[ccp4bb] molrep can't find *_trfn_scr.crd
When doing a multi-copy search, after the first rotation is done it seems to be looking for a _121_molrep_trfn_scr.crd file in my /tmp directory that it can't find just prior to doing the "first monomer search". Anyone have any ideas? thanks FR - Francis 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
[ccp4bb] Correction: [ccp4bb] molrep can't find *_trfn_scr.crd
Actually the file exists but molrep seems to looking it in the wrong directory (looks like a concatenation of the /tmp dir).. See below #CCP4I TERMINATION STATUS 0 "Last system error message: No such file or directory MOLREP(ccp4): Open failed: File: /tmp/francisreyes/ Andrea__121_molrep_/tmp/francisreyes/ Andrea_121_molrep_trfn_scr.crd " #CCP4I TERMINATION TIME 29 Jun 2009 11:10:51 #CCP4I MESSAGE Task failed Thanks FR Begin forwarded message: From: Francis E Reyes Date: June 29, 2009 11:15:14 AM MDT To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] molrep can't find *_trfn_scr.crd Reply-To: Francis E Reyes When doing a multi-copy search, after the first rotation is done it seems to be looking for a _121_molrep_trfn_scr.crd file in my /tmp directory that it can't find just prior to doing the "first monomer search". Anyone have any ideas? thanks FR - Francis 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 - Francis 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
[ccp4bb] Balbes 1.0.0. different results between WWW Server and Local instalation
* * * Hello After a helpful hand (on a Sunday !!) from Dr. Fei Long I installed BALBES 1.0.0 in my Ubuntu Linux machine and in order to try it, I ran the same job I had previously ran in the WWW Server. Surprisingly the results were worse in my local BALBES. Results from the WWW: BALBES Version is 1.0.0.May_16_2009 JOB FOR STARTS AT Tue Jun 9 12:17:31 2009 # | Please cite the following paper in any publication | | arising from use of BALBES: | | F.Long, A.Vagin, P.Young and G.N.Murshudov | | "BALBES: a Molecular Replacement Pipeline" | | Acta Cryst. D64 125-132(2008) | # You have provide 2 sequences in the input sequence file number of amino acids in the sequence 1 is 65 number of amino acids in the sequence 2 is 146 #---# # Structure Factor Data Analysis # #---# Information about the structure to be solved |--| | SPACE GROUP | I 41 2 2 | ||-| | CELL LENGTH | 130.050 130.050 70.190 | ||-| | CELL ANGLE | 90.000 90.000 90.000 | ||-| | RESOLUTIN_MAX | 1.95 | ||-| | RESOLUTIN_MIN | 32.79 | ||-| | DATA_COMPL | 99.86% | ||-| | PSEUDO_TRANS | not detected | ||-| | ALPHA_TWIN | not detected | ||-| ? The best Q after all model calculations is 0.7060 SOLUTION SUMMARY #---# # A structure is suggested by BALBES # # Its probability to be a solution is 99.0% # #---# The solution model index is as1m1 |---| | ITS PDB FILE | results/refmac_final_result.pdb | |---| | ITS MTZ FILE | results/refmac_final_result.mtz | |---| | R_ini/R_fin | 0.5130/0.3820 | Rfree_ini/Rfree_fin | 0.4920/0.4070 | Results from local BALBES: BALBES Version is 1.0.0.Jun_1_2009 JOB FOR STARTS AT Sun Jun 28 22:51:50 2009 # | Please cite the following paper in any publication | | arising from use of BALBES: | | F.Long, A.Vagin, P.Young and G.N.Murshudov | | "BALBES: a Molecular Replacement Pipeline" | | Acta Cryst. D64 125-132(2008) | # You have provide 2 sequences in the input sequence file number of amino acids in the sequence 1 is 65 number of amino acids in the sequence 2 is 146 #---# # Structure Factor Data Analysis # #---# Information about the structure to be solved |--| | SPACE GROUP | I 41 2 2 | ||-| | CELL LENGTH | 130.050 130.050 70.190 | ||-| | CELL ANGLE | 90.000 90.000 90.000 | ||-| | RESOLUTIN_MAX | 1.95 | ||-| | RESOLUTIN_MIN | 32.79 | ||-| | DATA_COMPL | 99.86% | ||-| | PSEUDO_TRANS | not detected | ||-| | ALPHA_TWIN | not detected | ||
Re: [ccp4bb] Balbes 1.0.0. different results between WWW Server and Local instalation
Dear Victor, Thank you very much for telling us that. Although it is a little strange to me because the same set of the fortran programs and python scripts are used in both the webserver version and standalone version of BALBES (except one fortran program I will mention later), we try to find why that happened. >From what shows in your email, we see the final solution given by the web server version of BALBES is from the template search model, as1m1, which is an assembly model (built from two input sequences), while the result from the standalone version is from the model, sq1st2m1, which is a single molecule model (built from one input sequence). The former should be better than the latter. But we want to see why the standalone version failed to find that assembly search model. You run our web server on Jun 9 and now you run the standalone BALBES released on June 25. During that time TWO things changed in