[ccp4bb] DNA in coot
Hi all, I am refining a structue of protein-DNA complex with coot. I add DNA by adding ideal DNA/RNA in the other model. But I cannot edit chi angle of these nucletide, neither the mutate. When I press the mutate and my DNA, coot give amino acid not nucletide. Why? Thanks Lisa
[ccp4bb] DNA length for crystallization
Hi all, I have a DNA binding protein. I can get crystals when I mix 8-28 nt dsDNA with my protein. But neither of them has good diffraction. Some biochemical data said the longer of DNA, the tigher of the binding betwwen DNA and my protein. The binding is not sequence-specfic. Does anyone have suggestion of the optimization? What is the good length of DNA for crystallization? Thank you. Lisa
Re: [ccp4bb] DNA length for crystallization
Lisa, there isn't unfortunately a hard and fast rule for the length of DNA used in co-crystallisation. It usually is just a case of screening different lengths, permuting the sequence, and investigating overhangs or gaps in the DNA duplex. We generally work with oligos between 8 and 21 nts in length, but there are many examples of longer DNAs being co-crystallised, the nucleosome comes to mind as an extreme example. Do you know if the protein binds better to DNA containing secondary structure elements, such as hairpin loops? This can make a difference, especially when you don't have sequence-specificity. Tony O. --- Mobile Account --- On 15 Feb 2012, at 08:07, LISA science...@gmail.com wrote: Hi all, I have a DNA binding protein. I can get crystals when I mix 8-28 nt dsDNA with my protein. But neither of them has good diffraction. Some biochemical data said the longer of DNA, the tigher of the binding betwwen DNA and my protein. The binding is not sequence-specfic. Does anyone have suggestion of the optimization? What is the good length of DNA for crystallization? Thank you. Lisa
Re: [ccp4bb] DNA length for crystallization
Use 5' overhangs of two and make the DNA 10, 11, 15, 20, 21 25, 26, 30, or 31 bases in length. Count the overhangs in the length. If you don't know where to start, try 15, 25, and 26 first because they will make 2(1) screws, which are good for crystals. James On Feb 15, 2012, at 1:06 AM, LISA wrote: Hi all, I have a DNA binding protein. I can get crystals when I mix 8-28 nt dsDNA with my protein. But neither of them has good diffraction. Some biochemical data said the longer of DNA, the tigher of the binding betwwen DNA and my protein. The binding is not sequence-specfic. Does anyone have suggestion of the optimization? What is the good length of DNA for crystallization? Thank you. Lisa
Re: [ccp4bb] DNA length for crystallization
This paper is a favorite:http://www.ncbi.nlm.nih.gov/pubmed/2160019 J Mol Biol. 1990 May 5;213(1):159-66. Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site. The use of modular DNA. On Feb 15, 2012, at 12:06 AM, LISA wrote: Hi all, I have a DNA binding protein. I can get crystals when I mix 8-28 nt dsDNA with my protein. But neither of them has good diffraction. Some biochemical data said the longer of DNA, the tigher of the binding betwwen DNA and my protein. The binding is not sequence-specfic. Does anyone have suggestion of the optimization? What is the good length of DNA for crystallization? Thank you. Lisa
Re: [ccp4bb] surface residue mutation
Dear All, One of the most efficient methods to change space group and packing without having to change the sequence is to change the length of N and/or C terminal tags. An example that I am familiar with is given by the following PDB codes. 1JIZ, 1RMZ, 1JK3, 1UTT, 1UTZ, 2WOA, 2W0D, 1ROS, 1OS9, 3BA0 It includes 1 surface residue mutation, but the rest are small variations in length. Complexation with any ligand that may protrude is also likely to work. Enrico. On Wed, 15 Feb 2012 01:35:36 +0100, Bernhard Rupp (Hofkristallrat a.D.) hofkristall...@gmail.com wrote: http://services.mbi.ucla.edu/SER/ but no space group predictions are possible. BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Prem Kaushal Sent: Tuesday, February 14, 2012 3:36 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] surface residue mutation Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://www-dsv.cea.fr/en/institutes/institute-of-biology-and-technology-saclay-ibitec-s/unites-de-recherche/department-of-molecular-engineering-of-proteins-simopro/molecular-toxinology-and-biotechnology-laboratory-ltmb/crystallogenesis-e.-stura http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] DNA in coot
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hello Lisa, which version of coot do you use? Maybe it is outdated and that function not yet properly implemented. I can confirm Bill's comment, and we work with coot 0.6.2. Cheers, Tim On 02/15/2012 09:01 AM, LISA wrote: Hi all, I am refining a structue of protein-DNA complex with coot. I add DNA by adding ideal DNA/RNA in the other model. But I cannot edit chi angle of these nucletide, neither the mutate. When I press the mutate and my DNA, coot give amino acid not nucletide. Why? Thanks Lisa - -- - -- 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/ iD8DBQFPO4qsUxlJ7aRr7hoRAnEhAJwOJyQQxLO9N/7Hjusb2d4ATTwz/QCeNbNP XzRMHtlwg/Fka/9ks/7HtBo= =vqOw -END PGP SIGNATURE-
[ccp4bb] EMBO practical course on Solution Scattering from Biological Macromolecules in Hamburg, October 17-24, 2012
EMBO practical course on Solution Scattering from Biological Macromolecules The course will take place at EMBL Hamburg, Germany, on October 17-24, 2012. Organizers: D. Svergun, M. Roessle, M. Petoukhov, A. Kikhney (European Molecular Biology Laboratory, Hamburg Outstation) The course aims at young biochemists/biophysicists and researchers active in related structural methods (protein crystallography, NMR, electron microscopy) with little or no experience in solution scattering. The course will cover basics of SAXS/SANS, instrumentation, data collection, modelling techniques and complementary use with other methods. The students will be encouraged to bring their own samples to perform synchrotron solution scattering experiments on-site and the results will be used for practical tutorials and discussed on the last day of the course. There is no registration fee and accommodation and subsistence are covered for accepted applicants. Scientists from industry will be required to pay a fee of Euro 1000. Limited travel grants are available to selected participants coming from labs in countries in need of scientific strengthening. The detailed course information and the online application form are available at http://events.embo.org/12-sas/ Please note that there will be an additional possibility for a combination of SAXS with sample optimization and crystallization experiments, supported by the EU program P-Cube (www.p-cube.eu). The selected participants will be offered the opportunity to send their samples in advance to the EMBL Hamburg high-throughput crystallization platform (http://www.embl-hamburg.de/facilities/htpx/index.html). Depending on the crystallization results, P-Cube may sponsor optimization of the sample conditions, use of the EMBL Sample Preparation and Characterization (SPC) facility (http://www.embl-hamburg.de/facilities/spc/index.html) and more extensive SAXS experiments during the Course. Travel support may also be offered for some of the P-cube sponsored participants. Contact e-mail: r.meij...@embl-hamburg.de. The deadline for applications is July 31st, 2012. The applications will be considered by a Selection Committee and the results of the selection procedure will be sent to applicants by email within about four weeks of the closing date. If you have any questions about the course, please post them to http://www.saxier.org/forum/viewtopic.php?t=1404 Best regards, The Organizing Committee -- Giancarlo Tria European Molecular Biology Laboratory - Hamburg Outstation c/o DESY Building 25A, Notkestrasse 85, D-22603 Hamburg, Germany Tel.: +49-40-89902-170 Fax: +49-40-89902-149 http://www.embl-hamburg.de/biosaxs/tria.html
