Re: [ccp4bb] Unidentified density
Dear Sudhir, A blob surrounded by three Asp residues looks like a positively charged ion. What else (buffer, additives) is in your crystallization drop? I would also try to fit cysteine/serine. At 2 Å you will quickly see whether or not this is correct. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sudhir Kumar Sent: Friday, February 10, 2012 7:06 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Unidentified density Dear all, I have a 2 A structure of an enzyme which show 12 molecules per asymmetric unit. While placing waters i found out some blobs where i could not model any ligand. It is surrounded by 3 Asp molecules. I have attached screenshot of the blob at 1.5 sigma cutoff 2fo-fc density. Crystallization condition had following precipitants: PEG 1000, Peg 3350, MPD, Ethylene glycol,and protein contained Glycerol. My protein is known to bind Cysteine/Serine, though i haven't added any in crystallization buffer. any suggestion what it might be are welcome. thanks in advance -- best regards Sudhir Kumar Research Scholar C/O Dr. S. Gourinath Structural Biology Laboratory SLS, JNU, New Delhi-110067
Re: [ccp4bb] unidentified density
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hallo , first build a poly-ALA stretch. In coot or O this is conveniently achieved using baton-build mode. This should improve the phases. Then look at the side chains.Turbo-frodo has got somehting like a slider that shows the sequence on screen and which helps you identify bulky side chains. The pattern of a few bulky and non-bulkyside chains might already be sufficient to dock the sequence into the density. Also take the environment into account and think about what interactions between side chains of peptide and protein are plausible. You can also take the results from secondary structure prediction into account (e.g. http://toolkit.tuebingen.mpg.de/hhpred) - the density you show looks like an alpha-helix, and according to hhpred the stretch of sequence below contains two helices. Cheers, Tim On 02/10/2012 08:35 AM, intekhab alam wrote: Hi all I have a 3A dataset for a protein-protein complex. I have successfully build the first protein and refined it to R/Rfree 24/28. I can see some density for my second protein but the density is a bit noisy. I have attached the coot image of the density. I want to model the aminoacid having sequence as given peptide: MGKKGKNKKGRGRPGVFRTRGLTDEEYDEFKKRRESRGGKYSIDDYLADREREEELLERDEEEAIFGDGFGLE 1.Based on map features which segemnt should i start with. 2. Is there anyway that i can build the best fit segment of my second protein. I tried autobuild but it failed to build any peptide for my second protein. Your help is highly appreciated. regards - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPNOmBUxlJ7aRr7hoRArQMAJkBfnu04rQX2nBwgGN13qiwg68JzgCg0aL9 6C2LEaLZA37XVmQ+siX8yrg= =+AF/ -END PGP SIGNATURE-
[ccp4bb] Application deadline for MX-beamtime at HZB-BESSY March , 1. 2012 !
Next MX-proposal application deadline: March 1, 2012 is approaching See also: http://www.bessy.de/boat/http://www.bessy.de/boat/www/ We kindly invite new MX-proposals for beamtime applications for the next beamtime period. In order to apply for beamtime, please register at the HZB on-line access tool BOAT (http://www.bessy.de/boat/http://www.bessy.de/boat/www/) and submit a new beamtime application proposal. HZB provides beamtime at the MX-beamlines 14.1, 14.2 and 14.3. The requested beamtime is granted based on the reviewed proposal and reports from previous research activities. Reported results from previous beamtimes stated in the Experimental Reports form will affect the chances for future beamtimes significantly. Please make sure to include them if available. Experimental setup: BL14.1 setup: - Photon flux: 1.4x10¹¹ Phot/sx100mAx0.05%BW at sample position (0.1-20 sec exposure time per frame) - User defined beam shaping from 30µm-100µm diameter possible - MX-225 X-ray detector, 92mm-720mm max. distance from the sample - Microdiffractometer (MD2) with Mini-kappa goniometer MK3 (STAC controlled) - Automatic sample changer (CATS), 90 sample storage capacity (SPINE-Pin EMBL sample magazine compatibility) - 96-well crystallization plate scanning operational upon request - fourty-core XEON-CPU server, with fibre channel SAN up-link data processing environment - EDNA installed and available - Common MX-software installed including XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, etc. - Automated data processing with iXDS and XDSAPP available - Remotely controlled cryo-shutter for crystal annealing - Bruker AXS X-Flash XRF detector We are offering the hard- and software environment for carrying out: - UVRIP experiments at BL14.1. For further information, please visit: