[ccp4bb] Ample fails
Dear all, I'm trying to use Ample from CCP4 (6.4) to solve a structure of 2x197 residues from 2.0 A data. After the generation of initial (120) models by Rosetta (3.3) Ample stops with the error-message below. Could anyone provide me information about the origin of this error and possibly how to solve the problem? Thanks in advance for your replies! Best wishes, Florian *** * Information from CCP4Interface script *** The program run with command: /home/flo/crystallography/ccp4-6.4.0/bin/ccp4-python -u /home/flo/crystallography/ccp4-6.4.0/bin/ample -mtz /home/flo/crystallography/Virchow/ESRF_20140707/CM133A/ccp4/pos5wedge3scaled.mtz -fasta /home/flo/Desktop/arch.seq -name MVD0 -run_dir /home/flo/crystallography/Virchow/ESRF_20140707/pos7au/ccp4 -nproc 3 -F F_XDSdataset -SIGF SIGF_XDSdataset -FREE FreeR_flag -models_dir /home/flo/crystallography/Virchow/ESRF_20140707/pos7au/ccp4/ROSETTA_MR_0/models/models_1 -early_terminate True -use_shelxe True -use_arpwarp False has failed with error message Traceback (most recent call last): File /home/flo/crystallography/ccp4-6.4.0/bin/ample, line 757, in ensemble.create_ensembles( amopt.d ) File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/ensemble.py, line 85, in create_ensembles ensembles = ensemble_models( amoptd['spicker_results'][ cluster-1 ].pdb_list, amoptd, ensemble_id=cluster ) File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/ensemble.py, line 58, in ensemble_models percent=amoptd['percent'] ) File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/ample_ensemble.py, line 168, in generate_ensembles self.make_truncated_ensembles() File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/ample_ensemble.py, line 312, in make_truncated_ensembles clusterer.generate_distance_matrix( self.truncated_models[tcount] ) File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/cluster_with_MAX.py, line 55, in generate_distance_matrix retcode = ample_util.run_command( cmd, logfile=log_name ) File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/ample_util.py, line 274, in run_command p = subprocess.Popen( cmd, stdin=stdin, stdout=logf, stderr=subprocess.STDOUT, cwd=directory, **kwargs ) File /home/flo/crystallography/ccp4-6.4.0/lib/python2.7/subprocess.py, line 711, in __init__ errread, errwrite) File /home/flo/crystallography/ccp4-6.4.0/lib/python2.7/subprocess.py, line 1308, in _execute_child raise child_exception OSError: [Errno 8] Exec format error *** -- Dr. Florian Sauer Rudolf Virchow Zentrum für Experimentelle Biomedizin Josef-Schneider-Str. 2, Haus D15 97080 Würzburg
Re: [ccp4bb] Ample fails
Dear Florian, Sorry to hear that Ample isn't working for you. I haven't seen this error before, but my initial guess would be that you're trying to run Ample on Windows? If so then unfortunately the current release of Ample isn't supported on Windows. Most of the work to port Ample to Windows has been carried out, and it will be made available in a forthcoming release of CCP4, but until then the only solution is to run Ample on Linux or Mac OSX. If you're not running on Windows, please could you send me (off list) the file debug.log, which should have been created in the directory: /home/flo/crystallography/Virchow/ESRF_20140707/pos7au/ccp4/ROSETTA_MR_0 (the directory may be named ROSETTA_MR_0, ROSETTA_MR_1, ROSETTA_MR_2... etc. depending on how many runs you have attempted.) Just for information, at 197 residues your protein is on the large side (although well within the bounds of what Ample is capable of solving), but 120 models is far fewer then we would normally recommend. The usual recommendation is to make at least 1000 models. Best wishes, Jens From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Florian Sauer [florian.sau...