[ccp4bb] X-ray crystallography manager position - The Institute of Cancer Research
The Institute of Cancer Research (University of London) Section of Structural Biology, Chelsea, London X-RAY CRYSTALLOGRAPHY MANAGER The Institute of Cancer Research (a College of the University of London) is a world-class cancer research organisation. In the 2008 Higher Education Funding Councils' Research Assessment Exercise, The ICR received the highest percentage of research assessed as four stars, world leading, in the areas of biological sciences and cancer studies. We came top of the Times Higher Education overall table of HEIs that submitted to more than one UoA - ranking it as the UK's leading academic research centre. Research in the Section of Structural Biology is aimed at understanding the structural basis for the function and regulation of proteins and complexes implicated in cancer. Our research programmes are complemented by close links with groups elsewhere in The Institute, and provide the mechanistic and structural framework for developing new therapeutics targeted at cancer. The Section is well equipped for structural biology with state of the art X-ray equipment, robotics and computational infrastructure. We seek to appoint an X-ray Crystallography Manager to facilitate the research programmes of the Section of Structural Biology and other groups within the Institute, particularly the Section of Cancer Therapeutics. The key roles of the post will be (i) to take direct responsibility for the overall crystallographic and crystallisation infrastructure of the Section, (ii) to coordinate and conduct crystallographic data collection experiments at synchrotron radiation sources, (iii) to assist PhD students and post-doctoral scientists to determine structures using X-ray crystallography, and (iv) contribute to the scientific output of the Section through protein crystal structure determination and analysis. The successful applicant will hold a PhD (or equivalent) in protein crystallography and have considerable post-doctoral experience. The position is non-timed limited, and the appointment will be at the Senior Scientific Officer level with a starting salary in the range £37,241 to £44,336 p.a. with a start date of 1st July or as soon as possible thereafter. Appointment will require clear evidence of contribution to published work. Informal enquires may be made to the Section Chairman, Professor David Barford (david.barf...@icr.ac.uk). Details of the Sections’ research activities can be found on http://www.icr.ac.uk/research/research_sections/structural_biology/index.shtml Please do not send your application direct to Professor Barford, CVs must be submitted in line with the instructions below. For further particulars and details of how to apply, please visit our website at www.icr.ac.uk. Alternatively you may call our 24 hour recruitment line on 020 7153 5475 quoting reference number C356. Closing date: 15th June 2010 __ Professor David Barford Section of Structural Biology Institute of Cancer Research Chester Beatty Laboratories 237 Fulham Road London, SW3 6JB United Kingdom Tel. +44 (0)20 7153 5420 FAX +44 (0)20 7153 5457 PA (Sonia Malkani) +44 (0)20 7153 5443 E. mail: david.barf...@icr.ac.uk http://www.icr.ac.uk/research/research_sections/structural_biology/teams/barford_team/index.shtml The Institute of Cancer Research: Royal Cancer Hospital, a charitable Company Limited by Guarantee, Registered in England under Company No. 534147 with its Registered Office at 123 Old Brompton Road, London SW7 3RP. This e-mail message is confidential and for use by the addressee only. If the message is received by anyone other than the addressee, please return the message to the sender by replying to it and then delete the message from your computer and network.