BALBES (in both web server and standalone versions). (1) We updated the database within BALBES. Your email shows (a)the web server running is via BALBES of Version 1.0.0.May_16_2009, which is version 1.0.0 with the database updated on May 16 2009 (b) your local running is via BALBES of Version 1.0.0.Jun_1_2009, which is version 1.0.0 with the database updated On June 1 2009. (2) We changed a fortran program 'get_structure_DB', which is responsible for building template search models, on Jun 14-15 2009 Both two changes should enhance the performance of BALBES instead of deteriorating it. But you results show the opposite. Could it be possible for you to provide us the files recording the running processes, i,e, the files called "Process_information.txt" under subdirectory ../results/ for both runs so that we can see what sets of template search models were given in both versions of BALBES and then what went wrong in the standalone version you downloaded ? You can send the files to any of one of us (Garib, Alexei, or me), Thank you very much. Fei 2009/6/29 Victor Alves : > Hello > > > > After a helpful hand (on a Sunday !!) from Dr. Fei Long I installed BALBES > 1.0.0 in my Ubuntu Linux machine and in order to try it, I ran the same job > I had previously ran in the WWW Server. > > Surprisingly the results were worse in my local BALBES. > > > > Results from the WWW: > > > > BALBES Version is 1.0.0.May_16_2009 > > JOB FOR STARTS AT Tue Jun 9 12:17:31 2009 > > > > # > > | Please cite the following paper in any publication | > > | arising from use of BALBES: | > > | F.Long, A.Vagin, P.Young and G.N.Murshudov | > > | "BALBES: a Molecular Replacement Pipeline" | > > | Acta Cryst. D64 125-132(2008) | > > # > > You have provide 2 sequences in the input sequence file > > number of amino acids in the sequence 1 is 65 > > number of amino acids in the sequence 2 is 146 > > > > #---# > > # Structure Factor Data Analysis # > > #---# > > Information about the structure to be solved > > |--| > > | SPACE GROUP | I 41 2 2 | > > ||-| > > | CELL LENGTH | 130.050 130.050 70.190 | > > ||-| > > | CELL ANGLE | 90.000 90.000 90.000 | > > ||-| > > | RESOLUTIN_MAX | 1.95 | > > ||-| > > | RESOLUTIN_MIN | 32.79 | > > ||-| > > | DATA_COMPL | 99.86% | > > ||-| > > | PSEUDO_TRANS | not detected | > > ||-| > > | ALPHA_TWIN | not detected | > > ||-| > > > > ? > > > > The best Q after all model calculations is 0.7060 > > > > SOLUTION SUMMARY > > #---# > > # A structure is suggested by > BALBES # > > # Its probability to be a solution is > 99.0% # > > #---# > > The solution model index is as1m1 > > |---| > > | ITS PDB FILE | > results/refmac_final_result.pdb | > > |---| > > | ITS MTZ FILE | > results/refmac_final_result.mtz
[ccp4bb] How to express a >95KD FAD protein
Dear all, I know that there are a lot of experts having experience in expressing a big protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding protein (Human protein). I tried very hard but have a bad luck over the past 1.5 years. I list what I did briefly so far. I am looking forward to getting suggestions from you. Thanks a ton in advance. E.Coli 1. Express full-length in pET28a (N-6xhis) No expression 2. Express full-length in pET21b (no-tag) Inclusion body 3. N and C terminus truncated construct Inclusion body (tagged and non-tagged) 4. Co-expression with GroEL/S Inclusion body 5. Co-expression with with cofactors Inclusion body 6. In pMAL vector Not sure Yeast: (No tag) 7. In pPICZb vector in Pichia No expression 8. In GHB30 vector in Saccharomyces cerevisiae No expression I also tried to use different ways to break cells, use detergent, express at low temperature (down to 15 degree), modify IPTG concentration and so on. I do not know what else I can try. Please give me suggestions. Thank you very much. Best wishes Wei Yong
Re: [ccp4bb] How to express a >95KD FAD protein
Hi Wei Yong, Sounds like a tricky situation. A couple of things come to mind: 1) Have you tried expression from a synthetic gene? Sometimes the mRNA is unstable and improving mRNA stability through optimization (synthetic gene) helps. 2) Are you able to look at either human isoforms or orthologs from other species? 3) Have you tried expressing with an N-terminal SUMO tag? I have seen expression with an N-terminal SUMO tag in some cases where expression was previously undetected. 4) Have you tried cell-free expression? 