@virchow.uni-wuerzburg.de] Sent: 11 July 2014 08:37 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Ample fails Dear all, I'm trying to use Ample from CCP4 (6.4) to solve a structure of 2x197 residues from 2.0 A data. After the generation of initial (120) models by Rosetta (3.3) Ample stops with the error-message below. Could anyone provide me information about the origin of this error and possibly how to solve the problem? Thanks in advance for your replies! Best wishes, Florian *** * Information from CCP4Interface script *** The program run with command: /home/flo/crystallography/ccp4-6.4.0/bin/ccp4-python -u /home/flo/crystallography/ccp4-6.4.0/bin/ample -mtz /home/flo/crystallography/Virchow/ESRF_20140707/CM133A/ccp4/pos5wedge3scaled.mtz -fasta /home/flo/Desktop/arch.seq -name MVD0 -run_dir /home/flo/crystallography/Virchow/ESRF_20140707/pos7au/ccp4 -nproc 3 -F F_XDSdataset -SIGF SIGF_XDSdataset -FREE FreeR_flag -models_dir /home/flo/crystallography/Virchow/ESRF_20140707/pos7au/ccp4/ROSETTA_MR_0/models/models_1 -early_terminate True -use_shelxe True -use_arpwarp False has failed with error message Traceback (most recent call last): File /home/flo/crystallography/ccp4-6.4.0/bin/ample, line 757, in ensemble.create_ensembles( amopt.d ) File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/ensemble.py, line 85, in create_ensembles ensembles = ensemble_models( amoptd['spicker_results'][ cluster-1 ].pdb_list, amoptd, ensemble_id=cluster ) File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/ensemble.py, line 58, in ensemble_models percent=amoptd['percent'] ) File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/ample_ensemble.py, line 168, in generate_ensembles self.make_truncated_ensembles() File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/ample_ensemble.py, line 312, in make_truncated_ensembles clusterer.generate_distance_matrix( self.truncated_models[tcount] ) File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/cluster_with_MAX.py, line 55, in generate_distance_matrix retcode = ample_util.run_command( cmd, logfile=log_name ) File /home/flo/crystallography/ccp4-6.4.0/share/ample/python/ample_util.py, line 274, in run_command p = subprocess.Popen( cmd, stdin=stdin, stdout=logf, stderr=subprocess.STDOUT, cwd=directory, **kwargs ) File /home/flo/crystallography/ccp4-6.4.0/lib/python2.7/subprocess.py, line 711, in __init__ errread, errwrite) File /home/flo/crystallography/ccp4-6.4.0/lib/python2.7/subprocess.py, line 1308, in _execute_child raise child_exception OSError: [Errno 8] Exec format error *** -- Dr. Florian Sauer Rudolf Virchow Zentrum für Experimentelle Biomedizin Josef-Schneider-Str. 2, Haus D15 97080 Würzburg
[ccp4bb] PhD-position in enzyme crystallography
Dear all, a PhD-position is available immediately in the laboratory of Prof. Dietmar A. Plattner at the University of Freiburg, Germany. We are seeking a highly motivated student with previous experience in enzyme handling (purification, characterization) and, ideally, protein X-ray crystallography. The successful candidate will find a fully equipped research environment with regular access to European synchrotrons (mainly ESRF). Our group is focused on the structure-functional investigation of heme or copper-containing oxidoreductases which are of high interest in biotechnology and organic synthesis (e.g. cell-free regio- and stereoselective hydroxylation of unfunctionalized hydrocarbons with self-sufficient peroxygenases). For more information on the position and the research group please visit https://portal.uni-freiburg.de/plattner/front-page?set_language=en https://portal.uni-freiburg.de/plattner/resolveuid/b1328bcc0bec660bedf3f83672a9b152 Freiburg is a beautiful town in southwestern Germany, located in the Dreiländereck near the borders of France and Switzerland and - of course - at the foot of the Black Forest. Inquiries and applications, including CV and the contact data of two referees, should be sent directly to Prof. Plattner (dplattatchemie.uni-freiburg.de). Eric Strittmatter Albert-Ludwigs-Universität Freiburg Institute of Organic Chemistry Biochemistry Albertstraße 21, D-79104 Freiburg Germany Mail: eric.strittmat...@ocbc.uni-freiburg.de
Re: [ccp4bb] CCP4-JHBuild: how to build both static and shared libs?
On Tue, Jul 08, 2014 at 07:50:42AM +0100, Alastair Fyfe wrote:
Is there a JHBuild setting for requesting both static and shared libs? The
default seems to only build *.so(s) and setting _build_static=True in
conf.py only yields *.a(s).
For libraries that use autotools for building (that includes mmdb, libccp4,
clipper) you may change --disable-static to --enable-static in conf.py.