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
Dear Vinson, I would agree with you on choice B. There are probably many ways to look at it. Here are two that come to me at the moment. 1. If the reaction is reversible, then there's no opportunity to put energy into the system to reduce its overall entropy. So a reversible epimerase would be like a Maxwell's demon, violating the second law of thermodynamics. 2. Reversible reactions obey the principle of microscopic reversibility, i.e. the reaction mechanism and the transition states are the same in both directions. There's no way for an enzyme to selectively reduce the transition state barrier going in one direction but not the other. Regards, Randy Read On 18 May 2010, at 08:31, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the ΔG of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
Barring some grammatical errors, you've pretty much summed it up. James On May 18, 2010, at 12:31 AM, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the ΔG of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson
Re: [ccp4bb] different compilers for ccp4 code
Hi Francois configure linux_intel_compilers should do the trick. We distribute ccp4 built with the intel compilers for OS X. Part of this is that the macs cover a much smaller range of cpus than linux boxes, so optimisation is less of a problem. If you want speed make sure that you are compiling for your architecture. My feeling, and I haven't checked this for some time, is that the intel compilers are a bit faster, 5-10% max, but that the gnu compilers have been closing the gap. A pointed out by the previous post the intel compilers basically have an assert that goes if CPU==AMD then stop. A number of years ago somebody published how to remove this from the code, and more recently intel lost a restrictive practice law case over it. So hopefully it will go away. Charles Ballard CCP4 ps- a lot of ccp4 is highly io dependent, so fast disks with decent cache can make a lot of difference On 18 May 2010, at 01:58, Francois Berenger wrote: Hello, This is not what you asked for, but I think it is good to know: Intel compilers don't seem to like non-Intel CPUs: http://www.agner.org/optimize/blog/read.php?i=49 Regards, F. Terry Lang wrote: Hey Everyone, I am considering switching from gcc to the Intel compiler in the hopes of making some of calculations run a bit faster. Has anyone ever tried compiling the ccp4 code base with the Intel compilers? Is there a difference in speed? What about in the reproducibility of the calculations? Any changes in statistics? Any information would be greatly appreciated! Thanks, Terry
[ccp4bb] Cut ligand or keep in whole piece when only part of it can be resolved
Hi everyone, I have two types of ligands cocrystallized with my protein, ligand 1 and ligand 2. I am only interested in resolving ligand 2. In my electron density map, only part of ligand 1 can be identified. Is it ok for me to cut ligand 1 so only the identifiable part is left or should I keep the whole piece? Thanks in advance. Jay
Re: [ccp4bb] updated mtz file or original one in refmac5
I just checked a recent refmac job and it seems that in the output mtz the Fobs has indeed changed. what's more interesting, the number of missing reflections has changed too (disturbingly, it decreased so that the dataset looks more complete 97.07% to 97.17% in this case). But if the same overall anisotropic B scaling is done every time, there seems to be no harm, right? Ed. On Tue, 2010-05-18 at 05:10 +0100, Frank von Delft wrote: Hi Jay No, don't use the new one: the F's in there have been scaled by the overall anisotropic B-factors. (At least, they used to be, a few years ago.) Definitely go with the old one, every time. The output mtz has the coefficients for the maps, that's all. Cheers phx. On 17/05/2010 18:26, Ian Tickle wrote: Hi Jay I always use the original, I only use the new one for maps deposition of Fcalc etc. But I don't think it does any harm to use the new one, all the info is copied over. HTH. Cheers -- Ian On Mon, May 17, 2010 at 5:25 PM, Jay Panccp4p...@gmail.com wrote: Hello every one, I am just starting to use refmac to do refinement. There is an mtz output file each time. Should I use this one for further refinement or should I use the original mtz file (the one after scaling)? Thanks. Jay -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] updated mtz file or original one in refmac5