[ccp4bb] Coot, restraints and anchors
Dear CCP4bb, Is there any way to define a hydrogen bond as a restraint for real space refinement in Coot? It would be really useful for e.g. nucleotides where you might hypothesize or know that specific hydrogen bonds are formed. Sincerely, Morten -- Morten K Grøftehauge, PhD Pohl Group Durham University -- Morten K Grøftehauge, PhD Pohl Group Durham University
Re: [ccp4bb] Coot, restraints and anchors
You can try the scripts user-define-restraints.scm/user_defined_restraints.py which allow you to specify restraints. These are not available in the distribution (yet) but from google code: http://code.google.com/p/coot/source/browse/trunk/scheme/user-define-restraints.scm http://code.google.com/p/coot/source/browse/trunk/python/user_defined_restraints.py (*) There is more on non-bonded restraints (including H-bonds) in the pipeline... B (*) just realised the name mismatch, so this may change at some point... Dear CCP4bb, Is there any way to define a hydrogen bond as a restraint for real space refinement in Coot? It would be really useful for e.g. nucleotides where you might hypothesize or know that specific hydrogen bonds are formed. Sincerely, Morten -- Morten K Grøftehauge, PhD Pohl Group Durham University -- Morten K Grøftehauge, PhD Pohl Group Durham University No virus found in this message. Checked by AVG - www.avg.com http://www.avg.com Version: 2012.0.1913 / Virus Database: 2112/4811 - Release Date: 02/15/12
[ccp4bb] how to fix c-beta deviations
Dear all, i am quite new to refinement and after refinement using refmac i have a resonable R-factor and R-free (22 and 27%), but Molprobity analysis shows c-beta devaition s as 23. How can i fix this? -- B. Anuradha Research scholar, CAS in crystallography and Biophysics, University of Madras.
Re: [ccp4bb] protein degradation
Late induction for short time. Then immediately purify it cut down on any unnecessary steps eg shorter spin all on ice or coldroom etc. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Feb 15, 2012, at 8:20, Sivasankar Putta sivasankarpu...@iisertvm.ac.inmailto:sivasankarpu...@iisertvm.ac.in wrote: Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta proteingel.pdf
Re: [ccp4bb] surface residue mutation
Wukovitz Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly. On 02/14/12 18:36, Prem Kaushal wrote: Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Problem with COOT and MSE (SeMET ) residues
Hi Laurie, Not much, I did not get any useful feedback so I emailed Paul Emsley directly. Chris On Tue, 2012-02-14 at 22:43 -0500, Laurie Betts wrote: Problem with COOT and MSE (SeMET ) residues -- Dr. Christopher Browning Post-Doctor to Prof. Petr Leiman EPFL BSP-416 1015 Lausanne Switzerland Tel: 0041 (0) 02 16 93 04 40
Re: [ccp4bb] protein degradation
Hi, you may also check things like chemical degradation in SDS buffer as part of the analysis. Esspecially your degradation pattern is very much constant throughout your whole purification procedure. Christian Am Mittwoch 15 Februar 2012 14:09:19 schrieb Sivasankar Putta: Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in* E. Coli.* The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta -- Christian Roth Institut für Bioanalytische Chemie Biotechnologisch-Biomedizinisches Zentrum Fakultät für Chemie und Mineralogie Universität Leipzig Deutscher Platz 5 04103 Leipzig Telefon: +49 (0)341 97 31316 Fax: +49 (0)341 97 31319
[ccp4bb] Fwd: [ccp4bb] protein degradation
try experimenting with different, especially protease-deficient, E coli strains to express the protein and try different methods to lyse the bacteria (sonication, french-press, emulsification, bead-beater, mortar pestle under liquid nitrogen). on the other hand, if you are lucky, you are just proteolysing some surface loops and can still purify and crystallise the protein. This was done on purpose for the cap-binding complex, see: Crystal structure of the human nuclear cap binding complex. Mazza C, Ohno M, Segref A, Mattaj IW, Cusack S. Mol Cell. 2001 Aug;8(2):383-96. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 15 Feb 2012, at 14:09, Sivasankar Putta wrote: Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta proteingel.pdf
Re: [ccp4bb] protein degradation
Hi Sivasankar: Are you sure it is due to the protein degradation? Maybe you can try to do a western blot or others to check if it is the product of degradation. By the way, where you put the 6 histag, N- or C-terminal? If it is at the N terminal, maybe it is the truncation version of your protein. After looking at the gel, it seems your sample was over-load or had lots of unspecific binding to the column. Maybe you can add salt (250 mM NaCl, final concentration) and small amount of imidazle in the sample before you load onto the column (for example, 20 mM Imidazole final concentration). One small trick you can try is wash the cell with the buffer containing PMSF once before lysising the cell. Yu Xiaodi Date: Wed, 15 Feb 2012 18:39:19 +0530 From: sivasankarpu...@iisertvm.ac.in Subject: [ccp4bb] protein degradation To: CCP4BB@JISCMAIL.AC.UK Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta
Re: [ccp4bb] protein degradation
You can also try putting a different affinity tag on the other terminus, and use that as a second step. JPK On Wed, Feb 15, 2012 at 11:25 AM, Xiaodi Yu uppsala@hotmail.com wrote: Hi Sivasankar: Are you sure it is due to the protein degradation? Maybe you can try to do a western blot or others to check if it is the product of degradation. By the way, where you put the 6 histag, N- or C-terminal? If it is at the N terminal, maybe it is the truncation version of your protein. After looking at the gel, it seems your sample was over-load or had lots of unspecific binding to the column. Maybe you can add salt (250 mM NaCl, final concentration) and small amount of imidazle in the sample before you load onto the column (for example, 20 mM Imidazole final concentration). One small trick you can try is wash the cell with the buffer containing PMSF once before lysising the cell. Yu Xiaodi Date: Wed, 15 Feb 2012 18:39:19 +0530 From: sivasankarpu...@iisertvm.ac.in Subject: [ccp4bb] protein degradation To: CCP4BB@JISCMAIL.AC.UK Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] surface residue mutation
Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted? JPK On Wed, Feb 15, 2012 at 8:05 AM, David Schuller dj...@cornell.edu wrote: Wukovitz Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly. On 02/14/12 18:36, Prem Kaushal wrote: Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] surface residue mutation