http://www.helmholtz-berlin.de/forschung/funkma/soft-matter/forschung/bessy-mx/ancillary-facilities/uvrip_en.html - In situ crystal screening experiments, please visit: http://www.helmholtz-berlin.de/forschung/funkma/soft-matter/forschung/bessy-mx/ancillary-facilities/insitu-screening_en.html BL14.2 setup: - Photon flux: 1.9x10¹¹ Phot/sx100mAx0.05%BW at sample position (3-20 sec exposure time per frame) - MX-225 X-ray detector, 45mm-380mm distance from the sample, 30 deg 2-Theta possible - mardtb goniometer installed - fourty-core XEON-CPU server, with fibre channel SAN up-link data processing environment - EDNA installed and available - Common MX-software installed includingXDS, IMOSFLM, CCP4, Phenix, SHELXC-E, etc. - Automated data processing with iXDS and XDSAPP available - Remotely controlled cryo-shutter for crystal annealing - Bruker AXS X-Flash XRF detector - Pressure chamber for noble gas derivatization (Xe, Kr available upon request) - Ultra high performance stereo microscope Leica M205A, 20-255x zoom, 8 Mpixel CCD-camera BL14.3 setup: - Photon flux: 4x10exp10 Phot/sx100mAx0.05%BW at sample position (3-20 sec exposure time per frame) - SX-165 X-ray detector, 45mm-380mm distance from the sample, 30 deg 2-Theta possible - mardtb goniometer installed - fourty-core XEON-CPU server, with fibre channel SAN up-link data processing environment - EDNA installed and available - Common MX software installed including HKL2000, XDS, IMOSFLM, CCP4, Phenix, SHELXC-E, etc. - Automated data processing with ixds and xdsi available - Remotely controlled cryo-shutter for crystal annealing - HC-1c dehydration device installed, HC1-beamtime upon request - Pressure chamber for noble gas derivatization (Xe, Kr available upon request) - Ultra high performance stereo microscope Leica M205A, 20-255x zoom, 8 Mpixel CCD-camera S1-biolab facilities: - Protein production and purification - Nanoliter 96 well crystallization plate formulation and storage at 5 °C and 20 °C - Biophysical characterization with real time PCR (thermofluor assay) Please visit our web page www.helmholtz-berlin.de/bessy-mxhttp://www.helmholtz-berlin.de/bessy-mx to gain updated information about our experimental setup and other requirements. Uwe Mueller, Manfred Weiss and the HZB BESSY-MX group Dr. Uwe Mueller Soft Matter and Functional Materials Macromolecular Crystallography (BESSY-MX) | Group leader Elektronenspeicherring BESSY II Albert-Einstein-Str. 15, D-12489 Berlin, Germany Fon: +49 30 8062 14974 Fax: +49 30 8062 14975 url: www.helmholtz-berlin.de/bessy-mxhttp://www.helmholtz-berlin.de/bessy-mx email:u...@helmholtz-berlin.demailto:u...@helmholtz-berlin.de Helmholtz-Zentrum Berlin für Materialien und Energie GmbH Mitglied der Hermann von Helmholtz-Gemeinschaft Deutscher Forschungszentren e.V. Aufsichtsrat: Vorsitzender Prof. Dr. Dr. h.c. mult. Joachim Treusch, stv. Vorsitzende Dr. Beatrix Vierkorn-Rudolph Geschäftsführerin: Prof. Dr. Anke Rita Kaysser-Pyzalla Sitz Berlin, AG Charlottenburg, 89 HRB 5583 Postadresse: Hahn-Meitner-Platz 1 D-14109 Berlin http://www.helmholtz-berlin.de
[ccp4bb] job opportunity in molecular biophysics / structural biology
Dear colleagues, I would like to point your attention to the following job opportunity - professorship/director (W3) - which is jointly offered by the Forschungszentrum Juelich and Heinrich Heine University Duesseldorf, Germany: http://www.nature.com/naturejobs/science/jobs/243931 The focus of his/her work will be on the application and development of modern cryo-electron microscopy in biology and biomedicine. Deadline for applications will be March 30, 2012. Best regards Oliver PD Dr. Oliver H. Weiergräber Institute of Complex Systems ICS-6: Structural Biochemistry Tel.: +49 2461 61-2028 Fax: +49 2461 61-1448 --- --- Forschungszentrum Juelich GmbH 52425 Juelich Sitz der Gesellschaft: Juelich Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498 Vorsitzender des Aufsichtsrats: MinDir Dr. Karl Eugen Huthmacher Geschaeftsfuehrung: Prof. Dr. Achim Bachem (Vorsitzender), Karsten Beneke (stellv. Vorsitzender), Prof. Dr.-Ing. Harald Bolt, Prof. Dr. Sebastian M. Schmidt --- --- Kennen Sie schon unsere app? http://www.fz-juelich.de/app
Re: [ccp4bb] unidentified density
On 02/10/2012 07:35 AM, intekhab alam wrote: Hi all I have a 3A dataset for a protein-protein complex. I have successfully build the first protein and refined it to R/Rfree 24/28. I can see some density for my second protein but the density is a bit noisy. I have attached the coot image of the density. I want to model the aminoacid having sequence as given peptide: MGKKGKNKKGRGRPGVFRTRGLTDEEYDEFKKRRESRGGKYSIDDYLADREREEELLERDEEEAIFGDGFGLE 1.Based on map features which segemnt should i start with. 2. Is there anyway that i can build the best fit segment of my second protein. I tried autobuild but it failed to build any peptide for my second protein. Your help is highly appreciated. regards Try buccaneer - it should work very easily with that density.. Eleanor