Or even better, in user's configuration file (cj.rc) add a line:
autogenargs = autogenargs.replace('--disable-static','--enable-static')
Libraries with different build systems usually can build only one version
(static or shared) at time.
(I haven't noticed this post at first, it's nested in a thread
with different subject)
Marcin
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[ccp4bb] Disrupt beta sheet formation
Hi, I have a protein that is prone to aggregation. I think the problem may be caused by a stretch of six-residues at the N-terminus that is predicted to form a beta-strand. I think this beta-strand is open ended and may form beta- sheets (similar to amyloid fibers) leading to the aggregation I observe. Are there any chemicals that would disrupt short beta-sheet formation? Thanks. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 www.khayatlab.org
[ccp4bb] Structural Biology post-doc position, Portsmouth UK
Dear ccp4bb, I’ve just got my first grant from the BBSRC and need to recruit a post-doc. The project will involve protein expression using HEK293 followed by crystallisation/data collection and conducting binding assays (Biacore, ITC, AUC etc.). The details and how to apply can be found on the following link (job reference 10012179 under external vacancies (EU only)): https://port.engageats.co.uk Closing date is 27th July with interviews aimed for 11th August. I am happy to be contacted informally for further information. Thanks! Simon -- Dr. Simon Kolstoe Institute of Biomedical and Biomolecular Science, School of Biological Sciences, University of Portsmouth, King Henry Building, Portsmouth. PO1 2DY, UK Tel: 023 9284 2058
[ccp4bb] position lab manager
Dear Colleagues Please forward this to suited candidates, candidates must apply through the link, sorry for the Norwegian (Scandinavian'ish) requirement, the candidate needs to be able to navigate around in the purchase portal only offered in Norwegian. http://uio.easycruit.com/vacancy/1225339/70932?iso=no best regards Preben Centre for Molecular Medicine Norway (NCMM) Laboratory Manager/Principal Engineer The one-year position, with possibilities for extension to 3 more years, is available in the laboratory of Dr. J. Preben Morth from 15th of August. *Challenges:* The group is interested on the systems involved with pH homeostasis. The main techniques used are cloning, expression purification and crystallization. The position will also include work with mammalian cells and various imaging techniques. The lab manager will play an active role in laboratory research projects and maintaining group resources. *Laboratory tasks include:* * Cloning including primer design PCR reaction, subcloning in both bacterial and cell lines * Protein purification of both soluble and membrane proteins * Cell line transfection * Crystallisation, computer support. * Maintaining the laboratory self made resources such as competent cells. *Qualifications:* * Formal requirement with regard to education is a masters degree in biochemistry /molecular biology as minimum however a PhD would be preferred. Experience from similar work can replace this formal requirement. * Experience with protein chemistry, protein crystallography and biophysical methods is preferred * Work knowledge of Word and Excel * The candidate must have basic knowledge in Norwegian, both written and oral * The group has a number of international collaborations and the position requires applicants to be highly organized and fluent in English * Open and friendly nature, not afraid to learn something new -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Tel: +47 2284 0794
[ccp4bb] Post doctoral position at the protein crystallization core facility / Institute of Biotechnology, University of Helsinki
A postdoctoral position is available in the Biocenter Finland Crystallization Core Facility, in the research group of Dr. Tommi Kajander at the institute of Biotechnology at University of Helsinki, Finland. The position is funded for two years starting from September 2014. The position will include crystallization facility responsibilities, and structural biology-related research in the Kajander lab, and possibly partly on collaborators/clients projects, as agreed. The topics of research project(s) include structural aspects of neuronal protein-protein receptor complexes, and will be discussed in detail with the selected candidates. Core facility tasks include scientific support, and help in maintenance of the services of the protein crystallization facility (http://www.biocenter.helsinki.fi/bi/xray/automation/index.html), including advising clients on structural biology projects, and data collection on synchrotron sources (e.g. Diamond and ESRF). The successful candidate must be experienced in macromolecular crystallography (crystallization, synchrotrons, structure solution, refinement etc.), and have very good teamwork, communication, and social skills, and capability to guide students. Ability to work in organized fashion and fluent English language skills are also required. Knowledge of automation, high throughput techniques and/or molecular biology and protein purification and production experience are desirable/preferable. The institute has excellent infrastructure for science and the unit has two imaging systems (Rigaku Minstrel UV and Explora Nova (+4 C), mosquito LCP, and a Hamilton star dispensing robot. The unit also has a thermofluor instrument and access to a MALLS system for upstream sample analysis. We also have a Rigaku rotating anode source w/RAXIS IV+ detector. We have regular (monthly) access to synchrotron sources. Institute of Biotechnology also has state-of-the-art cryo-EM and NMR facilities, and e.g. Biacore and thermophoresis instruments are located in the next building. Applicants should send: 1) A cover letter briefly describing previous achievements, and motivation for the position; 2) Curriculum vitae with a list of publications 3) Contact information of two referees to email address: tommi.kajander(at)helsinki.fi. The deadline for applications is August 31, 2014, or until suitable candidate is found. Contact for more information: e-mail: tommi.kajander(at)helsinki.fi (phone: +358-50-4480991) http://www.biocenter.helsinki.fi/bi/kajander/ Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki, Finland
Re: [ccp4bb] Proper detwinning?