The most famous case I know of was the HIV protease. My grad school PI used to use it as an example in class. Science. 1992 Jun 5;256(5062):1445-8. Total chemical synthesis of a D-enzyme: the enantiomers of HIV-1 protease show reciprocal chiral substrate specificity [corrected]. Milton RC, Milton SC, Kent SB. http://www.ncbi.nlm.nih.gov/pubmed/1604320 Kelly *** Kelly Daughtry, Ph.D. Post-Doctoral Fellow, Raetz Lab Biochemistry Department Duke University Alex H. Sands, Jr. Building 303 Research Drive RM 250 Durham, NC 27710 P: 919-684-5178 *** On Wed, Feb 15, 2012 at 12:41 PM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted? JPK On Wed, Feb 15, 2012 at 8:05 AM, David Schuller dj...@cornell.edu wrote: Wukovitz Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly. On 02/14/12 18:36, Prem Kaushal wrote: Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] AW: [ccp4bb] surface residue mutation
Building on Alex's suggestion, here are some papers on rationally engineering new crystal contacts: via disulfides: http://www.pnas.org/content/103/44/16230.long http://onlinelibrary.wiley.com/doi/10.1002/pro.550/abstract;jsessionid=783A91 CC93571B0348F37A325929676D.d01t02 or via leucine zippers: http://onlinelibrary.wiley.com/doi/10.1110/ps.072851407/abstract Good luck, Chris. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of alexander.paut...@boehringer-ingelheim.com Sent: Wednesday, February 15, 2012 2:36 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] AW: [ccp4bb] surface residue mutation Hi Prem, besides trying surface entropy reduction you can also start by analyzing your crystal contacts and mutate residues therein. This was for example investigated in the 90's by GE Schulz and coworkers. Good Luck Alex Dr. Alexander Pautsch Boehringer Ingelheim Pharma GmbH Co. KG Dept. Lead Identific. and Optim. Sup. Ge Tel.: +49 (7351) 54-4683 Fax: +49 (7351) 54-97924 mailto:alexander.paut...@boehringer-ingelheim.com mailto:alexander.paut...@boehringer-ingelheim.com Boehringer Ingelheim Pharma GmbH Co. KG, Sitz: Ingelheim am Rhein; Registergericht Mainz: HR A 22206; Komplementär Boehringer Ingelheim Deutschland GmbH; Geschäftsführung: Dr. Engelbert Günster (Vorsitzender), Ursula Fuggis-Hahn, Ralf Gorniak, Michael Klein, Dr. Martin Wanning; Vorsitzender des Aufsichtsrates: Prof. Dr. Dr. Andreas Barner; Sitz: Ingelheim am Rhein; Registergericht Mainz: HR B 23260 Diese E-Mail ist vertraulich zu behandeln. Sie kann besonderem rechtlichen Schutz unterliegen. Wenn Sie nicht der richtige Adressat sind, senden Sie bitte diese E-Mail an den Absender zurück, löschen die eingegangene E-Mail und geben den Inhalt der E-Mail nicht weiter. Jegliche unbefugte Bearbeitung, Nutzung, Vervielfältigung oder Verbreitung ist verboten. / This e-mail is confidential and may also be legally privileged. If you are not the intended recipient please reply to sender, delete the e-mail and do not disclose its contents to any person. Any unauthorized review, use, disclosure, copying or distribution is strictly prohibited. Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Prem Kaushal Gesendet: Mittwoch, 15. Februar 2012 00:36 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] surface residue mutation Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem --
Re: [ccp4bb] surface residue mutation
On 02/15/12 12:41, Jacob Keller wrote: Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted? JPK What do you mean by Out There? If you mean in the PDB, then yes. As of two weeks ago, there are ~ 14 racemic structures deposited; most in space group P -1, with one outlier in space group I -4 C 2. This includes RNA, DNA, and PNA, but 6 entries are actually protein. The longest is over 80 residues. Theoretically, enantiomer-specific catalysis ought to be inverted, but most of the structures solved are not enzymes. kaliotoxin, plectasin, antifreeze protein, monellin, villin, and a designed peptide. On the other hand, if by out there you meant in nature outside of biochemistry and organic chemistry labs; then no, I am not aware of any all-D proteins. There are a few protein/peptides which include a small number of D-residues, which is marked up to nonribosomal synthesis. The first paper I managed to Google: http://jb.asm.org/content/185/24/7036.full Learning from Nature's Drug Factories: Nonribosomal Synthesis of Macrocyclic Peptides doi: 10.1128/JB.185.24.7036-7043.2003 J. Bacteriol.December 2003 vol. 185 no. 24 7036-7043 If racemic crystallization isn't exciting enough for you, look into quasi-racemic crystallization./ / On Wed, Feb 15, 2012 at 8:05 AM, David Schullerdj...@cornell.edu wrote: Wukovitz Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly. On 02/14/12 18:36, Prem Kaushal wrote: Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
[ccp4bb] All-D World
So who out there wants to start an all-D microbial culture by total synthesis, a la the bacterium with the synthetic genome a while back? Could it work, I wonder? I guess that would be a certain benchmark for Man's conquest of nature. JPK ps maybe if there is a broadly-acting amino-acid isomerase or set of isomerases of appropriate properties, this could be helpful for getting the culture started--or even for preying on the L world? On Wed, Feb 15, 2012 at 12:17 PM, David Schuller dj...@cornell.edu wrote: On 02/15/12 12:41, Jacob Keller wrote: Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted? JPK What do you mean by Out There? If you mean in the PDB, then yes. As of two weeks ago, there are ~ 14 racemic structures deposited; most in space group P -1, with one outlier in space group I -4 C 2. This includes RNA, DNA, and PNA, but 6 entries are actually protein. The longest is over 80 residues. Theoretically, enantiomer-specific catalysis ought to be inverted, but most of the structures solved are not enzymes. kaliotoxin, plectasin, antifreeze protein, monellin, villin, and a designed peptide. On the other hand, if by out there you meant in nature outside of biochemistry and organic chemistry labs; then no, I am not aware of any all-D proteins. There are a few protein/peptides which include a small number of D-residues, which is marked up to nonribosomal synthesis. The first paper I managed to Google: http://jb.asm.org/content/185/24/7036.full Learning from Nature's Drug Factories: Nonribosomal Synthesis of Macrocyclic Peptides doi: 10.1128/JB.185.24.7036-7043.2003 J. Bacteriol. December 2003 vol. 185 no. 24 7036-7043 If racemic crystallization isn't exciting enough for you, look into quasi-racemic crystallization. On Wed, Feb 15, 2012 at 8:05 AM, David Schuller dj...@cornell.edu wrote: Wukovitz Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly. On 02/14/12 18:36, Prem Kaushal wrote: Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Crystal Structures as Snapshots--Summary