[ccp4bb] PhD studentship opportunity in Newcastle University
BBSRC MRes/PhD Studentship: Structural studies of biofilm-associated proteins from human pathogens 4 Years (MRes Medical and Molecular Biosciences followed by a three-year PhD) or 3 Years PhD, depending on qualifications, funded by Faculty of Medical Sciences and BBSRC at the UK/EU rate. This project is focused on the characterisation of proteins associated with biofilm formation in the fungus Candida albicans and the Gram-positive bacterium Clostridium difficile. The project will involve protein expression, purification and structural determination via protein X-ray crystallography, combined with other structural and biochemical methods. The successful candidate should have an undergraduate degree awarded at an upper second or higher (or equivalent) in Biochemistry, Chemistry, Molecular Biology or related subject (for 4 year programme) or a Masters degree (for 3 year programme). Equivalent experience will also be considered. Candidates must complete the University's postgraduate application form. http://www.ncl.ac.uk/postgraduate/apply/ Form more details, please see: http://www.ncl.ac.uk/postgraduate/funding/search/list/cb071 or contact me via this email. Best regards Dr Paula Salgado
[ccp4bb] Best method for weighted averaging of Friedel pairs?
Dear all, I have a anomalous dataset, processed in HKL2000. Scalepack outputs a file containing the separately merged sets of the Friedel pairs I- and I+ and their standard deviations sigI+ and sigI-. Scalepack does not output the averaged intensities (Imean) and the standard deviations (sigIMean). The CCP4 program truncate that I use to convert the intensities to amplitudes requires Imean, I- and I+ and the respective standard deviations in its input file. I have now found at least three different methods to generate the averaged intensities from the Friedel pairs: 1) scalepack2mtz uses standard deviations for the weights: weights w = 1/sigI Imean = (w+*I+ + w-*I- ) / (w+ + w-) sigImean = 1 / (w+ + w-) 2) Method described in Biomolecular crystallography by Bernhard Rupp, p. 332/333 to average symmetry equivalent reflections uses variances for the weights: weight w = 1/sigI^2 Imean = (w+*I+ + w-*I- ) / (w+ + w-) sigImean = 1 / sqrt(w+ + w-) 3) Method used in cctbx function miller.set.average_bijvoet_mates() that calls generic merge.merge_equivalent_obs(): same as methods 2, except that sigImean is the larger of either a) sigImean = 1 / sqrt(w+ + w-) or b) sigImean = sqrt( wvariance ) where wvariance = (w+ + w-) / [ (w+ + w-)^2 - (w+^2 + w-^2) ] * [ w+*(F+ - Imean)^2 + w-*(F- - Imean)^2 ] What are the advantages and disadvantages of each method? Should method 1 be used at all? Some example from my dataset: Reflection (1, 1, 0), space group P3 2 1 I+: 23841.50 sigI+: 634.01 I-: 9628.57, sigI-: 264.75 Method 1: Imean=13815.32, sigImean=186.76 Method 2: Imean=11738.95, sigIMean=244.31 Method 3: Imean=11738.95, sigIMean=7106.47 Thanks a lot! Cheers, Markus -- Markus Meier, Ph.D. University of Manitoba Department of Chemistry 144 Dysart Road Winnipeg, MB, R3T 2N2, Canada Phone: +1 204 474 7172 Fax:+1 204 474 7608 E-mail: me...@cc.umanitoba.ca
[ccp4bb] Crystal Structures as Snapshots
Dear Crystallographers, I am looking for references which discuss the validity of the assertion that multiple crystal structures of the same or similar proteins can be considered freeze-frame snapshots of actual conformations assumed in solution. In a way, the assertion seems almost definitely true to me, but on the other hand, I could imagine some objections as well. Seems there should be some classic literature here... All the best, Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Crystal Structures as Snapshots
How could they not be snapshots of conformations adopted in solution? James On Feb 10, 2012, at 1:25 PM, Jacob Keller wrote: Dear Crystallographers, I am looking for references which discuss the validity of the assertion that multiple crystal structures of the same or similar proteins can be considered freeze-frame snapshots of actual conformations assumed in solution. In a way, the assertion seems almost definitely true to me, but on the other hand, I could imagine some objections as well. Seems there should be some classic literature here... All the best, Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Crystal Structures as Snapshots