The decrease in missing reflections are due to the fact that the output file does not include the missing reflections that are lower resolution than the lowest resolution observed reflection. Thus, this file is no longer uniqueified and then refmac reports a higher completeness since it no longer counts the missing low resolution reflections as missing. In addition, if you are using experimental phase restraints, these data columns are not included in the output. I would recommend always using the same input data file for each round of refinement. Regards, Mitch -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ed Pozharski Sent: Tuesday, May 18, 2010 6:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] updated mtz file or original one in refmac5 I just checked a recent refmac job and it seems that in the output mtz the Fobs has indeed changed. what's more interesting, the number of missing reflections has changed too (disturbingly, it decreased so that the dataset looks more complete 97.07% to 97.17% in this case). But if the same overall anisotropic B scaling is done every time, there seems to be no harm, right? Ed. On Tue, 2010-05-18 at 05:10 +0100, Frank von Delft wrote: Hi Jay No, don't use the new one: the F's in there have been scaled by the overall anisotropic B-factors. (At least, they used to be, a few years ago.) Definitely go with the old one, every time. The output mtz has the coefficients for the maps, that's all. Cheers phx. On 17/05/2010 18:26, Ian Tickle wrote: Hi Jay I always use the original, I only use the new one for maps deposition of Fcalc etc. But I don't think it does any harm to use the new one, all the info is copied over. HTH. Cheers -- Ian On Mon, May 17, 2010 at 5:25 PM, Jay Panccp4p...@gmail.com wrote: Hello every one, I am just starting to use refmac to do refinement. There is an mtz output file each time. Should I use this one for further refinement or should I use the original mtz file (the one after scaling)? Thanks. Jay -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] Antibody digest with Ficin
Hi All, Thanks to all that replied for the advice, suggestions etc. about my antibody. I will definitely try the suggestions. Christine From: Sheemei Lok [mailto:sheeme...@yahoo.com.sg] Sent: Monday, May 17, 2010 9:56 PM To: Harman, Christine Subject: Re: [ccp4bb] Antibody digest with Ficin Hi, I included 1mM DTT in the buffer and it got rid of the non-specific disulphide bridges. sheemei From: Harman, Christine christine.har...@fda.hhs.gov To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, 17 May 2010 15:37:54 Subject: [ccp4bb] Antibody digest with Ficin Hi All, I figured there are plenty of Fab/peptide structures out there that I may find a crystallographer/antibody expert out there who can give me some advice. I am interested in obtaining a mFab/peptide complex structure. I have reached the stage where I have successfully produced Fab from mouse IgG1 using immobilized ficin (Kit from Pierce). After ficin digest I purify the Fab with protein A column ( included in kit from Pierce) and it appears relatively pure with minor contamination of F(ab')2 and possibly some minor amount of fragmented Fc. I planned on doing IEC or SEC but FPLC was down and couldn't proceed to next step immediately. I concentrated/buffer exchanged my Fab fraction in PBS and stored at 4ºC over the weekend. The problem arose when I ran a gel I noticed that my Fab fraction which previously (sampled before diafiltration) ran ~50kDa band was now running as small smear around 100kDa. I have a suspicion that the concentrating and resuspending (for buffer exchange) may have some role and what might have happened could be related to possibly non-specific disulfide formation; however, was hoping someone may have some ideas or may have experienced this before. Given my antibody subtype being G1, I used ficin as opposed to papain and my experience is limited in knowing the types of fragments that are generated with ficin. Pierce claims their ficin kit produces Fab and F(ab')2; however, I was wondering if ficin may also produce Fab' given that at different concentrations of cysteine either Fab or F(ab')2 can be generated. Any thoughts, comments or ideas would be much appreciated. Much Thanks, Christine Harman
Re: [ccp4bb] updated mtz file or original one in refmac5
On Tue, 2010-05-18 at 07:03 -0700, Miller, Mitchell D. wrote: The decrease in missing reflections are due to the fact that the output file does not include the missing reflections that are lower resolution than the lowest resolution observed reflection. Thus, this file is no longer uniqueified and then refmac reports a higher completeness since it no longer counts the missing low resolution reflections as missing. In addition, if you are using experimental phase restraints, these data columns are not included in the output. I would recommend always using the same input data file for each round of refinement. Hmm... The listed resolution limits are the same. Using the original mtz file in every round is of course the best (and in fact with ccp4i it takes an extra effort to do otherwise. At least for me, the Re-run job button fan :)) -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
[ccp4bb] complex
Hello I am trying to complex two proteins of 57 and 18 kda and using pET 22b as an expression system. I purified both with His tag and the purity is quite nice after Ni-NTA (Buffer is Tris,Nacl and Imidazole). After purification with Ni_NTA i tried to improve my protein purity using gel filteration(superdex-200). I successfully got the 57 Kda with a high purity (Buffer is Tris and nacl) but with the smaller protein i didnt get anything after the column. when i tried dialysis to remove imidazole from this 18kda protein it degraded.i tried protease inhibitors as well but still the protein is not stabilised. Can anyone suggest me something to get out from this. I am stuck at this stage. suggestion are highly welcomed and appreciated. -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL
Re: [ccp4bb] complex
For gel exclusion chromatography it is generally adsvisable to include 100 mM NaCl or equivalent ionic strength in the elution buffer to suppress nonspecific adsorption. In addition the pH and ionic strength of the elution buffer should be compatible with your protein folding stability. It is possible that your protein requires significant ionic strength to remain soluble or properly folded. It seems likely you lost protein in GEC due to adsorption or precipitation. Try adjusting the pH and ionic strength of the elution/dialysis buffer appropriately. It is unlikely you are getting proteolysis in a highly purified protein sample. Cheers, Roger Rowlett Professor Dept. of Chemistry Colgate University On 5/18/10, intekhab alam faisal...@gmail.com wrote: Hello I am trying to complex two proteins of 57 and 18 kda and using pET 22b as an expression system. I purified both with His tag and the purity is quite nice after Ni-NTA (Buffer is Tris,Nacl and Imidazole). After purification with Ni_NTA i tried to improve my protein purity using gel filteration(superdex-200). I successfully got the 57 Kda with a high purity (Buffer is Tris and nacl) but with the smaller protein i didnt get anything after the column. when i tried dialysis to remove imidazole from this 18kda protein it degraded.i tried protease inhibitors as well but still the protein is not stabilised. Can anyone suggest me something to get out from this. I am stuck at this stage. suggestion are highly welcomed and appreciated. -- INTEKHAB ALAM LABORATORY OF STRUCTURAL BIOINFORMATICS KOREA UNIVERSITY, SEOUL -- Sent from my mobile device
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
I think that it's possible to do a mutation that affects only one way of the reaction. You can mutate a residue that makes contacts only with the product of the direct way or only of the reverse way. Maia Randy Read wrote: Dear Vinson, I would agree with you on choice B. There are probably many ways to look at it. Here are two that come to me at the moment. 1. If the reaction is reversible, then there's no opportunity to put energy into the system to reduce its overall entropy. So a reversible epimerase would be like a Maxwell's demon, violating the second law of thermodynamics. 2. Reversible reactions obey the principle of microscopic reversibility, i.e. the reaction mechanism and the transition states are the same in both directions. There's no way for an enzyme to selectively reduce the transition state barrier going in one direction but not the other. Regards, Randy Read On 18 May 2010, at 08:31, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the ΔG of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk mailto:rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
You could make use of product binding energy to drive the reaction forward while the substrate/product is bound to the enzyme. But enzymes that pull that trick are barely enzymes - they stay stuck to the first product they make until something else uses some energy to release it. You can't change the equilibrium constant of the overall reaction - that would violate basic thermodynamics. This sounds a lot like a homework question I would assign my undergrads! Phoebe Original message Date: Tue, 18 May 2010 09:39:49 -0600 From: Maia Cherney ch...@ualberta.ca Subject: Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one? To: CCP4BB@JISCMAIL.AC.UK I think that it's possible to do a mutation that affects only one way of the reaction. You can mutate a residue that makes contacts only with the product of the direct way or only of the reverse way. Maia Randy Read wrote: Dear Vinson, I would agree with you on choice B. There are probably many ways to look at it. Here are two that come to me at the moment. 1. If the reaction is reversible, then there's no opportunity to put energy into the system to reduce its overall entropy. So a reversible epimerase would be like a Maxwell's demon, violating the second law of thermodynamics. 2. Reversible reactions obey the principle of microscopic reversibility, i.e. the reaction mechanism and the transition states are the same in both directions. There's no way for an enzyme to selectively reduce the transition state barrier going in one direction but not the other. Regards, Randy Read On 18 May 2010, at 08:31, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the ΔG of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson -- Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical Research Tel: + 44 1223 336500 Wellcome Trust/MRC Building Fax: + 44 1223 336827 Hills RoadE-mail: rj...@cam.ac.uk mailto:rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
