Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
Hi all, I would like to ask some questions regarding this thread.. 1) What is exactly meant by "Fourier transformed electron density"?- according to my knowldege performing a fourier transform on the electron density gives you the structure factor back. So, how does it related to what Prof. James H called "non-lattice-convoluted pattern"? It will be really nice if somebody can explain the thing in a " decoded" language?! And also any articles focusing on the concepts discussed in the entire thread will be very helpful Regards, ARKO On Sat, Jan 14, 2012 at 12:42 AM, Dale Tronrud wrote: > I think you have to be a little more clear as to what you mean > by an "electron density map". If you mean our usual maps that we > calculate all the time the Patterson map is just the usual Patterson > map. It also repeats to infinity, with the infinitely long Patterson > vectors (infinitely high frequency components) being required to > create the Bragg peaks. If you mean an electron density map of a > single object with finite bounds your Patterson map will also have > finite bounds, just with twice the radius. > > The Patterson boundary is not a sharp drop-off because there aren't > as many long vectors as short ones, but the distribution depends on > the exact shape of your object. Once you have a Patterson map that > has an isolated edge (no cross-vectors) back calculating the original > object is pretty easy. (Miao, et al, Annu. Rev. Phys. Chem. 2008, > 59:387-410) > > Dale Tronrud > > On 01/13/12 10:54, Jacob Keller wrote: > > I am trying to think, then, what would the Patterson map of a > > Fourier-transformed electron density map look like? Would you get the > > shape/outline of the object, then a sharp drop-off, presumably? Is > > this used to orient molecules in single-particle FEL diffraction > > experiments? > > > > JPK > > > > On Fri, Jan 13, 2012 at 12:33 PM, Dale Tronrud > > wrote: > >> > >> > >> On 01/13/12 09:53, Jacob Keller wrote: > >>> No, I meant the non-lattice-convoluted pattern--the pattern arising > >>> from the Fourier-transformed electron density map--which would > >>> necessarily become more complicated with larger molecular size, as > >>> there is more information to encode. I think this will manifest in > >>> what James H called a smaller "grain size." > >> > >> I've been thinking about these matters recently and had a nifty > >> insight about exactly this matter. (While this idea is new to me > >> I doubt it is new for others.) > >> > >> The lower limit to the size of the features in one of these > >> "scattergrams" is indicated by the scattergram's highest frequency > >> Fourier component. Its Fourier transform is the Patterson map. > >> While we usually think of the Patterson map as describing interatomic > >> vectors, it is also the frequency space for the diffraction pattern. > >> For a noncrystalline object the highest frequency component corresponds > >> to the longest Patterson vector or, in other words, the diameter of > >> the object! The bigger the object, the higher the highest frequency > >> of the scattergram, and the smaller its features. > >> > >> Dale Tronrud > >> > >>> > >>> JPK > >>> > >>> On Fri, Jan 13, 2012 at 11:41 AM, Yuri Pompeu > wrote: > to echo Tim's question: > If by pattern you mean the position of the spots on the film, I dont > think they would change based on the complexity of the macromolecule being > studied. As far I know it, the position of the spots are dictated by the > reciprocal lattice points > (therefore the real crystal lattice) (no?) > The intensity will, obviously, vary dramatically... > ps. Very interesting (cool) images James!!! > >>> > >>> > >>> > > > > > > > -- *ARKA CHAKRABORTY* *CAS in Crystallography and Biophysics* *University of Madras* *Chennai,India*
Re: [ccp4bb] on the electronic density of several maps
Fenghui, What is your resolution? If your having trouble distinguishing between pro and leu I am guessing it is worse than 2.8 A. You may not be able to model side chains confidently with lower resolution data. You may have to make a call on wether or not to model side chains, and if your model is interesting enough to pursue even without side chains.