Dear Crystallographers, thanks for all of the responses and conversation. I have culled together the various references which have been sent on the BB and which I have come up with, and posted them below. Worthy of special mention, I think, is the first one (Lange et al), in which 46 (!) different crystal structures are pitted against a lot of RDC NMR data, and the match seems to be excellent (although it seems you probably have to know both methods fairly well to evaluate this properly.) Anyway, for asserting that variances between crystal structures at least in some cases represent differences between physiologically-relevant states in solution, the Lange paper is really on the mark. Thanks again, Jacob 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. Kondrashov, D.A., Zhang, W., Aranda, R.t., Stec, B., and Phillips, G.N., Jr. (2008). Sampling of the native conformational ensemble of myoglobin via structures in different crystalline environments. Proteins 70, 353-362. Zhang, X. J., Wozniak, J. A., and Matthews, B. W. (1995) Protein flexibility and adaptability seen in 25 crystal forms of T4 lysozyme, Journal of molecular biology 250, 527-552. 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 Mancini EJ, Kainov DE, Grimes JM, Tuma R, Bamford DH, Stuart DI (2004) Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation. Cell 118(6):743-55 http://www.cell.com/abstract/S0092-8674(04)00837-2 Nature. 2009 Dec 3;462(7273):669-73. Hidden alternative structures of proline isomerase essential for catalysis. Fraser JS, Clarkson MW, Degnan SC, Erion R, Kern D, Alber T. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] All-D World
The structure of an all D-amino acid version of the HIV-1 protease was solved in 1992 (see Milton, Milton, and Kent, 1992, Science, 256:1445-1448). The D-enzyme was seen to have a structure that is the mirror image of the L-enzyme, and showed specificity for the enantiomeric form of the chiral substrate. P. Shing Ho, PhD Professor Chair Department of Biochemistry Molecular Biology Colorado State University Fort Collins, CO 80524-1870 On 2/15/12 11:28 AM, Jacob Keller j-kell...@fsm.northwestern.edu wrote: So who out there wants to start an all-D microbial culture by total synthesis, a la the bacterium with the synthetic genome a while back? Could it work, I wonder? I guess that would be a certain benchmark for Man's conquest of nature. JPK ps maybe if there is a broadly-acting amino-acid isomerase or set of isomerases of appropriate properties, this could be helpful for getting the culture started--or even for preying on the L world? On Wed, Feb 15, 2012 at 12:17 PM, David Schuller dj...@cornell.edu wrote: On 02/15/12 12:41, Jacob Keller wrote: Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted? JPK What do you mean by Out There? If you mean in the PDB, then yes. As of two weeks ago, there are ~ 14 racemic structures deposited; most in space group P -1, with one outlier in space group I -4 C 2. This includes RNA, DNA, and PNA, but 6 entries are actually protein. The longest is over 80 residues. Theoretically, enantiomer-specific catalysis ought to be inverted, but most of the structures solved are not enzymes. kaliotoxin, plectasin, antifreeze protein, monellin, villin, and a designed peptide. On the other hand, if by out there you meant in nature outside of biochemistry and organic chemistry labs; then no, I am not aware of any all-D proteins. There are a few protein/peptides which include a small number of D-residues, which is marked up to nonribosomal synthesis. The first paper I managed to Google: http://jb.asm.org/content/185/24/7036.full Learning from Nature's Drug Factories: Nonribosomal Synthesis of Macrocyclic Peptides doi: 10.1128/JB.185.24.7036-7043.2003 J. Bacteriol. December 2003 vol. 185 no. 24 7036-7043 If racemic crystallization isn't exciting enough for you, look into quasi-racemic crystallization. On Wed, Feb 15, 2012 at 8:05 AM, David Schuller dj...@cornell.edu wrote: Wukovitz Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly. On 02/14/12 18:36, Prem Kaushal wrote: Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Crystal Structures as Snapshots--Summary
I feel compelled to throw a few references into the ring. NFAT is a protein where you get a good sampling of snapshots: 1. Folded up as a monomer when interacting with partner proteins: http://www.ncbi.nlm.nih.gov/pubmed/9510247 http://www.ncbi.nlm.nih.gov/pubmed/16873067 2. Extended as a dimer: http://www.ncbi.nlm.nih.gov/pubmed/12949493 3. Folded up as a monomer when interacting with a partner protein which happens to be itself as an extended dimer: http://www.ncbi.nlm.nih.gov/pubmed/18462673 4. Wrapped around DNA as a monomer without partners:. http://www.ncbi.nlm.nih.gov/pubmed/14643663 In this last reference you get a sample of extended, wrapped around, and folded up all in the same unit cell! James On Feb 15, 2012, at 1:48 PM, Jacob Keller wrote: Dear Crystallographers, thanks for all of the responses and conversation. I have culled together the various references which have been sent on the BB and which I have come up with, and posted them below. Worthy of special mention, I think, is the first one (Lange et al), in which 46 (!) different crystal structures are pitted against a lot of RDC NMR data, and the match seems to be excellent (although it seems you probably have to know both methods fairly well to evaluate this properly.) Anyway, for asserting that variances between crystal structures at least in some cases represent differences between physiologically-relevant states in solution, the Lange paper is really on the mark. Thanks again, Jacob 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. Kondrashov, D.A., Zhang, W., Aranda, R.t., Stec, B., and Phillips, G.N., Jr. (2008). Sampling of the native conformational ensemble of myoglobin via structures in different crystalline environments. Proteins 70, 353-362. Zhang, X. J., Wozniak, J. A., and Matthews, B. W. (1995) Protein flexibility and adaptability seen in 25 crystal forms of T4 lysozyme, Journal of molecular biology 250, 527-552. 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 Mancini EJ, Kainov DE, Grimes JM, Tuma R, Bamford DH, Stuart DI (2004) Atomic snapshots of an RNA packaging motor reveal conformational changes linking ATP hydrolysis to RNA translocation. Cell 118(6):743-55 http://www.cell.com/abstract/S0092-8674(04)00837-2 Nature. 2009 Dec 3;462(7273):669-73. Hidden alternative structures of proline isomerase essential for catalysis. Fraser JS, Clarkson MW, Degnan SC, Erion R, Kern D, Alber T. *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] choice of wavelength
All, I am curious to hear what our CCP4 community thoughts are I have a marginally diffracting protein crystal (3-3.5 Angstrom resolution) and would like to squeeze in a few tenth of angstrom. Given that I am working on crystal quality improvement, would different wavelengths make any difference in resolution, for example 0.9 vs. 1.0 Angstrom at synchrotron? Thanks. Seungil Seungil Han, Ph.D. Pfizer Inc. Eastern Point Road, MS8118W-228 Groton, CT 06340 Tel: 860-686-1788, Fax: 860-686-2095 Email: seungil@pfizer.com
[ccp4bb] ccp4bb] All-D World