[ccp4bb] CryoEM postdoc position available
POSTDOCTORAL FELLOW/RESEARCH ASSOCIATE POSITION UNIV. OF PITTSBURGH A postdoctoral fellow/research associate position is available immediately in the laboratory of Dr. Zhang at the University of Pittsburgh for up to 4 years funded by an NIH grant. The research project focuses on structural characterization of HIV-1 antivirals in complex with HIV-1 capsid assemblies (Byeon et. al 2009, Cell, 139, 780-790) using cryo-electron microscopy (cryoEM) and cryo-electron tomography. The successful candidates will learn and be expected to responsible for the following: * High resolution imaging using cryoEM * Image processing and 3D reconstruction * Structural analysis and molecular modeling * Publishing research papers and reports Motivated candidates with strong interest in cryoEM and solid background in biophysics/physics or material sciences and engineering are all encouraged to apply. Research experience in HRTEM, cryoEM and image processing is plus. Interested candidates should send an application, including a CV, areas of expertise and interests, publications list, and names and contact information for 3 references to Dr. Peijun Zhang at p...@pitt.edu. Our laboratory is within the Dept. of Structural Biology, fully equipped for all aspects of molecular biology and protein biochemistry and state-of-the-art electron microscopes, including a FEI Polara 300kv FEG with liquid helium specimen stage and a TF20 FEG, both with high-resolution 4kx4k CCD cameras, a T12spirit with 2kx2k camera, and FEI vitrobot for cryoEM specimen preparation. The laboratory also has access to the protein expression and purification facilities, state-of-the-art NMR spectrometers and X-ray crystallography instrumentation, imaging and computing resources within the department. For more details about our research program, please refer to our website: http://www.structbio.pitt.edu/drupal-5/?q=peijun-zhang Pittsburgh is an excellent working and living environment for individuals or families, and a competitive stipend package will be provided. Necessary visa application materials will be provided for foreign applicants. --- Peijun Zhang, PhD Dept. of Structural Biology University of Pittsburgh School of Medicine 3501 5th Ave, Pittsburgh, PA 15260 (412)383-5907 (phone) (412)648-9008 (fax) p...@pitt.edu http://www.structbio.pitt.edu/webusers/pzhang/
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
Hi, I'm more of a Fourier coefficient kind of guy, but I thought that a ΔG of zero simply corresponded to an equilibrium constant of one. You can certainly have reversible reactions with other equilibrium constants. In fact I think irreversible reactions are simply ones where the equilibrium constant is so far to one side that, in practice, the reaction always goes all the way to product. As Randy pointed out the enzyme cannot change the ΔG (or the equilibrium constant). You could drive a reaction out of equilibrium by coupling it to some other reaction which itself is way out of equilibrium (such as ATP hydrolysis in the cell) but I don't think that's a simple mutation of your enzyme. ;-) Dale Tronrud On 05/18/10 00:31, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the ΔG of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
Vinson, As Dale and Randy pointed out, you cannot change the ΔG of a reaction by mutation: enzyme, which is a catalyst, affects only the activation barrier (ΔE double-dagger). You can just make it a better (or worse) catalyst which would allow the reaction to flow faster (or slower) towards equilibrium. Nature solves this problem very elegantly by taking a readily reversible enzyme, like an epimerase or isomerase, and coupling it to a much less reversible reaction which removes product quickly. Hence, the mass action is only in one direction. An example of such an arrangement is the triose phosphate isomerase (TIM)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction pair. TIM is readily reversible (DHA = G3P), but G3P is rapidly converted to 1,3-diphosphoglycerate by GAPDH. The oxidation and phosphorylation reactions of GAPDH now make TIM work in one direction. Since many epimerases are very optimized enzymes, why not consider making a fusion with a second enzyme (like a reductase) to make the system flow in one direction. Of course, this depends on what you want to do with the product. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On May 18, 2010, at 11:54 AM, Dale Tronrud wrote: Hi, I'm more of a Fourier coefficient kind of guy, but I thought that a ΔG of zero simply corresponded to an equilibrium constant of one. You can certainly have reversible reactions with other equilibrium constants. In fact I think irreversible reactions are simply ones where the equilibrium constant is so far to one side that, in practice, the reaction always goes all the way to product. As Randy pointed out the enzyme cannot change the ΔG (or the equilibrium constant). You could drive a reaction out of equilibrium by coupling it to some other reaction which itself is way out of equilibrium (such as ATP hydrolysis in the cell) but I don't think that's a simple mutation of your enzyme. ;-) Dale Tronrud On 05/18/10 00:31, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the ΔG of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
