb] DM - NCS averaging after phaser molecular replacement run-confused about coefficients
Hi everyone, I have a phaser molecular replacement solution for my membrane protein which crystallized in spacegroup P3. The diffraction data is good to about 3.3 A. The model I used had 39% homology to the given protein. A solvent content analysis suggests that there probably are three dimers in the ASU ( to give about 67% solvent) Phaser sucessfuly found two dimers in the ASU with good density and a third dimer with very weak almost non-existent density. I am now trying to do some NCS-averaging using DM to see If I can improve the desnity for dimer 3 and have a question about the different co-efficients I should be using for the averaging DM run. Question1: The phaser output mtz file has a PHWT and a PHIC . For carrying out DM with flattening, averaging and histogram matching which phases do I use PHIC or PHWT along with observed Fo. Also for the weight do I use the FOM. Question2: The output mtz from DM run either way above , now has a PHIDM and a PHWT along with a FWT and FC. Which coefficients should I be using to get a map after DM for building. FWT and PHWT or Fo and PHIDM or FWT and PHIDM. Thank you for your help Hari
Re: [ccp4bb] Off topic: Real funny until someone loses a Happy Meal
As opposed to putting McDonald's food into their stomachs? On Apr 4, 2008, at 9:33 AM, Ezra Peisach wrote: I saw that toy - Personally, I do not like the idea of encouraging children to aim a light (low powered led) directly at one's eye... Ezra P Hubbard wrote: I took my 5 year old niece to a McDonald's (in England) for a happy meal today - Spiderwick Chronicles promotion. The toy's light effect reminded me of a visual aid I saw to help introduce X-ray diffraction and the crystal lattice to students. My niece was more interested in using the visual effect to see if her toy was a goodie or baddie. Cheers! Going green? See the top 12 foods to eat organic. http://green.msn.com/galleries/photos/photos.aspx?gid=164ocid=T003MSN51N1653A
[ccp4bb] Error in configuration of hydroxyproline in HYP.cif
Dear All, My colleague Erhard Hohenester has noticed that there is an error in the cif definition file for hydroxyproline distributed with CCP4 version 6.02 and used by Refmac, Coot (and Phenix). In collagen a hydroxyl group is bonded to the CD atom of the prolyl ring to give an R configuration. [IUPAC: (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid] The monomer dictionary file HYP.cif incorrectly specifies the S configuration about the CD atom. In HYP.cif the chirality is specified by the sign of the chiral volume formed by a scalar triple product: HYP chir_01 CA N CB C negativ HYP chir_02 CG CB OD CDpositiv The second line should be: HYP chir_02 CG CB OD CDnegativ Alternatively, one could change the atom order. Peter -- Peter Brick, Division of Cell and Molecular Biology, Blackett Laboratory, Imperial College, Prince Consort Road, London SW7 2BW, UK Tel: 020-7594-7704 Fax: 020-7589-0191
Re: [ccp4bb] DM - NCS averaging after phaser molecular replacement run-confused about coefficients
On Apr 4 2008, hari jayaram wrote: Hi everyone, I have a phaser molecular replacement solution for my membrane protein which crystallized in spacegroup P3. The diffraction data is good to about 3.3 A. The model I used had 39% homology to the given protein. A solvent content analysis suggests that there probably are three dimers in the ASU ( to give about 67% solvent) Phaser sucessfuly found two dimers in the ASU with good density and a third dimer with very weak almost non-existent density. I am now trying to do some NCS-averaging using DM to see If I can improve the desnity for dimer 3 and have a question about the different co-efficients I should be using for the averaging DM run. Question1: The phaser output mtz file has a PHWT and a PHIC . For carrying out DM with flattening, averaging and histogram matching which phases do I use PHIC or PHWT along with observed Fo. Also for the weight do I use the FOM. Question2: The output mtz from DM run either way above , now has a PHIDM and a PHWT along with a FWT and FC. Which coefficients should I be using to get a map after DM for building. FWT and PHWT or Fo and PHIDM or FWT and PHIDM. Thank you for your help Hari In the MTZ file produced by Phaser, the FWT/PHWT pair provide the 2mFo-DFc map coefficients that should reduce model bias. I'd suggest starting DM from these coefficients, which you can do by adding FDM=FWT PHIDM=PHWT to the LABIN command for DM. (If you want to do this from the current ccp4i GUI for DM, the only way is to use the RunView Com File command.) After running DM, the map coefficients to view in coot are FDM/PHIDM. Good luck! Randy Read