On Fri, Feb 10, 2012 at 12:29 PM, James Stroud xtald...@gmail.com wrote: How could they not be snapshots of conformations adopted in solution? Packing billions of copies of an irregularly-shaped protein into a compact lattice and freezing it to 100K isn't necessarily representative of solution, especially when your solution contains non-physiological amounts of salt and various organics (and possibly non-physiological pH too). -Nat
Re: [ccp4bb] Crystal Structures as Snapshots
How could they not be snapshots of conformations adopted in solution? Let me clarify--sorry about that. Consider several structures of the same protein solved under different conditions, or several homologs solved under similar conditions, or both. Further, let's say some structural element, perhaps a helix, assumes different mannerisms in the various structures. Can I reasonably assert that these structures are snapshots of the protein which would have existed under physiological conditions, and assemble the structures to a unifying conception of the helical motion, or must I assume these are artifacts of bizarre solution conditions, and one has nothing to do with the other? Or perhaps every case/protein is unique, in which case no general rule can be followed, amounting approximately to the same thing? Jacob
Re: [ccp4bb] Crystal Structures as Snapshots
Hi Jacob, Lorena Beese has a few systems where snapshots of reaction mechanisms have been looked at structurally. Here are two such papers: Long, SB, Casey, P., Beese, LS (2002) The reaction path of protein farnesyltransferase at atomic resolution. Nature Oct 10; 419(6907):645-50. http://www.ncbi.nlm.nih.gov/pubmed?term=The%20reaction%20path%20of%20protein%20farnesyltransferase%20at%20atomic%20resolution J. R. Kiefer, C. Mao, J. C. Braman and L. S. Beese (1998) “Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal” Nature 6664:304-7. http://www.ncbi.nlm.nih.gov/pubmed?term=Visualizing%20DNA%20replication%20in%20a%20catalytically%20active%20Bacillus%20DNA%20polymerase%20crystal Cheers, -bob On Fri, Feb 10, 2012 at 3:25 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Dear Crystallographers, I am looking for references which discuss the validity of the assertion that multiple crystal structures of the same or similar proteins can be considered freeze-frame snapshots of actual conformations assumed in solution. In a way, the assertion seems almost definitely true to me, but on the other hand, I could imagine some objections as well. Seems there should be some classic literature here... All the best, Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Crystal Structures as Snapshots
On 02/10/2012 03:25 PM, Jacob Keller wrote: Dear Crystallographers, I am looking for references which discuss the validity of the assertion that multiple crystal structures of the same or similar proteins can be considered freeze-frame snapshots of actual conformations assumed in solution. In a way, the assertion seems almost definitely true to me, but on the other hand, I could imagine some objections as well. Seems there should be some classic literature here... How could that possibly be the case when any structure is an average of all the unit cells of the crystal over the timespan of the diffraction experiment? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Crystal Structures as Snapshots
I believe the most justifiable assumption one can make is that crystal structures are likely to represent the least soluble conformations of a protein under the conditions of crystallization (which might be a broad range of conditions, including physiological). This can be quite vexing if you are studying an allosteric protein and one of the two conformations is typically much less soluble than the other. BTDT. I'm sure others have had the same experience. Having said that, the solvent content of protein crystals (which is close to that of cellular conditions), the observation of enzymatic activity in many protein crystals, and the *general* concordance of XRD and NMR structures of proteins (when both have been determined) leads one to believe that XRD structures are likely representative of physiologically relevant conformations. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/10/2012 3:34 PM, Nat Echols wrote: On Fri, Feb 10, 2012 at 12:29 PM, James Stroud xtald...@gmail.com wrote: How could they not be snapshots of conformations adopted in solution? Packing billions of copies of an irregularly-shaped protein into a compact lattice and freezing it to 100K isn't necessarily representative of "solution", especially when your solution contains non-physiological amounts of salt and various organics (and possibly non-physiological pH too). -Nat
Re: [ccp4bb] Crystal Structures as Snapshots