If you change the reaction rate in one direction 1000 times slower than in the other direction, then the reaction becomes practically irreversible. And the system might not be at equilibrium. Maia R. M. Garavito wrote: Vinson, As Dale and Randy pointed out, you cannot change the ΔG of a reaction by mutation: enzyme, which is a catalyst, affects only the activation barrier (ΔE double-dagger). You can just make it a better (or worse) catalyst which would allow the reaction to flow faster (or slower) towards equilibrium. Nature solves this problem very elegantly by taking a readily reversible enzyme, like an epimerase or isomerase, and coupling it to a much less reversible reaction which removes product quickly. Hence, the mass action is only in one direction. An example of such an arrangement is the triose phosphate isomerase (TIM)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction pair. TIM is readily reversible (DHA = G3P), but G3P is rapidly converted to 1,3-diphosphoglycerate by GAPDH. The oxidation and phosphorylation reactions of GAPDH now make TIM work in one direction. Since many epimerases are very optimized enzymes, why not consider making a fusion with a second enzyme (like a reductase) to make the system flow in one direction. Of course, this depends on what you want to do with the product. Cheers, Michael // /R. Michael Garavito, Ph.D./ /Professor of Biochemistry Molecular Biology/ /513 Biochemistry Bldg. / /Michigan State University / /East Lansing, MI 48824-1319/ /Office:// //(517) 355-9724 Lab: (517) 353-9125/ /FAX: (517) 353-9334Email: rmgarav...@gmail.com mailto:garav...@gmail.com/ // On May 18, 2010, at 11:54 AM, Dale Tronrud wrote: Hi, I'm more of a Fourier coefficient kind of guy, but I thought that a ΔG of zero simply corresponded to an equilibrium constant of one. You can certainly have reversible reactions with other equilibrium constants. In fact I think irreversible reactions are simply ones where the equilibrium constant is so far to one side that, in practice, the reaction always goes all the way to product. As Randy pointed out the enzyme cannot change the ΔG (or the equilibrium constant). You could drive a reaction out of equilibrium by coupling it to some other reaction which itself is way out of equilibrium (such as ATP hydrolysis in the cell) but I don't think that's a simple mutation of your enzyme. ;-) Dale Tronrud On 05/18/10 00:31, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the ΔG of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson
Re: [ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
Sounds like a good explanation. Thank you. Maia Dale Tronrud wrote: If you change the reaction rate in one direction 1000 times slower then the reaction rate in the other direction will also be 1000 times slower and the equilibrium will be in exactly the same place. You can't make the transition state less stable when approached from the left without making it less stable when approached from the right. Dale Tronrud On 05/18/10 12:34, Maia Cherney wrote: If you change the reaction rate in one direction 1000 times slower than in the other direction, then the reaction becomes practically irreversible. And the system might not be at equilibrium. Maia R. M. Garavito wrote: Vinson, As Dale and Randy pointed out, you cannot change the ΔG of a reaction by mutation: enzyme, which is a catalyst, affects only the activation barrier (ΔE double-dagger). You can just make it a better (or worse) catalyst which would allow the reaction to flow faster (or slower) towards equilibrium. Nature solves this problem very elegantly by taking a readily reversible enzyme, like an epimerase or isomerase, and coupling it to a much less reversible reaction which removes product quickly. Hence, the mass action is only in one direction. An example of such an arrangement is the triose phosphate isomerase (TIM)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction pair. TIM is readily reversible (DHA = G3P), but G3P is rapidly converted to 1,3-diphosphoglycerate by GAPDH. The oxidation and phosphorylation reactions of GAPDH now make TIM work in one direction. Since many epimerases are very optimized enzymes, why not consider making a fusion with a second enzyme (like a reductase) to make the system flow in one direction. Of course, this depends on what you want to do with the product. Cheers, Michael // /R. Michael Garavito, Ph.D./ /Professor of Biochemistry Molecular Biology/ /513 Biochemistry Bldg. / /Michigan State University / /East Lansing, MI 48824-1319/ /Office:// //(517) 355-9724 Lab: (517) 353-9125/ /FAX: (517) 353-9334Email: rmgarav...@gmail.com mailto:garav...