[ccp4bb] Off-topic: fungus contamination of SF9 cells
Dear all, sorry for this off topic question. I am using baculovirus/insect cell expression and I have had persisting problems with contamination that seems to be some kind of fungus. I have a picture attached showing the filamentous structure of it. This contamination has persisted for a few months now and always seems to occur when we go to large 2.8 L flasks (however, there was also one instant where this was not the case). We have autoclaved all flasks for 90 minutes each and still we get contamination. Does anybody have experience with this kind of contamination? And what would you suggest for us to do? We will (again) disinfect our incubator and the hood. Also we are thinking about using an antimycotic/antifungal drug to get rid of this problem. Thanks a lot for any suggestion! Filipp
Re: [ccp4bb] Off-topic: fungus contamination of SF9 cells
Hi Filipp, Start a new culture from your frozen cell bank using disposable plasticwares. If it still happens and assuming the working environment is clean and operators follow the SOP, you may have to get a new cell stock from somewhere else. We had a contamination once and traced back to its origin. It actually took a couple of weeks for the contamination to be visible. Whatever they were, just grew very slowly in the insect cell media. I guess you may not notice the contamination in smaller flasks when you pass the cells frequently. Letting the culture grow longer in large flasks accumulates the contaminants. Using AA adds cost. Better to make sure operators clean their gloves with ethanol. Very interesting picture. It could be a PhD thesis to figure out what it is. Good luck! Chun Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 [EMAIL PROTECTED] www.accelagen.com -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Filipp Frank Sent: Friday, April 04, 2008 12:49 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Off-topic: fungus contamination of SF9 cells Dear all, sorry for this off topic question. I am using baculovirus/insect cell expression and I have had persisting problems with contamination that seems to be some kind of fungus. I have a picture attached showing the filamentous structure of it. This contamination has persisted for a few months now and always seems to occur when we go to large 2.8 L flasks (however, there was also one instant where this was not the case). We have autoclaved all flasks for 90 minutes each and still we get contamination. Does anybody have experience with this kind of contamination? And what would you suggest for us to do? We will (again) disinfect our incubator and the hood. Also we are thinking about using an antimycotic/antifungal drug to get rid of this problem. Thanks a lot for any suggestion! Filipp
Re: [ccp4bb] off topic - C-ter cleavable his tag
Hi, Generally speaking, these days you don't need a special vector to introduce a C-His (or an N-His, BAP, STREP, STREPII, or any short tag for that matter), since the sequence of the tag + cleavage site is short enough to be engineered directly into your PCR amplicon. There are numerous ways to cleave C-terminal tags, however most of them leave several residues behind, due to the specificity determinants of (most) proteases being upstream of the cut site. If you absolutely have to use C-terminal His, consider not removing it at all since the difference between a site-specific protease 'leftover' and a 6-His stretch isn't very significant (although of course the effect on crystallization can be profound!). There are several somewhat esoteric ways to get rid of His-tags at the C-terminus cleanly - these involve treatment with carboxypeptidases and other similar tricks. Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Vijay Kumar Sent: Friday, April 04, 2008 11:40 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off topic - C-ter cleavable his tag Hi, I am looking for a vector with a cleavable C-terminal his tag for prokaryotic protein expression. I found pET-52b but it has also got a N-terminal cleavable strep-tag which I cannot avoid. Thanks Vijay