[ccp4bb] 5BrU
Does anyone have the dictionary file for BrU, as in the brominated ribonucleotide? The distributed files include the deoxy version, 5-bromo-2'-deoxyuridine-5'-monophosphate. Best wishes, Pete
Re: [ccp4bb] on the electronic density of several maps
You might want to look at some images of side chain electron density. http://www.ruppweb.org/garland/gallery/Ch2/index_2.htm BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dialing Pretty Sent: Friday, January 13, 2012 2:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] on the electronic density of several maps Dear All, For the electronic density of LEU and Pro in the electronic density map, which is much stronger? For the electronic density of LEU and Lys in the electronic density map, which is much stronger? The reason I ask the above questions is I need to distinguish them in the electronic density map. I am looking forward to getting your reply. Fenghui
Re: [ccp4bb] on the electronic density of several maps
It's not the strength of the electron density it's the shape that is important. Dale Tronrud On 01/13/12 14:21, Dialing Pretty wrote: > > Dear All, > > For the electronic density of LEU and Pro in the electronic density map, > which is much stronger? > > For the electronic density of LEU and Lys in the electronic density map, > which is much stronger? > > The reason I ask the above questions is I need to distinguish them in > the electronic density map. > > I am looking forward to getting your reply. > > Fenghui > > > >
[ccp4bb] on the electronic density of several maps
Dear All, For the electronic density of LEU and Pro in the electronic density map, which is much stronger? For the electronic density of LEU and Lys in the electronic density map, which is much stronger? The reason I ask the above questions is I need to distinguish them in the electronic density map. I am looking forward to getting your reply. Fenghui
Re: [ccp4bb] RMSD of side chains
On Fri, 2012-01-13 at 10:40 -0800, Ethan Merritt wrote: > Which of these two statements would be more useful: > 1) The RMSD for sidechain atoms between apo and holo was 0.678 Å. > or > 2) Only two residues exhibited a significant change of > conformation: Perhaps the same is true for the backbone. RMSD is uninterpretable in general, since one collapses information about the distribution of positional shifts of hundreds of atoms into one number. On the other hand, looking at phi/psi angles helps to pinpoint the regions that undergo conformational changes. -- Hurry up, before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] RMSD of side chains
> Let me put it this way. Suppose you were reading a paper about someone > else's structures. Which of these two statements would be more useful: > 1) The RMSD for sidechain atoms between apo and holo was 0.678 Å. > or > 2) Only two residues exhibited a significant change of conformation: >the Asn XXX carboxamide flipped 180 degrees allowing ND to act as >H-bond donor to ligand atom FOO; the Lys YYY sidechain occluded >the ligand binding site in the apo structure but extends into the >solvent when the ligand is bound. Certainly, in a paper, specific and detailed information regarding differences in particular side chain conformations would be more useful and biologically relevant than merely quoting an overall global measure of side chain dissimilarities. I am sure that no-one would think of making standalone statements such as "the average RMSD of side chain atoms is 0.678Å". However, it can be useful to calculate side chain RMSD for various purposes, whether in a publication, or simply for personal use during the course of a comparative structural analysis. It is more meaningful if side chain RMSD is calculated using a local coordinate frame (i.e. after local superposition), resulting in a measure which is independent of global conformation. This is only meaningful in cases where the backbone of the compared structures is locally very similar. For example, if this is performed for sequence-identical structures in different global conformations, then it is possible to use local side chain RMSD to identify and visualise the side chain conformational (dis)similarity between corresponding residues. Such information can be hard for humans to otherwise discern visually, since our interpretation is not independent of the superposition used to visualise the structures. Such information allows us to easily tell whether observed differences are due to differences in the backbone conformation, or differences in side chains, and can be easily visualised by colouring residues according to the side chain dissimilarity score. Certainly, if structures are practically near-identical in net local backbone structure, then interesting information can be achieved by moving to the higher level of structural resolution of side chain atoms. Furthermore, if wanting to compare a class of structures, all of which have highly conserved backbones, information regarding the overall similarity of side chain conformations may help in identifying or quantifying intraclass (dis)similarities. > For residues where the two sequences are > not identical, how do you even calculate an RMSD for sidechain atoms? For sure, comparing side chains becomes less meaningful if the sequences are not identical - this would not be useful in most cases. Nevertheless, if the backbone is practically identical then some researchers performing structural comparisons may want to achieve some way of visualising differences in general side chain orientations. This can be achieved by considering the difference between average positions of side chain atoms between the compared residues, after local backbone superposition. Whilst interpretation would have to be thought about very carefully, such analyses can provide useful information that would be very hard for us to otherwise obtain (e.g. regarding side chain signalling patterns). Considering side chain RMSD requires careful consideration, but can provide useful information. Regards Rob On 13 Jan 2012, at 18:40, Ethan