Interesting to juxtapose these two responses: James Stroud: How could they not be snapshots of conformations adopted in solution? David Schuller: How could that possibly be the case when any structure is an average of all the unit cells of the crystal over the timespan of the diffraction experiment? JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Crystal Structures as Snapshots
So the implication is that some of these treatments might allow the protein to overcome energetic barriers that are prohibitive in solution--after the protein is already in the solid state and not in solution any more? Another view is that crystallization is a result of stabilizing conformations that are accessible in solution. On the point of physiological relevance, it wasn't mentioned in the original question. James On Feb 10, 2012, at 1:34 PM, Nat Echols wrote: On Fri, Feb 10, 2012 at 12:29 PM, James Stroud xtald...@gmail.com wrote: How could they not be snapshots of conformations adopted in solution? Packing billions of copies of an irregularly-shaped protein into a compact lattice and freezing it to 100K isn't necessarily representative of solution, especially when your solution contains non-physiological amounts of salt and various organics (and possibly non-physiological pH too). -Nat
Re: [ccp4bb] Crystal Structures as Snapshots
Packing billions of copies into a compact lattice Not so compact there is 40-80% water freezing it to 100K We have frozen many times protein solutions in liquid nitrogen and then thaw and were working OK non-physiological amounts of salt and various organics What is the amount of salt and osmotic pressure in the cell?? non-physiological pH too What is the non-physiological pH too? I am sure that some enzymes they are not working in pH 7. Also most of the proteins they have crystallized in pH close to 7 so I would not say non-physiological. George PS There are lots of solution NMR structures as well supporting the physiological crystal structures -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols Sent: Friday, February 10, 2012 10:35 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal Structures as Snapshots On Fri, Feb 10, 2012 at 12:29 PM, James Stroud xtald...@gmail.com wrote: How could they not be snapshots of conformations adopted in solution? Packing billions of copies of an irregularly-shaped protein into a compact lattice and freezing it to 100K isn't necessarily representative of solution, especially when your solution contains non-physiological amounts of salt and various organics (and possibly non-physiological pH too). -Nat
[ccp4bb] Creating new project directories with ccp4i
Hi, I have run ccp4 without issue on my OS X machine until recently. For some reason I am not able to create new project directories, and therefore am not able to run routines. The message that I am getting is the following: ERROR saving parameters to file .../.CCP4/unix/directories.def I have seen previous posts regarding this problem, but I am still not able to determine how to resolve the issue. Thanks. Chad
Re: [ccp4bb] Crystal Structures as Snapshots
The contrast seems to boil down to the semantics of the word snapshot. In my definition, I assume that the uncertainty of a structure is an intrinsic quality of the structure and thus included in the meaning of snapshot. Part of that uncertainty comes from averaging. James On Feb 10, 2012, at 1:51 PM, Jacob Keller wrote: Interesting to juxtapose these two responses: James Stroud: How could they not be snapshots of conformations adopted in solution? David Schuller: How could that possibly be the case when any structure is an average of all the unit cells of the crystal over the timespan of the diffraction experiment? JPK
Re: [ccp4bb] Crystal Structures as Snapshots
On Friday, February 10, 2012 12:51:03 pm Jacob Keller wrote: Interesting to juxtapose these two responses: James Stroud: How could they not be snapshots of conformations adopted in solution? David Schuller: How could that possibly be the case when any structure is an average of all the unit cells of the crystal over the timespan of the diffraction experiment? This pair of perspectives is the starting point for the introductory rationale I usually present for TLSMD analysis. The crystal structure is a snapshot, but just like a photographic snapshot it contains blurry parts where the camera has captured a superposition of microconformations. When you photograph an object in motion, those microconformations correspond to a trajectory purely along time. In a crystallographic experiment, the microconformations correspond to samples from a trajectory in solution. Separation in time has been transformed into separation in space (from one unit cell to another). A TLSMD model tries to reproduce the observed blurring by modeling it a samples from a trajectory described by TLS displacement. The issue of averaging over the timespan of the diffraction experiment is relevant primarily to individual atomic vibrations, not so much to what we normally mean by conformations of overall protein structure. Ethan -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Crystal Structures as Snapshots