5) You might also consider insect cell expression or expression from mammalian cells 6) Do you get enough protein in some cases that you might try to attempt some refolding from inclusion bodies? Just a couple of avenues to think along. Good luck! Raji On Jun 29, 2009, at 5:54 PM, Yong, Wei wrote: Dear all, I know that there are a lot of experts having experience in expressing a big protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding protein (Human protein). I tried very hard but have a bad luck over the past 1.5 years. I list what I did briefly so far. I am looking forward to getting suggestions from you. Thanks a ton in advance. E.Coli 1. Express full-length in pET28a (N-6xhis) No expression 2. Express full-length in pET21b (no- tag)Inclusion body 3. N and C terminus truncated construct Inclusion body (tagged and non-tagged) 4. Co-expression with GroEL/ S Inclusion body 5. Co-expression with with cofactors Inclusion body 6. In pMAL vector Not sure Yeast: (No tag) 7. In pPICZb vector in Pichia No expression 8. In GHB30 vector in Saccharomyces cerevisiae No expression I also tried to use different ways to break cells, use detergent, express at low temperature (down to 15 degree), modify IPTG concentration and so on. I do not know what else I can try. Please give me suggestions. Thank you very much. Best wishes Wei Yong
Re: [ccp4bb] How to express a >95KD FAD protein
a couple of quick things. This sounds like more of a protein-folding issue. The lack of expression is probably caused by the cells clearing of aggregated protein. You want to have conditions where the protein can properly fold. 1) have you considered periplasmic expression by adding a periplasmic targetting sequence to the N-terminal? It is generally thought, perhaps anecdotally, that the periplasm is better equipped to handle proteins likely to misfold (and presumably cause inclusion bodies). 2) Perhaps a heat shock of your cells prior to induction. Incubate the cells briefly at 42 degrees. This will upregulate the cell's expression of chaperones and other heat shock proteins which may help avoid the inclusion body. 3) Are there any cysteines which might be forming non-specific disulfides? Perhaps this can be resolved by mutagenesis. 4) Last resort. have you tried a snap refold of the inclusion bodies? Dissolve the pellet in urea. Dialyse it away and see if any amount of the protein has refolded spontaneously. good luck Simon Yong, Wei wrote: Dear all, I know that there are a lot of experts having experience in expressing a big protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding protein (Human protein). I tried very hard but have a bad luck over the past 1.5 years. I list what I did briefly so far. I am looking forward to getting suggestions from you. Thanks a ton in advance. E.Coli 1. Express full-length in pET28a (N-6xhis) No expression 2. Express full-length in pET21b (no-tag) Inclusion body 3. N and C terminus truncated construct Inclusion body (tagged and non-tagged) 4. Co-expression with GroEL/S Inclusion body 5. Co-expression with with cofactors Inclusion body 6. In pMAL vector Not sure Yeast: (No tag) 7. In pPICZb vector in Pichia No expression 8. In GHB30 vector in Saccharomyces cerevisiae No expression I also tried to use different ways to break cells, use detergent, express at low temperature (down to 15 degree), modify IPTG concentration and so on. I do not know what else I can try. Please give me suggestions. Thank you very much. Best wishes Wei Yong
Re: [ccp4bb] How to express a >95KD FAD protein
In addition to other excellent suggestions voiced here (insect cells, refolding, etc.) you can attempt expression in constituitive mode - using a mild always-on promoter. Provided that your protein is not too toxic to the cells this may result in gradual accumulation of folded material rather than sudden accumulation of misfolded junk. Artem "Nothing is built on stone; all is built on sand, but we must build as if the sand were stone" Jorge Luis Borges -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Yong, Wei Sent: Monday, June 29, 2009 4:55 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to express a >95KD FAD protein Dear all, I know that there are a lot of experts having experience in expressing a big protein in E.coli or yeast. My project is about a 95kd covalently-FAD-binding protein (Human protein). I tried very hard but have a bad luck over the past 1.5 years. I list what I did briefly so far. I am looking forward to getting suggestions from you. Thanks a ton in advance. E.Coli 1. Express full-length in pET28a (N-6xhis) No expression 2. Express full-length in pET21b (no-tag) Inclusion body 3. N and C terminus truncated construct Inclusion body (tagged and non-tagged) 4. Co-expression with GroEL/S Inclusion body 5. Co-expression with with cofactors Inclusion body 6. In pMAL vector Not sure Yeast: (No tag) 7. In pPICZb vector in Pichia No expression 8. In GHB30 vector in Saccharomyces cerevisiae No expression I also tried to use different ways to break cells, use detergent, express at low temperature (down to 15 degree), modify IPTG concentration and so on. I do not know what else I can try. Please give me suggestions. Thank you very much. Best wishes Wei Yong