@gmail.com/ // On May 18, 2010, at 11:54 AM, Dale Tronrud wrote: Hi, I'm more of a Fourier coefficient kind of guy, but I thought that a ΔG of zero simply corresponded to an equilibrium constant of one. You can certainly have reversible reactions with other equilibrium constants. In fact I think irreversible reactions are simply ones where the equilibrium constant is so far to one side that, in practice, the reaction always goes all the way to product. As Randy pointed out the enzyme cannot change the ΔG (or the equilibrium constant). You could drive a reaction out of equilibrium by coupling it to some other reaction which itself is way out of equilibrium (such as ATP hydrolysis in the cell) but I don't think that's a simple mutation of your enzyme. ;-) Dale Tronrud On 05/18/10 00:31, Vinson LIANG wrote: Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the ΔG of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson
[ccp4bb] Native Gel Theory and Practice
Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Kabat antibody numbering
Hi there CCP4ers, I need to number a PDB file following the Kabat numbering. Is there any script to do this numbering automatically? Thanks a lot in advanced. Best, Ariel -- * Ariel Talavera Pérez, PhD Nanobiology Group Center of Molecular Immunology calle 216 esq. A 15, Atabey, Playa Havana 11600, CUBA tel: (53-7) 214 3178 fax: (53-7) 272 0644 email: talav...@cim.sld.cu *
[ccp4bb] Should I be worried about negative electron density?
Hello Everyone, I have a reasonably well fitted electron density map through molecular replacement. However, there is always some red region left no matter how hard I tried when the mtz file is loaded into Coot. Is this because my model is still not good enough or it’s natural to most model fittings. In another word, should I be worried about the red region? Thanks in advance. Jay
Re: [ccp4bb] Should I be worried about negative electron density?
On May 18, 2010, at 4:13 PM, Jay Pan wrote: Hello Everyone, I have a reasonably well fitted electron density map through molecular replacement. However, there is always some red region left no matter how hard I tried when the mtz file is loaded into Coot. Is this because my model is still not good enough or it’s natural to most model fittings. Negative density (with absolute value above 2 sigma) sitting on atoms is much more of a concern than random negative peaks floating in the solvent breeze, as it indicates misplacement of the model. In another word, should I be worried about the red region? Thanks in advance. I've alway found it very helpful to worry about absolutely everything obsessively to the point where it causes endoderm to bleed. Which is why, I suppose, Paul chose red. Jay
[ccp4bb] electron microscopy: where open access fails
Dear colleagues, whereas data sharing for most crystallographers appears to be a no-brainer, making coordinates and (most of the time, hopefully) structure factors available, it seems the electron microscopists are drastically lagging behind when it comes to making data available. Many cryoEM structures are still being published without the corresponding maps being deposited in the EM database. In one particular case, I was interested in looking at a cryoEM map from a paper published in a well-renowned open access journal starting. The paper contains the EMDB accession codes for the maps, but these maps appear to be 'on hold' since over a year. Enquiry with the authors delivered a firm 'no' in releasing the maps: they claim it is OK to keep the maps on hold for 2 years, simply because the EMDB gives the option to do so. A further enquiry with the journal editors led to no avail: despite the clear journal policy on sharing both manuscripts and data, they were also unable to force the authors to release their maps, now ~13 months after publication of the paper. The fact that this was in an open access journal makes this all the more shocking. It is striking to see how much still has to be done to lift the cryoEM world up to the same standards as the crystallographic community (when it comes to sharing data, at least). Structures can simply be published without anybody being able to check the validity, let alone use it for obvious experiments such as docking crystal structures, integrative protein structure modeling, etc. With many structural targets going towards bigger and more challenging, combining cryoEM data with crystal structures is going to become more and more important. What can we, crystallographers, do to stimulate data-sharing in the cryoEM world? (My apologies to the cryoEM people on this bulletin board: if you have been making your maps available, you'll agree that clearly not everybody does...) Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] electron microscopy: where open access fails