Just to clarify - I actually think the original assumption that Jacob posted is generally reasonable. But it needn't necessarily follow that the conformation we see in crystal structures is always representative of the solution state; given the extreme range of conditions in which crystals grow, I would be surprised if there weren't counter-examples. I'm not familiar enough with the literature on domain swapping (e.g. diptheria toxin) to know if any of those structures are crystal packing artifacts. On Fri, Feb 10, 2012 at 1:04 PM, George gkontopi...@vet.uth.gr wrote: Packing billions of copies into a compact lattice Not so compact there is 40-80% water freezing it to 100K We have frozen many times protein solutions in liquid nitrogen and then thaw and were working OK non-physiological amounts of salt and various organics What is the amount of salt and osmotic pressure in the cell?? non-physiological pH too What is the non-physiological pH too? I am sure that some enzymes they are not working in pH 7. Also most of the proteins they have crystallized in pH close to 7 so I would not say non-physiological. George PS There are lots of solution NMR structures as well supporting the physiological crystal structures -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols Sent: Friday, February 10, 2012 10:35 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal Structures as Snapshots On Fri, Feb 10, 2012 at 12:29 PM, James Stroud xtald...@gmail.com wrote: How could they not be snapshots of conformations adopted in solution? Packing billions of copies of an irregularly-shaped protein into a compact lattice and freezing it to 100K isn't necessarily representative of solution, especially when your solution contains non-physiological amounts of salt and various organics (and possibly non-physiological pH too). -Nat
Re: [ccp4bb] Crystal Structures as Snapshots
Along the lines of Roger's second point, there was a very nice paper a few years back that found very good agreement between the conformational ensemble sampled by ubiquitin in solution (by NMR) with the ensemble of conformations observed in a large number of crystal structures: Lange OF, Lakomek NA, Farès C, Schröder GF, Walter KF, Becker S, Meiler J, Grubmüller H, Griesinger C, de Groot BL. Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution. Science. 2008 Jun 13;320(5882):1471-5. PubMed PMID: 18556554. Best, Damian Ekiert On Feb 10, 2012, at 12:50 PM, Roger Rowlett wrote: I believe the most justifiable assumption one can make is that crystal structures are likely to represent the least soluble conformations of a protein under the conditions of crystallization (which might be a broad range of conditions, including physiological). This can be quite vexing if you are studying an allosteric protein and one of the two conformations is typically much less soluble than the other. BTDT. I'm sure others have had the same experience. Having said that, the solvent content of protein crystals (which is close to that of cellular conditions), the observation of enzymatic activity in many protein crystals, and the *general* concordance of XRD and NMR structures of proteins (when both have been determined) leads one to believe that XRD structures are likely representative of physiologically relevant conformations. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/10/2012 3:34 PM, Nat Echols wrote: On Fri, Feb 10, 2012 at 12:29 PM, James Stroud xtald...@gmail.com wrote: How could they not be snapshots of conformations adopted in solution? Packing billions of copies of an irregularly-shaped protein into a compact lattice and freezing it to 100K isn't necessarily representative of solution, especially when your solution contains non-physiological amounts of salt and various organics (and possibly non-physiological pH too). -Nat
Re: [ccp4bb] Crystal Structures as Snapshots