Merritt wrote: > On Friday, January 13, 2012 09:07:07 am Appu kumar wrote: >> Firstly thanks to Robert Nicholls for making me aware of the software >> necessary for side chain RMSD calculation. I have installed and now going >> through manual to use it for exploiting the structural differences. Thanks >> a lot. >> >> Secondly, for Ethan Merritt, I am seeking the information for comparing the >> side chains RMSD for better comparison of structure. Please correct me if i >> am wrong, i want to elaborate more on what i am thinking. If we have refine >> the structure well so that issue of rotamers are fixed > > Sorry, I don't know what you mean when you say "the issue of rotamers are > fixed". > >> , then it is >> possible to take the advantage of side chain orientation for correctly >> understanding the trivial differences between homologous proteins and such >> differences harbouring good piece information for understanding protein >> structure-function relationship. Any kind of suggestion would be highly >> appreciated. > > Let me put it this way. Suppose you were reading a paper about someone > else's structures. Which of these two statements would be more useful: > 1) The RMSD for sidechain atoms between apo and holo was 0.678 Å. > or > 2) Only two residues exhibited a significant change of conformation: > the Asn XXX carboxamide flipped 180 degrees allowing ND to act as > H-bond donor to ligand atom FOO; the Lys Y
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
I think you have to be a little more clear as to what you mean by an "electron density map". If you mean our usual maps that we calculate all the time the Patterson map is just the usual Patterson map. It also repeats to infinity, with the infinitely long Patterson vectors (infinitely high frequency components) being required to create the Bragg peaks. If you mean an electron density map of a single object with finite bounds your Patterson map will also have finite bounds, just with twice the radius. The Patterson boundary is not a sharp drop-off because there aren't as many long vectors as short ones, but the distribution depends on the exact shape of your object. Once you have a Patterson map that has an isolated edge (no cross-vectors) back calculating the original object is pretty easy. (Miao, et al, Annu. Rev. Phys. Chem. 2008, 59:387-410) Dale Tronrud On 01/13/12 10:54, Jacob Keller wrote: > I am trying to think, then, what would the Patterson map of a > Fourier-transformed electron density map look like? Would you get the > shape/outline of the object, then a sharp drop-off, presumably? Is > this used to orient molecules in single-particle FEL diffraction > experiments? > > JPK > > On Fri, Jan 13, 2012 at 12:33 PM, Dale Tronrud > wrote: >> >> >> On 01/13/12 09:53, Jacob Keller wrote: >>> No, I meant the non-lattice-convoluted pattern--the pattern arising >>> from the Fourier-transformed electron density map--which would >>> necessarily become more complicated with larger molecular size, as >>> there is more information to encode. I think this will manifest in >>> what James H called a smaller "grain size." >> >> I've been thinking about these matters recently and had a nifty >> insight about exactly this matter. (While this idea is new to me >> I doubt it is new for others.) >> >> The lower limit to the size of the features in one of these >> "scattergrams" is indicated by the scattergram's highest frequency >> Fourier component. Its Fourier transform is the Patterson map. >> While we usually think of the Patterson map as describing interatomic >> vectors, it is also the frequency space for the diffraction pattern. >> For a noncrystalline object the highest frequency component corresponds >> to the longest Patterson vector or, in other words, the diameter of >> the object! The bigger the object, the higher the highest frequency >> of the scattergram, and the smaller its features. >> >> Dale Tronrud >> >>> >>> JPK >>> >>> On Fri, Jan 13, 2012 at 11:41 AM, Yuri Pompeu wrote: to echo Tim's question: If by pattern you mean the position of the spots on the film, I dont think they would change based on the complexity of the macromolecule being studied. As far I know it, the position of the spots are dictated by the reciprocal lattice points (therefore the real crystal lattice) (no?) The intensity will, obviously, vary dramatically... ps. Very interesting (cool) images James!!! >>> >>> >>> > > >
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
I am trying to think, then, what would the Patterson map of a Fourier-transformed electron density map look like? Would you get the shape/outline of the object, then a sharp drop-off, presumably? Is this used to orient molecules in single-particle FEL diffraction experiments? JPK On Fri, Jan 13, 2012 at 12:33 PM, Dale Tronrud wrote: > > > On 01/13/12 09:53, Jacob Keller wrote: >> No, I meant the non-lattice-convoluted pattern--the pattern arising >> from the Fourier-transformed electron density map--which would >> necessarily become more complicated with larger molecular size, as >> there is more information to encode. I think this will manifest in >> what James H called a smaller "grain size." > > I've been thinking about these matters recently and had a nifty > insight about exactly this matter. (While this idea is new to me > I doubt it is new for others.) > > The lower limit to the size of the features in one of these > "scattergrams" is indicated by the scattergram's highest frequency > Fourier component. Its Fourier transform is the Patterson map. > While we usually think of the Patterson map as describing interatomic > vectors, it is also the frequency space for the diffraction pattern. > For a noncrystalline object the highest frequency component corresponds > to the longest Patterson vector or, in other words, the diameter of > the object! The bigger the object, the higher the highest frequency > of the scattergram, and the smaller its features. > > Dale Tronrud > >> >> JPK >> >> On Fri, Jan 13, 2012 at 11:41 AM, Yuri Pompeu wrote: >>> to echo Tim's question: >>> If by pattern you mean the position of the spots on the film, I dont think >>> they would change based on the complexity of the macromolecule being >>> studied. As far I know it, the position of the spots are dictated by the >>> reciprocal lattice points >>> (therefore the real crystal lattice) (no?) >>> The intensity will, obviously, vary dramatically... >>> ps. Very interesting (cool) images James!!! >> >> >> -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] RMSD of side chains