Weds., Feb. 15th 2012 EBI Good evening, PDB entries with spg P 1- or I -4 C 2 are 3al1 3boi 2gpm 1krl 2gq7 2gq6 2gq5 2gq4 2g32 1pup 3e7r 1vtu 3odv 3trw 3try you can get information on each from pdbe pdbe.org/entry i.e. pdbe.org/3al1 etc. Miri, PDBe On Wed, 15 Feb 2012, Jacob Keller wrote: So who out there wants to start an all-D microbial culture by total synthesis, a la the bacterium with the synthetic genome a while back? Could it work, I wonder? I guess that would be a certain benchmark for Man's conquest of nature. JPK ps maybe if there is a broadly-acting amino-acid isomerase or set of isomerases of appropriate properties, this could be helpful for getting the culture started--or even for preying on the L world? On Wed, Feb 15, 2012 at 12:17 PM, David Schuller dj...@cornell.edu wrote: On 02/15/12 12:41, Jacob Keller wrote: Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted? JPK What do you mean by Out There? If you mean in the PDB, then yes. As of two weeks ago, there are ~ 14 racemic structures deposited; most in space group P -1, with one outlier in space group I -4 C 2. This includes RNA, DNA, and PNA, but 6 entries are actually protein. The longest is over 80 residues. Theoretically, enantiomer-specific catalysis ought to be inverted, but most of the structures solved are not enzymes. kaliotoxin, plectasin, antifreeze protein, monellin, villin, and a designed peptide. On the other hand, if by out there you meant in nature outside of biochemistry and organic chemistry labs; then no, I am not aware of any all-D proteins. There are a few protein/peptides which include a small number of D-residues, which is marked up to nonribosomal synthesis. The first paper I managed to Google: http://jb.asm.org/content/185/24/7036.full Learning from Nature's Drug Factories: Nonribosomal Synthesis of Macrocyclic Peptides doi: 10.1128/JB.185.24.7036-7043.2003 J. Bacteriol. December 2003 vol. 185 no. 24 7036-7043 If racemic crystallization isn't exciting enough for you, look into quasi-racemic crystallization. On Wed, Feb 15, 2012 at 8:05 AM, David Schuller dj...@cornell.edu wrote: Wukovitz Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly. On 02/14/12 18:36, Prem Kaushal wrote: Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** Weds. Feb. 15th, 2012 EBI
Re: [ccp4bb] All-D World
Hi Jacob: After giving this a great deal of reflection ….. I realized that you would face the same paradox that God had to resolve six thousand years ago at the Dawn of Creation, i.e., He needed D-deoxyribose DNA to code for L-amino acid proteins, and vice versa. Likewise, you would probably be faced with a situation where you need L-deoxyribose DNA to code for D-amino acid proteins, so once again, you need a ribozyme self-replicase to escape the Irreducible Complexity(™). (The Central Dogma at least is achiral.) At least it can be done six thousand years, which isn't unreasonable for a Ph.D. thesis project (especially when combined with an M.D.), and you, unlike Him, have access to a Sigma catalogue. All the best, Bill William G. Scott Professor Department of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA 228 Sinsheimer Laboratories University of California at Santa Cruz Santa Cruz, California 95064 USA On Feb 15, 2012, at 10:28 AM, Jacob Keller wrote: So who out there wants to start an all-D microbial culture by total synthesis, a la the bacterium with the synthetic genome a while back? Could it work, I wonder? I guess that would be a certain benchmark for Man's conquest of nature. JPK ps maybe if there is a broadly-acting amino-acid isomerase or set of isomerases of appropriate properties, this could be helpful for getting the culture started--or even for preying on the L world? On Wed, Feb 15, 2012 at 12:17 PM, David Schuller dj...@cornell.edu wrote: On 02/15/12 12:41, Jacob Keller wrote: Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted? JPK What do you mean by Out There? If you mean in the PDB, then yes. As of two weeks ago, there are ~ 14 racemic structures deposited; most in space group P -1, with one outlier in space group I -4 C 2. This includes RNA, DNA, and PNA, but 6 entries are actually protein. The longest is over 80 residues. Theoretically, enantiomer-specific catalysis ought to be inverted, but most of the structures solved are not enzymes. kaliotoxin, plectasin, antifreeze protein, monellin, villin, and a designed peptide. On the other hand, if by out there you meant in nature outside of biochemistry and organic chemistry labs; then no, I am not aware of any all-D proteins. There are a few protein/peptides which include a small number of D-residues, which is marked up to nonribosomal synthesis. The first paper I managed to Google: http://jb.asm.org/content/185/24/7036.full Learning from Nature's Drug Factories: Nonribosomal Synthesis of Macrocyclic Peptides doi: 10.1128/JB.185.24.7036-7043.2003 J. Bacteriol. December 2003 vol. 185 no. 24 7036-7043 If racemic crystallization isn't exciting enough for you, look into quasi-racemic crystallization. On Wed, Feb 15, 2012 at 8:05 AM, David Schuller dj...@cornell.edu wrote: Wukovitz Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly. On 02/14/12 18:36, Prem Kaushal wrote: Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Fwd: HR3699, Research Works Act
If you agree, please signing the petition below. You need to register on the link below before you can sign this petition. Registration and signing the petition took about a minute or two. Cheers, Raji -- Forwarded message -- From: Seth Darst da...@mail.rockefeller.edu Date: Tue, Feb 14, 2012 at 12:40 PM Subject: HR3699, Research Works Act To: Rep. Caroline Maloney has not backed off in her attempt to put forward the interests of Elsevier and other academic publishers. If you oppose this measure, please sign this petition on the official 'we the people' White House web site. It needs 23,000 signatures before February 22nd and only 1100 so far. Please forward far and wide. Oppose HR3699, the Research Works Act HR 3699, the Research Works Act will be detrimental to the free flow of scientific information that was created using Federal funds. It is an attempt to put federally funded scientific information behind pay-walls, and confer the ownership of the information to a private entity. This is an affront to open government and open access to information created using public funds. This link gets you to the petition: https://wwws.whitehouse.gov/petitions#!/petition/oppose-hr3699-research-works-act/vKMhCX9k -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] choice of wavelength
No impact ? Longer wavelength more absorption more damage. But between the choices given no problem. Spread of spots might be better with 1.0 versus 0.9 but that depends on your cell and also how big your detector is. Given your current resolution none of the mentioned issues are deal breakers. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Feb 15, 2012, at 18:08, Jacob Keller j-kell...@fsm.northwestern.edu wrote: I would say the better practice would be to collect higher multiplicity/completeness, which should have a great impact on maps. Just watch out for radiation damage though. I think the wavelength will have no impact whatsoever. JPK On Wed, Feb 15, 2012 at 4:23 PM, Seungil Han shan06...@gmail.com wrote: All, I am curious to hear what our CCP4 community thoughts are I have a marginally diffracting protein crystal (3-3.5 Angstrom resolution) and would like to squeeze in a few tenth of angstrom. Given that I am working on crystal quality improvement, would different wavelengths make any difference in resolution, for example 0.9 vs. 1.0 Angstrom at synchrotron? Thanks. Seungil Seungil Han, Ph.D. Pfizer Inc. Eastern Point Road, MS8118W-228 Groton, CT 06340 Tel: 860-686-1788, Fax: 860-686-2095 Email: seungil@pfizer.com -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] choice of wavelength