Isn't calcium-calmodulin one of the archetypical examples of the crystal structure probably not representing the solution structure (perhaps because the crystallization pH = 4.5)? Look at that linker helix--how stable can that be in solution? I don't think a single one of the NMR ca-calmodulin structures/conformers has the central helix like that. Jacob On Fri, Feb 10, 2012 at 3:31 PM, Nat Echols nathaniel.ech...@gmail.com wrote: Just to clarify - I actually think the original assumption that Jacob posted is generally reasonable. But it needn't necessarily follow that the conformation we see in crystal structures is always representative of the solution state; given the extreme range of conditions in which crystals grow, I would be surprised if there weren't counter-examples. I'm not familiar enough with the literature on domain swapping (e.g. diptheria toxin) to know if any of those structures are crystal packing artifacts. On Fri, Feb 10, 2012 at 1:04 PM, George gkontopi...@vet.uth.gr wrote: Packing billions of copies into a compact lattice Not so compact there is 40-80% water freezing it to 100K We have frozen many times protein solutions in liquid nitrogen and then thaw and were working OK non-physiological amounts of salt and various organics What is the amount of salt and osmotic pressure in the cell?? non-physiological pH too What is the non-physiological pH too? I am sure that some enzymes they are not working in pH 7. Also most of the proteins they have crystallized in pH close to 7 so I would not say non-physiological. George PS There are lots of solution NMR structures as well supporting the physiological crystal structures -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols Sent: Friday, February 10, 2012 10:35 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal Structures as Snapshots On Fri, Feb 10, 2012 at 12:29 PM, James Stroud xtald...@gmail.com wrote: How could they not be snapshots of conformations adopted in solution? Packing billions of copies of an irregularly-shaped protein into a compact lattice and freezing it to 100K isn't necessarily representative of solution, especially when your solution contains non-physiological amounts of salt and various organics (and possibly non-physiological pH too). -Nat -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Crystal Structures as Snapshots
Hi Nat, there are a number of viruses in which a domain swap occurs inside the capsid, with the hinge sequence being highly conserved among their respective families. Perhaps I'm missing your point, but I won't attribute that kind of domain swap to any sort of crystal packing artifact. Jon 2012/2/10 Nat Echols nathaniel.ech...@gmail.com Just to clarify - I actually think the original assumption that Jacob posted is generally reasonable. But it needn't necessarily follow that the conformation we see in crystal structures is always representative of the solution state; given the extreme range of conditions in which crystals grow, I would be surprised if there weren't counter-examples. I'm not familiar enough with the literature on domain swapping (e.g. diptheria toxin) to know if any of those structures are crystal packing artifacts. On Fri, Feb 10, 2012 at 1:04 PM, George gkontopi...@vet.uth.gr wrote: Packing billions of copies into a compact lattice Not so compact there is 40-80% water freezing it to 100K We have frozen many times protein solutions in liquid nitrogen and then thaw and were working OK non-physiological amounts of salt and various organics What is the amount of salt and osmotic pressure in the cell?? non-physiological pH too What is the non-physiological pH too? I am sure that some enzymes they are not working in pH 7. Also most of the proteins they have crystallized in pH close to 7 so I would not say non-physiological. George PS There are lots of solution NMR structures as well supporting the physiological crystal structures -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols Sent: Friday, February 10, 2012 10:35 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal Structures as Snapshots On Fri, Feb 10, 2012 at 12:29 PM, James Stroud xtald...@gmail.com wrote: How could they not be snapshots of conformations adopted in solution? Packing billions of copies of an irregularly-shaped protein into a compact lattice and freezing it to 100K isn't necessarily representative of solution, especially when your solution contains non-physiological amounts of salt and various organics (and possibly non-physiological pH too). -Nat -- Dr. Jon Agirre Postdoctoral Scientist - Protein and Virus X-ray Crystallography Group Biophysics Unit (CSIC-UPV/EHU) +0034946013357
Re: [ccp4bb] Crystal Structures as Snapshots
Hi Jacob, For Ca2+-CaM, and flexible proteins in general, the average conformation in solution may differ from the most crystallizable conformation. However, any crystallized conformation had to be sampled in solution at some point in order to form a crystal, and thus the crystal structure tells us something about the range of conformations accessible to the protein under the crystallization conditions. In Ca2+-CaM, the presence of MPD is probably more responsible for the continuous central helix than the pH, but early analysis of the thermal factors in that region of the crystal structure predicted flexibility in the center of this helix that was subsequently observed by NMR to be a flexible linker region. More generally, I'd argue that crystal disorder is a subset of solution motion: i.e. disorder observed in crystalline protein almost certainly corresponds to motions that occur in solution (perhaps with altered amplitude), but not all solution motions are observed as disorder in the crystal. Best regards, Mark Mark A. Wilson Associate Professor Department of Biochemistry/Redox Biology Center University of Nebraska N118 Beadle Center 1901 Vine Street Lincoln, NE 68588 (402) 472-3626 mwilso...