On Friday, January 13, 2012 09:07:07 am Appu kumar wrote: > Firstly thanks to Robert Nicholls for making me aware of the software > necessary for side chain RMSD calculation. I have installed and now going > through manual to use it for exploiting the structural differences. Thanks > a lot. > > Secondly, for Ethan Merritt, I am seeking the information for comparing the > side chains RMSD for better comparison of structure. Please correct me if i > am wrong, i want to elaborate more on what i am thinking. If we have refine > the structure well so that issue of rotamers are fixed Sorry, I don't know what you mean when you say "the issue of rotamers are fixed". > , then it is > possible to take the advantage of side chain orientation for correctly > understanding the trivial differences between homologous proteins and such > differences harbouring good piece information for understanding protein > structure-function relationship. Any kind of suggestion would be highly > appreciated. Let me put it this way. Suppose you were reading a paper about someone else's structures. Which of these two statements would be more useful: 1) The RMSD for sidechain atoms between apo and holo was 0.678 Å. or 2) Only two residues exhibited a significant change of conformation: the Asn XXX carboxamide flipped 180 degrees allowing ND to act as H-bond donor to ligand atom FOO; the Lys YYY sidechain occluded the ligand binding site in the apo structure but extends into the solvent when the ligand is bound. Your comparison apparently involves a pair of homologs rather than a pair of holo/apo structures, but I suggest to you that RMSD is even more useless in this case. For residues where the two sequences are not identical, how do you even calculate an RMSD for sidechain atoms? Ethan > > Thank you > Appu > > On 13 January 2012 21:53, Ethan Merritt wrote: > > > On Friday, 13 January 2012, Appu kumar wrote: > > > Dear ccp4 users, > > >Would you please guide me how to calculate > > > the RMSD of side chains alone without considering C-alpha backbone. > > > Is/are there any program/programs availble which do this job. I want > > > to know the RMSD of side chains for protein comparison. > > > > What is the question that you are trying to answer? > > If you are going to disregard the mainchain position, then > > I would guess that you'd be better off comparing rotamer > > classes than comparing coordinates. > > > >Ethan > > > > > > > > > > Thank you in advance. > > > Appu > > > > > > > > -- Ethan A Merritt Biomolecular Structure Center, K-428 Health Sciences Bldg University of Washington, Seattle 98195-7742
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
On 01/13/12 09:53, Jacob Keller wrote: > No, I meant the non-lattice-convoluted pattern--the pattern arising > from the Fourier-transformed electron density map--which would > necessarily become more complicated with larger molecular size, as > there is more information to encode. I think this will manifest in > what James H called a smaller "grain size." I've been thinking about these matters recently and had a nifty insight about exactly this matter. (While this idea is new to me I doubt it is new for others.) The lower limit to the size of the features in one of these "scattergrams" is indicated by the scattergram's highest frequency Fourier component. Its Fourier transform is the Patterson map. While we usually think of the Patterson map as describing interatomic vectors, it is also the frequency space for the diffraction pattern. For a noncrystalline object the highest frequency component corresponds to the longest Patterson vector or, in other words, the diameter of the object! The bigger the object, the higher the highest frequency of the scattergram, and the smaller its features. Dale Tronrud > > JPK > > On Fri, Jan 13, 2012 at 11:41 AM, Yuri Pompeu wrote: >> to echo Tim's question: >> If by pattern you mean the position of the spots on the film, I dont think >> they would change based on the complexity of the macromolecule being >> studied. As far I know it, the position of the spots are dictated by the >> reciprocal lattice points >> (therefore the real crystal lattice) (no?) >> The intensity will, obviously, vary dramatically... >> ps. Very interesting (cool) images James!!! > > >
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
No, I meant the non-lattice-convoluted pattern--the pattern arising from the Fourier-transformed electron density map--which would necessarily become more complicated with larger molecular size, as there is more information to encode. I think this will manifest in what James H called a smaller "grain size." JPK On Fri, Jan 13, 2012 at 11:41 AM, Yuri Pompeu wrote: > to echo Tim's question: > If by pattern you mean the position of the spots on the film, I dont think > they would change based on the complexity of the macromolecule being studied. > As far I know it, the position of the spots are dictated by the reciprocal > lattice points > (therefore the real crystal lattice) (no?) > The intensity will, obviously, vary dramatically... > ps. Very interesting (cool) images James!!! -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
to echo Tim's question: If by pattern you mean the position of the spots on the film, I dont think they would change based on the complexity of the macromolecule being studied. As far I know it, the position of the spots are dictated by the reciprocal lattice points (therefore the real crystal lattice) (no?) The intensity will, obviously, vary dramatically... ps. Very interesting (cool) images James!!!