Well, but there is more scattering with lower energy as well. The salient parameter should probably be scattering per damage. I remember reading some systematic studies a while back in which wavelength choice ended up being insignificant, but perhaps there is more info now, or perhaps I am remembering wrong? Jacob On Wed, Feb 15, 2012 at 5:14 PM, Bosch, Juergen jubo...@jhsph.edu wrote: No impact ? Longer wavelength more absorption more damage. But between the choices given no problem. Spread of spots might be better with 1.0 versus 0.9 but that depends on your cell and also how big your detector is. Given your current resolution none of the mentioned issues are deal breakers. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Feb 15, 2012, at 18:08, Jacob Keller j-kell...@fsm.northwestern.edu wrote: I would say the better practice would be to collect higher multiplicity/completeness, which should have a great impact on maps. Just watch out for radiation damage though. I think the wavelength will have no impact whatsoever. JPK On Wed, Feb 15, 2012 at 4:23 PM, Seungil Han shan06...@gmail.com wrote: All, I am curious to hear what our CCP4 community thoughts are I have a marginally diffracting protein crystal (3-3.5 Angstrom resolution) and would like to squeeze in a few tenth of angstrom. Given that I am working on crystal quality improvement, would different wavelengths make any difference in resolution, for example 0.9 vs. 1.0 Angstrom at synchrotron? Thanks. Seungil Seungil Han, Ph.D. Pfizer Inc. Eastern Point Road, MS8118W-228 Groton, CT 06340 Tel: 860-686-1788, Fax: 860-686-2095 Email: seungil@pfizer.com -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] choice of wavelength
Acta Cryst. (1997). D53, 734-737[ doi:10.1107/S0907444997007233 ] The Ultimate Wavelength for Protein Crystallography? I. Polikarpov, A. Teplyakov and G. Oliva http://scripts.iucr.org/cgi-bin/paper?gr0657 may give some insights. To the OP, have you solved the structure? In some cases, seeing the packing at low resolution can give you ideas on how to change the construct to obtain higher diffracting crystals. F On Feb 15, 2012, at 4:21 PM, Jacob Keller wrote: Well, but there is more scattering with lower energy as well. The salient parameter should probably be scattering per damage. I remember reading some systematic studies a while back in which wavelength choice ended up being insignificant, but perhaps there is more info now, or perhaps I am remembering wrong? Jacob On Wed, Feb 15, 2012 at 5:14 PM, Bosch, Juergen jubo...@jhsph.edu wrote: No impact ? Longer wavelength more absorption more damage. But between the choices given no problem. Spread of spots might be better with 1.0 versus 0.9 but that depends on your cell and also how big your detector is. Given your current resolution none of the mentioned issues are deal breakers. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Feb 15, 2012, at 18:08, Jacob Keller j-kell...@fsm.northwestern.edu wrote: I would say the better practice would be to collect higher multiplicity/completeness, which should have a great impact on maps. Just watch out for radiation damage though. I think the wavelength will have no impact whatsoever. JPK On Wed, Feb 15, 2012 at 4:23 PM, Seungil Han shan06...@gmail.com wrote: All, I am curious to hear what our CCP4 community thoughts are I have a marginally diffracting protein crystal (3-3.5 Angstrom resolution) and would like to squeeze in a few tenth of angstrom. Given that I am working on crystal quality improvement, would different wavelengths make any difference in resolution, for example 0.9 vs. 1.0 Angstrom at synchrotron? Thanks. Seungil Seungil Han, Ph.D. Pfizer Inc. Eastern Point Road, MS8118W-228 Groton, CT 06340 Tel: 860-686-1788, Fax: 860-686-2095 Email: seungil@pfizer.com -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] choice of wavelength
Diffracted intensity goes up by the cube of the wavelength, but so does absorption and I don't know exactly about radiation damage. One interesting point is that on image plate and CCD detectors the signal is also proportional to photon energy, so doubling the wavelength gives 8 times diffraction intensity, but only 4 times the signal on integrating detectors (assuming the full photon energy is captured). So it would be interesting to see how the equation works out on the new counting detectors where the signal does not depend on photon energy. Another point to take into account is that beamlines can have different optimal wavelength ranges. Typically, your beamline guy/gal should be the one to ask. Maybe James Holton will chime in on this. Bart On 12-02-15 04:21 PM, Jacob Keller wrote: Well, but there is more scattering with lower energy as well. The salient parameter should probably be scattering per damage. I remember reading some systematic studies a while back in which wavelength choice ended up being insignificant, but perhaps there is more info now, or perhaps I am remembering wrong? Jacob On Wed, Feb 15, 2012 at 5:14 PM, Bosch, Juergenjubo...@jhsph.edu wrote: No impact ? Longer wavelength more absorption more damage. But between the choices given no problem. Spread of spots might be better with 1.0 versus 0.9 but that depends on your cell and also how big your detector is. Given your current resolution none of the mentioned issues are deal breakers. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Feb 15, 2012, at 18:08, Jacob Kellerj-kell...@fsm.northwestern.edu wrote: I would say the better practice would be to collect higher multiplicity/completeness, which should have a great impact on maps. Just watch out for radiation damage though. I think the wavelength will have no impact whatsoever. JPK On Wed, Feb 15, 2012 at 4:23 PM, Seungil Hanshan06...@gmail.com wrote: All, I am curious to hear what our CCP4 community thoughts are I have a marginally diffracting protein crystal (3-3.5 Angstrom resolution) and would like to squeeze in a few tenth of angstrom. Given that I am working on crystal quality improvement, would different wavelengths make any difference in resolution, for example 0.9 vs. 1.0 Angstrom at synchrotron? Thanks. Seungil Seungil Han, Ph.D. Pfizer Inc. Eastern Point Road, MS8118W-228 Groton, CT 06340 Tel: 860-686-1788, Fax: 860-686-2095 Email: seungil@pfizer.com -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] All-D World
Well if you think about it technically God produced plants within three days which if far too little work for a thesis. Maybe it could count as preliminary results for one aim of a thesis proposal so it might be enough to get PhD candidacy depending on how demanding your committee is. You could always propose the remaining 5999 years and 362 days to do a massive data mining initiative. Thanks for the laugh. I needed it. Katherine On Wed, Feb 15, 2012 at 4:48 PM, William G. Scott wgsc...