Nat and Misha, Thank you for the suggestions. Xtriage does indeed detect twinning in P1, reporting similar values for |L|, L^2, and twin fraction as in P212121. The unit cell dimensions for the 2.0-A structure (P1) are: 72.050 105.987 201.142 89.97 89.98 89.94 P 1 The unit cell dimensions for the 2.8-A structure (P212121) are: 75.456 115.154 202.022 90.00 90.00 90.00 P 21 21 21 I have been processing in HKL2000, which only recognizes one set of unit cell parameters for each Bravais lattice (does anyone know how to change this?). Specifically, for a primitive monoclinic unit cell it estimates: 104.53 71.82 200.99 89.86 91.80 91.16 This is the unit cell which refined to Rwork/Rfree ~ 27%/34%. Indexing in mosflm gives three options for primitive monoclinic: 105.6 71.7 200.9 90.0 90.1 90.0 71.7 105.6 201.0 90.0 89.9 90.0 71.7 200.9 105.6 90.0 90.3 90.0 Attempting to integrate in any of these space groups leads to a fatal error in subroutine MASKIT. I can also use the index multiple lattices feature to get a whole slew of potential space group; however, integrating reflections leads to the same fatal error. Finally, Zanuda tells me that P212121 is the best space group, according to R-factors. However, I do not believe P212121 is the correct assignment. Best, Chris On 7/10/14, Isupov, Michail m.isu...@exeter.ac.uk wrote: I would recommend to run ZANUDA in the default mode from ccp4i or on CCP4 web server. ZANUDA has resolved several similar cases for me. Misha From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chris Fage [cdf...@gmail.com] Sent: 10 July 2014 01:14 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Proper detwinning? Hi Everyone, Despite modelling completely into great electron density, Rwork/Rfree stalled at ~38%/44% during refinement of my 2.0-angstrom structure (P212121, 4 monomers per asymmetric unit). Xtriage suggested twinning, with |L| = 0.419, L^2 = 0.245, and twin fraction = 0.415-0.447. However, there are no twin laws in this space group. I reprocessed the dataset in P21 (8 monomers/AU), which did not alter Rwork/Rfree, and in P1 (16 monomers/AU), which dropped Rwork/Rfree to ~27%/32%. Xtriage reported the pseudo-merohedral twin laws below. P21: h, -k, -l P1: h, -k, -l; -h, k, -l; -h, -k, l Performing intensity-based twin refinement in Refmac5 dropped Rwork/Rfree to ~27%/34% (P21) and ~18%/22% (P1). Would it be appropriate to continue with twin refinement in space group P1? How do I know I'm taking the right approach? Interestingly, I solved the structure of the same protein in P212121 at 2.8 angstroms from a different crystal. Rwork/Rfree bottomed out at ~21%/26%. One unit cell dimension is 9 angstroms greater in the twinned dataset than in the untwinned. Thank you for any suggestions! Regards, Chris
Re: [ccp4bb] Proper detwinning?