[ccp4bb] Crystallographer Position at Evotec
Crystallographer X-Ray Crystallographers within our Structural Biology Department Salary: £23,000 - £39,000 + benefits Location: Oxfordshire, UK Full time; Permanent Evotec (UK) Ltd is currently seeking X-Ray Crystallographers for our Structural Biology Department based in Oxfordshire. X-Ray Crystallography works closely with our Discovery Chemistry Department and with clients to develop novel small molecule drugs. The group is at the forefront of new science and technology, and is seeking to expand as business need grows. The successful candidates will be part of the Structural Biology group responsible for expression, purification, crystallisation and structure determination of proteins and protein-ligand complexes. Knowledge of state of the art crystallographic methods is a must, along with skills in molecular biology and protein purification. Expertise in protein-ligand complex crystallisation problems would be an advantage though not essential. You will have excellent written and verbal communication skills and will be a strong team player. Dynamic and innovative, you will be a self-starter with a flexible approach who enjoys a challenge. You will be PhD qualified or equivalent in Chemistry, Biochemistry, Molecular Biology or Biophysics, with experience in protein X-ray crystallography. Candidates will also be considered who have a Masters degree with extensive experience or equivalent. We offer competitive salaries plus extensive benefits including annual bonus, pension plan, private medical and dental cover. If you feel that your skills and experience match what we are looking for, please apply by emailing your CV, a brief statement of research accomplishments and interests to humanresources-abing...@evotec.com or to post it to: Evotec (UK) Ltd Human Resources 114 Milton Park Abingdon Oxfordshire OX14 4SA United Kingdom Evotec (UK) Ltd is a limited company registered in England and Wales. Registration number:2674265. Registered office: 114 Milton Park, Abingdon, Oxfordshire, OX14 4SA, United Kingdom.
Re: [ccp4bb] RMSD of side chains
Firstly thanks to Robert Nicholls for making me aware of the software necessary for side chain RMSD calculation. I have installed and now going through manual to use it for exploiting the structural differences. Thanks a lot. Secondly, for Ethan Merritt, I am seeking the information for comparing the side chains RMSD for better comparison of structure. Please correct me if i am wrong, i want to elaborate more on what i am thinking. If we have refine the structure well so that issue of rotamers are fixed, then it is possible to take the advantage of side chain orientation for correctly understanding the trivial differences between homologous proteins and such differences harbouring good piece information for understanding protein structure-function relationship. Any kind of suggestion would be highly appreciated. Thank you Appu On 13 January 2012 21:53, Ethan Merritt wrote: > On Friday, 13 January 2012, Appu kumar wrote: > > Dear ccp4 users, > >Would you please guide me how to calculate > > the RMSD of side chains alone without considering C-alpha backbone. > > Is/are there any program/programs availble which do this job. I want > > to know the RMSD of side chains for protein comparison. > > What is the question that you are trying to answer? > If you are going to disregard the mainchain position, then > I would guess that you'd be better off comparing rotamer > classes than comparing coordinates. > >Ethan > > > > > > Thank you in advance. > > Appu > > > >
Re: [ccp4bb] RMSD of side chains
On Friday, 13 January 2012, Appu kumar wrote: > Dear ccp4 users, >Would you please guide me how to calculate > the RMSD of side chains alone without considering C-alpha backbone. > Is/are there any program/programs availble which do this job. I want > to know the RMSD of side chains for protein comparison. What is the question that you are trying to answer? If you are going to disregard the mainchain position, then I would guess that you'd be better off comparing rotamer classes than comparing coordinates. Ethan > > Thank you in advance. > Appu >
[ccp4bb] Job posts - ICR, London, UK
Dear CCP4bb readers, a postdoctoral position and a scientific officer / higher scientific officer position are available at The Institute of Cancer Research (ICR, Chelsea, London, UK), to undertakes crystallographic, single particle electron microscopy analysis and biochemical analysis of large eukaryotic transcriptional complexes. We are particularly interested in studying macromolecular complexes that are involved in cancer development and several projects (which require different skills) are available. The Division of Structural Biology of the ICR has managed facilities for protein crystallography (Bruker Microstar and CCD detector and crystallisation robots), cryo-electron microscopy (FEI Tecnai F20 and T12), and protein production with expertise in multi-subunit expression (insect cell, yeast and bacterial expression including a 60 L fermentor). The Division is also well equipped with equipment for biophysical analysis (e.g. ITC, fluorescence, multi-angle light scattering). Applicants for the postdoctoral position should possess a PhD (or equivalent) in biochemistry or molecular biology with a sound knowledge of protein purification for structural biology analysis. Experience in recombinant protein production and purification as well as protein crystallography and/or single particle electron microscopy is essential. Experience in the biochemistry of multi-subunit protein complexes, yeast genetics or protein production in insect cells (MultiBac) would be an advantage. Appointment will be on Fixed Term Contract for 3 years in the first instance, with a starting salary in the range of £27,536 to £33,852 p.a. inclusive (based on previous post-doctoral experience). For further particulars and details of how to apply, please refer to this vacancy on our online application portal: Ref n. 1228522 Applicants for the scientific officer / higher scientific officer should possess a BSc. (or equivalent) in molecular biology or cell biology with sound knowledge of cloning techniques. Experience in molecular cloning and protein expression is essential. Experience in yeast genetics and/or or protein production in insect cells (MultiBac) would be an advantage. Appointment will be on Fixed Term Contract for 3 years in the first instance, with a starting salary in the range of £23,000 to £29,545 p.a. inclusive dependant on skills and experience. In addition to annual performance related pay awards we offer a generous leave entitlement of 25 days annual leave per annum which rises to a maximum of 30 days per annum related to length of service, as well as 8 bank/public holidays and 3 additional ICR-set privilege days per annum. For further particulars and details of how to apply, please refer to this vacancy on our online application portal: Ref n. 1228533 Closing date: 8th February 2012 Please direct informal inquiries to Dr. Alessandro Vannini at alessandro.vann...@icr.ac.uk Please DO NOT send your application to Dr. Alessandro Vannini; CVs must be submitted in line with the instructions above.