@unl.edu Jacob Keller j-kell...@fsm.northwestern.edu Sent by: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK 02/10/2012 03:37 PM Please respond to Jacob Keller j-kell...@fsm.northwestern.edu To CCP4BB@JISCMAIL.AC.UK cc Subject Re: [ccp4bb] Crystal Structures as Snapshots Isn't calcium-calmodulin one of the archetypical examples of the crystal structure probably not representing the solution structure (perhaps because the crystallization pH = 4.5)? Look at that linker helix--how stable can that be in solution? I don't think a single one of the NMR ca-calmodulin structures/conformers has the central helix like that. Jacob On Fri, Feb 10, 2012 at 3:31 PM, Nat Echols nathaniel.ech...@gmail.com wrote: Just to clarify - I actually think the original assumption that Jacob posted is generally reasonable. But it needn't necessarily follow that the conformation we see in crystal structures is always representative of the solution state; given the extreme range of conditions in which crystals grow, I would be surprised if there weren't counter-examples. I'm not familiar enough with the literature on domain swapping (e.g. diptheria toxin) to know if any of those structures are crystal packing artifacts. On Fri, Feb 10, 2012 at 1:04 PM, George gkontopi...@vet.uth.gr wrote: Packing billions of copies into a compact lattice Not so compact there is 40-80% water freezing it to 100K We have frozen many times protein solutions in liquid nitrogen and then thaw and were working OK non-physiological amounts of salt and various organics What is the amount of salt and osmotic pressure in the cell?? non-physiological pH too What is the non-physiological pH too? I am sure that some enzymes they are not working in pH 7. Also most of the proteins they have crystallized in pH close to 7 so I would not say non-physiological. George PS There are lots of solution NMR structures as well supporting the physiological crystal structures -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols Sent: Friday, February 10, 2012 10:35 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal Structures as Snapshots On Fri, Feb 10, 2012 at 12:29 PM, James Stroud xtald...@gmail.com wrote: How could they not be snapshots of conformations adopted in solution? Packing billions of copies of an irregularly-shaped protein into a compact lattice and freezing it to 100K isn't necessarily representative of solution, especially when your solution contains non-physiological amounts of salt and various organics (and possibly non-physiological pH too). -Nat -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Crystal Structures as Snapshots
Hi, There is a interesting paper/tool that might shed a little light on the debate here: The paper: http://www.ncbi.nlm.nih.gov/pubmed/19956261 The tool: http://ucxray.berkeley.edu/ringer/Documentation/ringerManual.htm#Utility As I remember, this tool claimed to be able to extract information about the subtle or hidden movements of side chains of an enzyme from high-res crystallographic data. One thing to note: the authors collected the dataset used in the Nature paper at RT. However their online manual said they also analyzed 402 hi-res structure in PDB (all kinds of growth conditions apparently, and most, if not all, were probably collected under cryo stream) and found an abundance of alternative side chain conformations. Are all these alternative conformations relevant to the proteins' native states under physiological conditions? I guess it must be case-by-case. JPK: you might find something you are looking for in the nature paper's reference. The part mentioning the myoglobin and RNase work seems promising. Good luck. Zhijie -- From: Jacob Keller j-kell...@fsm.northwestern.edu Sent: Friday, February 10, 2012 3:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal Structures as Snapshots Dear Crystallographers, I am looking for references which discuss the validity of the assertion that multiple crystal structures of the same or similar proteins can be considered freeze-frame snapshots of actual conformations assumed in solution. In a way, the assertion seems almost definitely true to me, but on the other hand, I could imagine some objections as well. Seems there should be some classic literature here... All the best, Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