Chris, To change the axis ordering for e.g. changing which cell edge is the P21 B axis use an hkl matrix command. Probably can do this via the Macros during scaling but I distrust this and just edit scl.in by hand and run it as scalepack scl.in For k,l,h reindexing use hkl matrix 0 1 0 0 0 1 1 0 0 For l, h, k reindexing use hkl matrix 0 0 1 1 0 0 0 1 0 Systematic absences would be an anecdotal indicator that it is/isn't P212121. That would show strong systematic absences for (h,0,0), (0,k,0), (0,0,l) reflections. (Or reflexions if one prefers). While not impossible I would think it statistically unlikely to observe such absences if the data was really P1, P2 or P21. Going back to the images to eyeball the actual reflections on the display can be pretty illuminating. I don't remember Scalepack giving much detail in postrefinement but paying attention to the positional chi^2 values during integration might give clues about how far from 90 those unit cell axes are wandering if you try integrating in different space groups. There's also a method (or was, last time I tried it) to change the way Scalepack postrefines unit cell dimensions (value per frame or value per crystal) which might also help. More hacking of scl.in might be required. However I'm usually pretty happy if my R-free drops 12% at 2.0 Angstrom resolution when going from P21 to P1. I would look for legitimate deviations between previously identical monomers in the map and probably consider using NCS to reduce the random deviation between monomers that actually are identical by symmetry. You may have assigned the crystallographic 21 down the wrong unit cell axis in that P21 test case. Phil Jeffrey Princeton On 7/11/14 7:33 PM, Chris Fage wrote: Nat and Misha, Thank you for the suggestions. Xtriage does indeed detect twinning in P1, reporting similar values for |L|, L^2, and twin fraction as in P212121. The unit cell dimensions for the 2.0-A structure (P1) are: 72.050 105.987 201.142 89.97 89.98 89.94 P 1 The unit cell dimensions for the 2.8-A structure (P212121) are: 75.456 115.154 202.022 90.00 90.00 90.00 P 21 21 21 I have been processing in HKL2000, which only recognizes one set of unit cell parameters for each Bravais lattice (does anyone know how to change this?). Specifically, for a primitive monoclinic unit cell it estimates: 104.53 71.82 200.99 89.86 91.80 91.16 This is the unit cell which refined to Rwork/Rfree ~ 27%/34%. Indexing in mosflm gives three options for primitive monoclinic: 105.6 71.7 200.9 90.0 90.1 90.0 71.7 105.6 201.0 90.0 89.9 90.0 71.7 200.9 105.6 90.0 90.3 90.0 Attempting to integrate in any of these space groups leads to a fatal error in subroutine MASKIT. I can also use the index multiple lattices feature to get a whole slew of potential space group; however, integrating reflections leads to the same fatal error. Finally, Zanuda tells me that P212121 is the best space group, according to R-factors. However, I do not believe P212121 is the correct assignment. Best, Chris On 7/10/14, Isupov, Michail m.isu...@exeter.ac.uk wrote: I would recommend to run ZANUDA in the default mode from ccp4i or on CCP4 web server. ZANUDA has resolved several similar cases for me. Misha From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chris Fage [cdf...@gmail.com] Sent: 10 July 2014 01:14 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Proper detwinning? Hi Everyone, Despite modelling completely into great electron density, Rwork/Rfree stalled at ~38%/44% during refinement of my 2.0-angstrom structure (P212121, 4 monomers per asymmetric unit). Xtriage suggested twinning, with |L| = 0.419, L^2 = 0.245, and twin fraction = 0.415-0.447. However, there are no twin laws in this space group. I reprocessed the dataset in P21 (8 monomers/AU), which did not alter Rwork/Rfree, and in P1 (16 monomers/AU), which dropped Rwork/Rfree to ~27%/32%. Xtriage reported the pseudo-merohedral twin laws below. P21: h, -k, -l P1: h, -k, -l; -h, k, -l; -h, -k, l Performing intensity-based twin refinement in Refmac5 dropped Rwork/Rfree to ~27%/34% (P21) and ~18%/22% (P1). Would it be appropriate to continue with twin refinement in space group P1? How do I know I'm taking the right approach? Interestingly, I solved the structure of the same protein in P212121 at 2.8 angstroms from a different crystal. Rwork/Rfree bottomed out at ~21%/26%. One unit cell dimension is 9 angstroms greater in the twinned dataset than in the untwinned. Thank you for any suggestions! Regards, Chris