Re: [ccp4bb] Metal won't strip from IMAC
Have run into a similar problem. Cleared the background color by running 2M NaOH together with 0.2M EDTA. Better replace BMT with TCEP (1 mM). Also keep in mind that adsorption is pH dependent, that is the higher the pH, the better is adsorption. Many proteins adsorb irreversibly above pH 7.0. If you reduce the pH, say to 5.2-5.5, not only you make adsorption less stronger (hence, column capacity may drop down), but you will at the same time prevent cysteine oxidation. You can also increase [imidazole] in the equilibration buffer to reduce adsorption. HTH, Nadir Pr. Nadir T. Mrabet Structural& Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabet medecine.uhp-nancy.fr On 13/01/2012 01:42, Michael Thompson wrote: Katherine, You are not alone. I have inadvertently destroyed a GE HisTrap column with high concentrations of proteins that contain many exposed cysteines. In my case the Co2+ resin turned a very dark purplish-brown and the protein appeared to have crashed out on the column. I didn't try to strip it, because I figured it was done for anyway, so I can't tell you any more about the problem. Here's how I explained it to myself (whether or not this is actually right I'm not 100% sure, but it makes sense in my head). The columns I was using have a maximum concentration of 5mM for DTT and 10mM for B-mercaptoethanol. So that seems like the column can handle 10mM thiol groups. If you have a protein with many cysteines and it is very highly concentrated (as was the case for me) then you are adding considerably more thiol groups to the solution. This abundance of thiols reduces the metal on the column, and disaster ensues. For me, repeating the same prep with less DTT (3mM vs. 5mM) in the buffer fixed the issue. If you are concerned about your protein oxidizing at lower concentrations of DTT or BME, the other alternative is to switch to TCEP. The IMAC columns can tolerate higher concentrations of TCEP, and it is a far superior reducing agent (more stable, more reductive, etc.)...but also a lot more expensive (although you can get away with using much less because it works so much better). HTH, Mike - Original Message - From: "Katherine Sippel" To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, January 12, 2012 4:01:10 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Metal won't strip from IMAC Hi all, I've run into a bit of a protein purification conundrum and wondered if anyone had encountered a similar situation. I've exercised all of my google-fu and can't find anything. It's a fairly straightforward setup; His-tagged protein and Talon Co2+ resin, load lysate, wash with 5 mM imidazole, elute with 150 mM imidazole. There is protein in the elution fractions as would be expected. The strangeness occurs when I try to regenerate the column. Using the standard protocol of 25 mM MES, 100 mM NaCl pH 5 doesn't change the color of the resin back to light pink the way it should with a regenerated column. I try stripping with the suggested 0.2M EDTA, still pink, 0.5M EDTA, still pink, 8 M urea plus 4% CHAPS and then EDTA, still pink, 1 M NaOH then EDTA, still pink. I've checked the resin using a Western (with a really specific monoclonal Ab) and it seems that my protein has somehow irreversibly bound to the column and is preventing the metal from releasing the sepharose. I've even tried competing the protein off with excess Co2+ and Mg2+ (the endogenous divalent bound cation). Clearly the solution is swapping to a Ni column, but this is really bugging me now. Has anyone run into this problem with IMAC before? Background: The protein does bind divalent cations (Mg and Mn) with low affinity (~1 mM) and has a ridiculous number of cysteines (10 in 416 residues total). There is 1 mM BME and 1 mM MgCl2 in all of the buffers. Thanks, Katherine
Re: [ccp4bb] JLigand distorts molecules
That is interesting. It works for me here and for few other people in other places. Can you exit and restart JLigand, can you send me a figure of what is happening? regards Garib On 13 Jan 2012, at 11:00, Wolfgang Skala wrote: > ccp4 is 6.2.0, > refmac5 is 5.7.0010 (the file you provided; formerly 5.6.0117), > libcheck is 5.2 (formerly 5.1.14), > dictionary is 5.28 > > I also tried each of the four refmac5-libcheck combinations, but without > success. > > yours > Wolfgang Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] Metal won't strip from IMAC