Re: [ccp4bb] Principal Scientist: Protein Crystallography position available - Australian Synchrotron
Principal Scientist - Protein Crystallography Australian Synchrotron The Australian Synchrotron, a major new national facility, provides researchers from Australia, NZ and overseas with a powerful new tool for scientific and industrial research. The facility has applications in fields as diverse as structural molecular biology, environmental science, materials science and medical diagnosis and therapy. The synchrotron operates a number of beamlines and laboratories. Background The Protein Crystallography beamlines (bending magnet) and undulator sources generate world class data, allowing users to map atom by atom, highly complex biomolecules. A recent highlight is the commissioning of a high-capacity robot, which can load and upload sensitive protein crystal samples in a fraction of the time required by human hands. The Position We are seeking a Principal Scientist to lead the development and operation of the Protein Crystallography beamline at the Australian Synchrotron (AS), ensuring it meets its' scientific objectives. As a recognised expert in their scientific field, you will drive all aspects of a research program, generating significant scientific and industrial outcomes. You will be required to display leadership abilities in managing and mentoring staff. You will be expected to provide expert advice, advocacy and direction to user groups and their communities. Occasional travel will be required. To be successful you will have: * Effective management skills and proven ability to liaise with people at all levels * Effective advocate for the vision and values of the AS * Scientific leadership and the ability to manage and mentor other scientists * Strong organisational and time management skills * Scientific ownership of relevant techniques, equipment and control systems * Expertise in relevant data processing and analysis software * Ability to manage the generation of scientific and industrial outcomes * Proven ability to drive and grow new science in collaboration with external groups * A PhD in a relevant field and several years post doctoral employment with extensive experience in a related field. To apply for this position please send your confidential resume and cover letter to h...@synchrotron.org.au or visit our website www.synchrotron.org.au for a full position description. Applications Close: Friday 3 July 2009 This message and any attachments may contain proprietary or confidential information. If you are not the intended recipient or you received the message in error, you must not use, copy or distribute the message. Please notify the sender immediately and destroy the original message. Thank you.
[ccp4bb] can I try crystallization in high salt?
Dear All, I have a peri domain protein that is stable in high salt concentration(500 mM), if I dialysis to a lower salt buffer and then concentrate, it'll preticipate out. If I need to crystallize it, can I use the high salt buffer? Is there any optimization kits that could help to increase the solubility? Thanks.
Re: [ccp4bb] can I try crystallization in high salt?
You can try including some salt to the reservoir after mixing protein and well solution. Joe rui wrote: Dear All, I have a peri domain protein that is stable in high salt concentration(500 mM), if I dialysis to a lower salt buffer and then concentrate, it'll preticipate out. If I need to crystallize it, can I use the high salt buffer? Is there any optimization kits that could help to increase the solubility? Thanks.
Re: [ccp4bb] can I try crystallization in high salt?
You may also apply salting-in: if the reservoir concentration is lower than that of the protein buffer, water may evaporate from the reservoir into the drop, lowering the protein's solubility and thus maybe grow crystals. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Mon, 29 Jun 2009, rui wrote: Dear All, I have a peri domain protein that is stable in high salt concentration(500 mM), if I dialysis to a lower salt buffer and then concentrate, it'll preticipate out. If I need to crystallize it, can I use the high salt buffer? Is there any optimization kits that could help to increase the solubility? Thanks.
[ccp4bb] How to get anomalous signal for Ca
Dear All: When I solved the crystal structure of protein, I found a metal binding in it. But however, I can not decide which type of atom it is. I tried ICP-AES and the result suggested the protein contained Ca. I tried to get anomalous signal at the wavelength of 2.07A. And I collected the 360 degree with the spacegroup P6 and the average redundancy is 19.6. But still I can not see the anomalous signal for that metal even I can see clearly the anomalous signal for sulfur. Does anybody have suggestions for me? Thanks in advance. 2009-06-30 xianchidong