When X-ray crystallographers not so many years ago thought they are the salt of the earth, of science and then some (well, some believe so to this date), the same attitude prevailed (data holds). Once every person with access to Google does it, that secrecy slowly disappears. What also helps is a few major fups, which promotes community response by those who have nothing to hide. Fups tend to happen at the transition from expert science to Google science. EM is probably not there yet. So, talk to you colleagues, question every structure, and make the argument that science without data is non-falsifiable in Popper's sense and thus en par with quackery and superstition. That should make you a lot of friends (or at least it will draw a response). Cheers, BR From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Filip Van Petegem Sent: Tuesday, May 18, 2010 4:48 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] electron microscopy: where open access fails Dear colleagues, whereas data sharing for most crystallographers appears to be a no-brainer, making coordinates and (most of the time, hopefully) structure factors available, it seems the electron microscopists are drastically lagging behind when it comes to making data available. Many cryoEM structures are still being published without the corresponding maps being deposited in the EM database. In one particular case, I was interested in looking at a cryoEM map from a paper published in a well-renowned open access journal starting. The paper contains the EMDB accession codes for the maps, but these maps appear to be 'on hold' since over a year. Enquiry with the authors delivered a firm 'no' in releasing the maps: they claim it is OK to keep the maps on hold for 2 years, simply because the EMDB gives the option to do so. A further enquiry with the journal editors led to no avail: despite the clear journal policy on sharing both manuscripts and data, they were also unable to force the authors to release their maps, now ~13 months after publication of the paper. The fact that this was in an open access journal makes this all the more shocking. It is striking to see how much still has to be done to lift the cryoEM world up to the same standards as the crystallographic community (when it comes to sharing data, at least). Structures can simply be published without anybody being able to check the validity, let alone use it for obvious experiments such as docking crystal structures, integrative protein structure modeling, etc. With many structural targets going towards bigger and more challenging, combining cryoEM data with crystal structures is going to become more and more important. What can we, crystallographers, do to stimulate data-sharing in the cryoEM world? (My apologies to the cryoEM people on this bulletin board: if you have been making your maps available, you'll agree that clearly not everybody does...) Filip Van Petegem -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
Re: [ccp4bb] Native Gel Theory and Practice
Dear Jacob, I offer you my opinion. Are you talking about electrophoresis? As far as I know it does not work for the mass. The velocity of a protein depends on the charge at a particular pH, the mass and shape of molecules etc. It's very difficult to take all these things into consideration. Otherwise this would be a very convenient method, much easier than the analytical centrifugation or gel-filtration that are usually used. However, electrophoresis does not work for mass determination. Besides, complex formation hugely depends on the protein concentration. If you dilute your mixture, your complexes might dissociate. There is equilibrium constant between different types of complexes. Maia Jacob Keller wrote: Dear Crystallographers, I am trying to optimize a native gel experiment of a two-protein complex, running the smallest-detectable amount of protein component A with varying amounts of component B. MWCharge MW/Charge A 22 -5-4308 B 17-24 -702 This experiment is partly to determine stoichiometry, but also to determine roughly the strength of the interaction. B definitely runs much faster than A alone, as predicted, but I am wondering what to expect with various oligomers. Should ABB run faster or slower than AB? What about AABB? Theoretically, AA should certainly run slower than A, and BB slower than B, simply because the mass/charge ratio is the same, but the overall mass is greater. But what happens when you have AAB, for example? There must be an equation relating the mass/charge and mass (and perhaps gel percentage) to the speed traveled in the gel--but what is the equation? Thanks for your consideration, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Is it possible to mutate a reversible epimerase into an inreversible one?
Dear all, Sorry for this silly biochemistory question. Thing is that I have a reversible epimerase and I want to mutate it into an inreversible one. However, I have been told that the ΔG of a reversible reaction is zero. Which direction the reaction goes depends only on the concentration of the substrate. So the conclusion is, A: I can mutate the epimerase into an inreversible one. But it has no influence on the reaction direction, and hence it has little mean. B: There is no way to change a reversible epimerase into an inversible one. Could somebody please give me some comment on the two conclution? Thank you all for your time. Best, Vinson