Hi Katherine, I recommend Zn-IDA Sepharose (Chelating Sepharose Fast Flow, GE Healthcare), which we have been using successfully for more than 20 years, since the early days of IMAC: Skerra et al. (1991) The functional expression of antibody Fv fragments in Escherichia coli: improved vectors and a generally applicable purification technique. (Nature) Biotechnology 9, 273-8. This metal/chelate combination has exquisite selectivity for the His6-tag, at least if operated with an imidazole concentration gradient. Importantly, Zn(II) typically forms reversible sulfide complexes and it is not redox-active (in contrast with Co, Cu, Ni)! In deviation of our old protocol I would just recommend to use a concentrated ZnSO4 stock solution (instead of ZnCl2), which is less prone to hydrolysis upon longer storage. Good luck, Arne Am 13.01.2012 um 01:01 schrieb Katherine Sippel: > Hi all, > > I've run into a bit of a protein purification conundrum and wondered if > anyone had encountered a similar situation. I've exercised all of my > google-fu and can't find anything. It's a fairly straightforward setup; > His-tagged protein and Talon Co2+ resin, load lysate, wash with 5 mM > imidazole, elute with 150 mM imidazole. There is protein in the elution > fractions as would be expected. The strangeness occurs when I try to > regenerate the column. Using the standard protocol of 25 mM MES, 100 mM NaCl > pH 5 doesn't change the color of the resin back to light pink the way it > should with a regenerated column. I try stripping with the suggested 0.2M > EDTA, still pink, 0.5M EDTA, still pink, 8 M urea plus 4% CHAPS and then > EDTA, still pink, 1 M NaOH then EDTA, still pink. I've checked the resin > using a Western (with a really specific monoclonal Ab) and it seems that my > protein has somehow irreversibly bound to the column and is preventing the > metal from releasing the sepharose. I've even tried competing the protein off > with excess Co2+ and Mg2+ (the endogenous divalent bound cation). > > Clearly the solution is swapping to a Ni column, but this is really bugging > me now. Has anyone run into this problem with IMAC before? > > Background: The protein does bind divalent cations (Mg and Mn) with low > affinity (~1 mM) and has a ridiculous number of cysteines (10 in 416 residues > total). There is 1 mM BME and 1 mM MgCl2 in all of the buffers. > > Thanks, > > Katherine + Prof. Dr. Arne Skerra + Lehrstuhl f. Biologische Chemie + Technische Universitaet Muenchen Emil-Erlenmeyer-Forum 5 + 85350 Freising-Weihenstephan + Germany Phone: +49 (0)8161 71-4351 + Fax: -4352 http://www.wzw.tum.de/bc + eMail: ske...@tum.de
Re: [ccp4bb] JLigand distorts molecules
ccp4 is 6.2.0, refmac5 is 5.7.0010 (the file you provided; formerly 5.6.0117), libcheck is 5.2 (formerly 5.1.14), dictionary is 5.28 I also tried each of the four refmac5-libcheck combinations, but without success. yours Wolfgang
Re: [ccp4bb] Molprobity Clashscore
Ok, I'm completed baffled... and have obviously started something unintentionally... NB: it was a joke! I was amused that Molprobity, after 'adding' hydrogens to my model, had 'improved' the clashscore of my model by an obviously unnecessary number of decimal places...! [0.0098 point apparently]. Just me apparently. Off to put my head in a cardboard box. T. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 On 1/12/12 11:14 PM, "Tim Fenn" wrote: >On Thu, Jan 12, 2012 at 8:11 AM, Pavel Afonine wrote: >> >>> Who needs hydrogens? >> >> >> may be you need to read this (for example): >> >> http://www.phenix-online.org/papers/dz5209_reprint.pdf >> > >While this reference is useful, it neglects the role of prior chemical >forces (vdW and electrostatics, for example) in positioning hydrogen >atoms. The X-ray/neutron data is often not sufficient to uniquely >define an atomic position (hydrogen or otherwise), which can be >especially problematic for atoms with several degrees of freedom, like >water or a hydroxyl hydrogen. Force fields have come a long way in >defining these forces with reasonable chemical accuracy in the past 10 >years, and there is work to show this does benefit X-ray/neutron >refinement (e.g. http://dx.doi.org/10.1016/j.str.2011.01.015) - >suggesting its worthwhile to include this information in X-ray target >functions. At the very least, it should not be left out of the >discussion, especially when hydrogen atoms are concerned!!! > >Regards, >Tim
Re: [ccp4bb] JLigand distorts molecules