@ucsc.edu wrote: Hi Jacob: After giving this a great deal of reflection ….. I realized that you would face the same paradox that God had to resolve six thousand years ago at the Dawn of Creation, i.e., He needed D-deoxyribose DNA to code for L-amino acid proteins, and vice versa. Likewise, you would probably be faced with a situation where you need L-deoxyribose DNA to code for D-amino acid proteins, so once again, you need a ribozyme self-replicase to escape the Irreducible Complexity(™). (The Central Dogma at least is achiral.) At least it can be done six thousand years, which isn't unreasonable for a Ph.D. thesis project (especially when combined with an M.D.), and you, unlike Him, have access to a Sigma catalogue. All the best, Bill William G. Scott Professor Department of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA 228 Sinsheimer Laboratories University of California at Santa Cruz Santa Cruz, California 95064 USA On Feb 15, 2012, at 10:28 AM, Jacob Keller wrote: So who out there wants to start an all-D microbial culture by total synthesis, a la the bacterium with the synthetic genome a while back? Could it work, I wonder? I guess that would be a certain benchmark for Man's conquest of nature. JPK ps maybe if there is a broadly-acting amino-acid isomerase or set of isomerases of appropriate properties, this could be helpful for getting the culture started--or even for preying on the L world? On Wed, Feb 15, 2012 at 12:17 PM, David Schuller dj...@cornell.edu wrote: On 02/15/12 12:41, Jacob Keller wrote: Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted? JPK What do you mean by Out There? If you mean in the PDB, then yes. As of two weeks ago, there are ~ 14 racemic structures deposited; most in space group P -1, with one outlier in space group I -4 C 2. This includes RNA, DNA, and PNA, but 6 entries are actually protein. The longest is over 80 residues. Theoretically, enantiomer-specific catalysis ought to be inverted, but most of the structures solved are not enzymes. kaliotoxin, plectasin, antifreeze protein, monellin, villin, and a designed peptide. On the other hand, if by out there you meant in nature outside of biochemistry and organic chemistry labs; then no, I am not aware of any all-D proteins. There are a few protein/peptides which include a small number of D-residues, which is marked up to nonribosomal synthesis. The first paper I managed to Google: http://jb.asm.org/content/185/24/7036.full Learning from Nature's Drug Factories: Nonribosomal Synthesis of Macrocyclic Peptides doi: 10.1128/JB.185.24.7036-7043.2003 J. Bacteriol. December 2003 vol. 185 no. 24 7036-7043 If racemic crystallization isn't exciting enough for you, look into quasi-racemic crystallization. On Wed, Feb 15, 2012 at 8:05 AM, David Schuller dj...@cornell.edu wrote: Wukovitz Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly. On 02/14/12 18:36, Prem Kaushal wrote: Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] All-D World
G-d is right-handed, so to speak: Ex 15:6 Thy right hand, O LORD, is become glorious in power: thy right hand, O LORD, hath dashed in pieces the enemy. Since we are made in His image, and our (chiral) molecules are the cause of making most of us right-handed, which enantiomer to use was not a real choice but rather flowed logically from His (right-handed) Essence. Our chirality is dictated by His, whatever that means! JPK On Wed, Feb 15, 2012 at 4:48 PM, William G. Scott wgsc...@ucsc.edu wrote: Hi Jacob: After giving this a great deal of reflection ….. I realized that you would face the same paradox that God had to resolve six thousand years ago at the Dawn of Creation, i.e., He needed D-deoxyribose DNA to code for L-amino acid proteins, and vice versa. Likewise, you would probably be faced with a situation where you need L-deoxyribose DNA to code for D-amino acid proteins, so once again, you need a ribozyme self-replicase to escape the Irreducible Complexity(™). (The Central Dogma at least is achiral.) At least it can be done six thousand years, which isn't unreasonable for a Ph.D. thesis project (especially when combined with an M.D.), and you, unlike Him, have access to a Sigma catalogue. All the best, Bill William G. Scott Professor Department of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA 228 Sinsheimer Laboratories University of California at Santa Cruz Santa Cruz, California 95064 USA On Feb 15, 2012, at 10:28 AM, Jacob Keller wrote: So who out there wants to start an all-D microbial culture by total synthesis, a la the bacterium with the synthetic genome a while back? Could it work, I wonder? I guess that would be a certain benchmark for Man's conquest of nature. JPK ps maybe if there is a broadly-acting amino-acid isomerase or set of isomerases of appropriate properties, this could be helpful for getting the culture started--or even for preying on the L world? On Wed, Feb 15, 2012 at 12:17 PM, David Schuller dj...@cornell.edu wrote: On 02/15/12 12:41, Jacob Keller wrote: Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted? JPK What do you mean by Out There? If you mean in the PDB, then yes. As of two weeks ago, there are ~ 14 racemic structures deposited; most in space group P -1, with one outlier in space group I -4 C 2. This includes RNA, DNA, and PNA, but 6 entries are actually protein. The longest is over 80 residues. Theoretically, enantiomer-specific catalysis ought to be inverted, but most of the structures solved are not enzymes. kaliotoxin, plectasin, antifreeze protein, monellin, villin, and a designed peptide. On the other hand, if by out there you meant in nature outside of biochemistry and organic chemistry labs; then no, I am not aware of any all-D proteins. There are a few protein/peptides which include a small number of D-residues, which is marked up to nonribosomal synthesis. The first paper I managed to Google: http://jb.asm.org/content/185/24/7036.full Learning from Nature's Drug Factories: Nonribosomal Synthesis of Macrocyclic Peptides doi: 10.1128/JB.185.24.7036-7043.2003 J. Bacteriol. December 2003 vol. 185 no. 24 7036-7043 If racemic crystallization isn't exciting enough for you, look into quasi-racemic crystallization. On Wed, Feb 15, 2012 at 8:05 AM, David Schuller dj...@cornell.edu wrote: Wukovitz Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067 predicts that the most probable space group for macromolecular crystallization is P -1 (P 1-bar). All you have to do to try it out is synthesize the all-D enantiomer of your protein and get it to fold properly. On 02/14/12 18:36, Prem Kaushal wrote: Hi We have a protein that crystallized in P21212 space group. We are looking for some different crystal forms. We tried few things did not work. Now we are thinking to mutate surface residues. Anybody aware of any software which can predict the mutations that might help in crystallizing protein in different space group, please inform me. Thanks in advance Prem -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu
Re: [ccp4bb] All-D World
Jacob, I wish it were that cheery. Do not forget the darker side of history. The prefix L- stands for levorotary. The levo comes from the Latin wording for left side. Left handedness is also known as sinistrality, from the Latin sinistra which also meant the left side, but over time took on the connotations that we currently associate with the word sinister. The latter word, of course, is generally associated with dark and evil. It is therefore erroneous to attribute the L amino acid to the Almighty. The L amino acid is in fact a diabolical corruption of cellular processes that begin with the D-nucleotide (D- meaning rotating to the right, but derived from dexter, meaning dextrous and skillful). The instrument which causes this perversion of God's perfect righteousness into a sign of evil deserves our strongest moral condemnation... I am referring, of course, to that devilish piece of cellular machinery known as the ribosome. The discovery of the ribosome was a significant blow to the success of what Charles Baudelaire famously called the devil's greatest trick. For years now, his acolytes have attempted to hide the truth about the ribosome by referring to its work with the neutral, innocent-sounding phrase translation. Don't be fooled, but instead pray for the development of the next generation of ribosome inhibitors, or at least dissolve the current generation in holy water before ingesting. -Eric On Feb 15, 2012, at 7:24 PM, Jacob Keller wrote: G-d is right-handed, so to speak: Ex 15:6 Thy right hand, O LORD, is become glorious in power: thy right hand, O LORD, hath dashed in pieces the enemy. Since we are made in His image, and our (chiral) molecules are the cause of making most of us right-handed, which enantiomer to use was not a real choice but rather flowed logically from His (right-handed) Essence. Our chirality is dictated by His, whatever that means! JPK On Wed, Feb 15, 2012 at 4:48 PM, William G. Scott wgsc...@ucsc.edu wrote: Hi Jacob: After giving this a great deal of reflection ….. I realized that you would face the same paradox that God had to resolve six thousand years ago at the Dawn of Creation, i.e., He needed D-deoxyribose DNA to code for L-amino acid proteins, and vice versa. Likewise, you would probably be faced with a situation where you need L-deoxyribose DNA to code for D-amino acid proteins, so once again, you need a ribozyme self-replicase to escape the Irreducible Complexity(™). (The Central Dogma at least is achiral.) At least it can be done six thousand years, which isn't unreasonable for a Ph.D. thesis project (especially when combined with an M.D.), and you, unlike Him, have access to a Sigma catalogue. All the best, Bill William G. Scott Professor Department of Chemistry and Biochemistry and The Center for the Molecular Biology of RNA 228 Sinsheimer Laboratories University of California at Santa Cruz Santa Cruz, California 95064 USA On Feb 15, 2012, at 10:28 AM, Jacob Keller wrote: So who out there wants to start an all-D microbial culture by total synthesis, a la the bacterium with the synthetic genome a while back? Could it work, I wonder? I guess that would be a certain benchmark for Man's conquest of nature. JPK ps maybe if there is a broadly-acting amino-acid isomerase or set of isomerases of appropriate properties, this could be helpful for getting the culture started--or even for preying on the L world? On Wed, Feb 15, 2012 at 12:17 PM, David Schuller dj...@cornell.edu wrote: On 02/15/12 12:41, Jacob Keller wrote: Are there any all-D proteins out there, of known structure or otherwise? If so, do enantiomer-specific catalyses become inverted? JPK What do you mean by Out There? If you mean in the PDB, then yes. As of two weeks ago, there are ~ 14 racemic structures deposited; most in space group P -1, with one outlier in space group I -4 C 2. This includes RNA, DNA, and PNA, but 6 entries are actually protein. The longest is over 80 residues. Theoretically, enantiomer-specific catalysis ought to be inverted, but most of the structures solved are not enzymes. kaliotoxin, plectasin, antifreeze protein, monellin, villin, and a designed peptide. On the other hand, if by out there you meant in nature outside of biochemistry and organic chemistry labs; then no, I am not aware of any all-D proteins. There are a few protein/peptides which include a small number of D-residues, which is marked up to nonribosomal synthesis. The first paper I managed to Google: http://jb.asm.org/content/185/24/7036.full Learning from Nature's Drug Factories: Nonribosomal Synthesis of Macrocyclic Peptides doi: 10.1128/JB.185.24.7036-7043.2003 J. Bacteriol. December 2003 vol. 185 no. 24 7036-7043 If racemic crystallization isn't exciting enough for you, look into quasi-racemic crystallization. On Wed, Feb 15, 2012 at 8:05 AM,
Re: [ccp4bb] DNA in coot
Hi Lisa, Please go check your PDB file. Are those bases written out like DT or THY or Td. Coot recognizes certain format for DNA bases but I forgot which one coot likes. I don't have my laptop with me right now. My guess would be Td. :) Best, Xun On Wednesday, February 15, 2012, LISA science...@gmail.com wrote: Hi all, I am refining a structue of protein-DNA complex with coot. I add DNA by adding ideal DNA/RNA in the other model. But I cannot edit chi angle of these nucletide, neither the mutate. When I press the mutate and my DNA, coot give amino acid not nucletide. Why? Thanks Lisa -- Department of Molecular and Structural Biochemistry North Carolina State University
Re: [ccp4bb] DNA length for crystallization
Hi Lisa, I will second James' suggestion. DNA packing seems really important, and making the DNA length as X half turns is usually good for packing (X=2, 3, 4 ...). Another thing you might want to try is Hoogstein base pairing. Cheers, Xun On Wednesday, February 15, 2012, James Stroud xtald...@gmail.com wrote: Use 5' overhangs of two and make the DNA 10, 11, 15, 20, 21 25, 26, 30, or 31 bases in length. Count the overhangs in the length. If you don't know where to start, try 15, 25, and 26 first because they will make 2(1) screws, which are good for crystals. James On Feb 15, 2012, at 1:06 AM, LISA wrote: Hi all, I have a DNA binding protein. I can get crystals when I mix 8-28 nt dsDNA with my protein. But neither of them has good diffraction. Some biochemical data said the longer of DNA, the tigher of the binding betwwen DNA and my protein. The binding is not sequence-specfic. Does anyone have suggestion of the optimization? What is the good length of DNA for crystallization? Thank you. Lisa -- Department of Molecular and Structural Biochemistry North Carolina State University
Re: [ccp4bb] DNA in coot
El 16/02/12 05:06, Xun Lu escribió: Hi Lisa, Please go check your PDB file. Are those bases written out like DT or THY or Td. Coot recognizes certain format for DNA bases but I forgot which one coot likes. I don't have my laptop with me right now. My guess would be Td. :) Best, Xun Hi, And that is compounded with the fact that depending on your installation, Coot may be using its own libraries or the CCP4 ones. And they may differ, especially if you're using the new dictionnaries for refmac5. Plus the fact that depending on your preferences Coot converts atoms to PDB v.2.x so they may not come back as you gave them to it. A mess... Paul, at the very least, it would be helpful if like in Lisa's case, an ideal DNA/RNA is created consistent with whatever libraries Coot is going to use for real space refining it. Best regards, -- Miguel On Wednesday, February 15, 2012, LISA science...@gmail.com mailto:science...@gmail.com wrote: Hi all, I am refining a structue of protein-DNA complex with coot. I add DNA by adding ideal DNA/RNA in the other model. But I cannot edit chi angle of these nucletide, neither the mutate. When I press the mutate and my DNA, coot give amino acid not nucletide. Why? Thanks Lisa -- Department of Molecular and Structural Biochemistry North Carolina State University -- Miguel Architecture et Fonction des Macromolécules Biologiques (UMR6098) CNRS, Universités d'Aix-Marseille I II Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France Tel: +33(0) 491 82 55 93 Fax: +33(0) 491 26 67 20 mailto:miguel.ortiz-lombar...@afmb.univ-mrs.fr http://www.afmb.univ-mrs.fr/Miguel-Ortiz-Lombardia