That is interesting. Do you use the latest ccp4 and dictionary that comes with it? What are the version of dictionary, libcheck, refmac Just typing refmac5 -i libcheck -i you should get version information. The version of the dictionary is on the top of a file head $CLIBD_MON/list/mon_lib_list.cif ccp4 version should be 6.2, refmac either 5.6 or after 5.7.007 libcheck 5.2 Dictionary 5.28 or later. regards Garib On 13 Jan 2012, at 10:13, Wolfgang Skala wrote: > Dear Garib, > > thanks for your reply. However, when I use the new versions, 3GP is an > apparently random polyhedron which does not resemble 3'-GMP at all. > > Yours > Wolfgang Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
Re: [ccp4bb] JLigand distorts molecules
Dear Garib, thanks for your reply. However, when I use the new versions, 3GP is an apparently random polyhedron which does not resemble 3'-GMP at all. Yours Wolfgang
[ccp4bb] RMSD of side chains
Dear ccp4 users, Would you please guide me how to calculate the RMSD of side chains alone without considering C-alpha backbone. Is/are there any program/programs availble which do this job. I want to know the RMSD of side chains for protein comparison. Thank you in advance. Appu
Re: [ccp4bb] Lithium versus Sodium
Dear Matt, We were working with Hal2p a lithium inhibited Ins-mono-PPase like protein 10 years ago. We had good biochemistry showing that lithium replaced mg at the active site. We grow crystals in presence of Li and we worked with 1.6 A diffraction data. Unfortunatelly we did not see any electron density peak, however, the "Li" site displayed nice tetrahedral coordination having four oxygen atoms as ligands. Armando El 12/01/2012, a las 18:16, Matthew Franklin escribió: > Hi Ed - > > There was a peak in the difference maps, as I recall. I believe it initially > got built as a water, but that proved to be too many electrons, giving a > negative peak. I removed the water, but it was clear that something needed > to be there, at which point I started casting about for alternative atoms, > and settled on lithium. I'm pretty sure I never tried to put sodium in there > and see if it refined. > > Caveat: this work was all done eight years ago, and I don't have access to > any of the files anymore. So I can't verify any of these statements! > However, I did deposit Fobs for these structures, if you care to make your > own maps... > > I just checked the structure in EDS, and the peak for the lithium is pretty > low, around 0.6 sigma. I would say that it looked better in the maps I was > using. > > Hope that helps, > Matt > > > On 1/12/12 11:22 AM, Ed Pozharski wrote: >> Matt, >> >> thank you, this is an excellent summary. One question remains - the >> lithium peak should be, afaiu, much lower than the water/sodium. Was >> there a peak in difference map or was placement based on identifying >> something that looked like a coordination site? >> >> Cheers, >> >> Ed. >> >> On Thu, 2012-01-12 at 10:23 -0500, Matthew Franklin wrote: >>> On 1/12/12 9:42 AM, Ed Pozharski wrote: On Thu, 2012-01-12 at 09:52 +, Patel, Joe wrote: > Do you have ultra-high resolution? Something I did not…. Are there > many examples in the pdb of proteins with Li+ refined? http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=n/a&template=het2pdb.html¶m1=_LI 39 in total. Some are fairly low resolution (2.8A), and only five are higher than 1.2A. I'd think that placing lithium ion should be based on some extra-crystallographic evidence, plus maybe some structural considerations such as correctly placed coordinating ligands. >>> Since I'm responsible for eight of those structures, I'll just say that >>> I thought fairly hard before building a lithium into that peak, knowing >>> that I couldn't really distinguish it from water or sodium. I was >>> working with a 1.7 A map, and I put the lithium there based on three >>> criteria: >>> >>> - the crystals grew in something like 2 M lithium sulfate, whereas the >>> only source of sodium would have been from the buffer or the protein >>> solution >>> >>> - there were two negatively charged residues coordinating the peak in >>> question, suggesting it was a cation >>> >>> - the bond distances were consistent with lithium coordination, for what >>> that's worth at this resolution >>> >>> That was the first structure (1TW7), and all of the others were treated >>> the same since it was the same crystals soaked with different compounds >>> in the same conditions. >>> >>> - Matt >>> >>> > > > -- > Matthew Franklin, Ph. D. > Senior Research Scientist > New York Structural Biology Center > 89 Convent Avenue, New York, NY 10027 > (646) 275-7165
