[ccp4bb] modified amino acids in the PDB
Dear colleagues, We deposited protein structures with modified lysine side chains and were surprised that the PDB treats the modification as an independent molecule, with a “LINK” record indicating the covalent bond – instead of defining a modified residue (that’s what we had uploaded to the PDB). Apparently, anything attached to an amino acid is considered an independent molecule (and the lysine just called a regular lysine) if it comprises more than 10 atoms (see below for the PDB guidelines). I think that’s kind of arbitrary and would give all modified residue also modified names – i.e. individual names for all modified lysines, as it is done for acetyl- or methyl-lysines, for example. I wonder what other people’s opinion is?! Best regards Clemens This is in accordance to the wwPDB annotation guidelines (http://www.wwpdb.org/procedure.html#toc_2). *Modified amino acids and nucleotides* If an amino acid or nucleotide is modified by a chemical group greater than 10 atoms, the residue will be split into two groups: the amino acid/nucleotide group and the modification. A link record will be generated between the amino acid/nucleotide group and the modification. For modified amino acids and nucleotides that were not split will follow standard atom nomenclature.
[ccp4bb] AW: [ccp4bb] help identifying ligand
Dear Ed, What is the pH of your crystallization buffer? If it is acidic, either the azide or the carboxylate may be protonated. Also the local environment of the carboxylate can make a hugh difference in PKa. You could also use some Bayesian logic: given the elongated linear density, what else of the available components of your crystallization drop would fit? Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward A. Berry Gesendet: Sonntag, 7. Juli 2013 22:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] help identifying ligand In a structure I'm refining, there are a couple of oblong blobs associated with carboxylates. (screenshots at http://sb20.lbl.gov/berry/ccp4/azide/) If I modeled with two waters, they refine too close together for normal H-bond, 2.3 to 2.5 A; and their density is connected. I considered one water with alternate locations, but the distal position wouldn't make much sense if the proximal water wasn't there. The density is the right size for azide, which was present in the medium, but I expect a chemist would find it unreasonable to have anionic azide (pKa of hydrazoic acid ~4.6) associating with a carboxylate. Would that make sense? or does anyone have other suggestions? (resolution is 2.2A, contour 0.25 e/A^3 or about 1.3 sigma) Thanks, Ed
Re: [ccp4bb] AW: [ccp4bb] help identifying ligand
Dear CCP4BB, The most likely components are those at the highest concentration in the crystallization or cryosolution. And a few wild ideas to continue the discussion that is very important as the ligands are always very difficult to identify. Example: If you have 1.5 M ammonium sulfate you should consider hydrated ammonium ions H3O+ + NH3 in equilib. H2O + NH4+ The pH will determine the equilibrium point and NH4+ would be a good ligand for a carboxylate. Assuming 200mM Li2SO4: A lithium ion (H20-Li-H20 with a Li-O distance of 2.14 Ang) Li+ is often associated with more that two H2O molecules with an angle of 105° not 180° but cannot be excluded in proximity of a carboxylate where the environ ment could be distorted (not very believable). (H2O, Na+ and Mg++ 10 electrons) water is always the most probable. 2 H2O in equilib. OH- + H3O+ Carboxylates are often destroyed by radiation damage. The most probable ligand will be at high concentration in the mother liquor the moment the crystal was flashcooled. This is rarely the case for typically 0.02% azide (I would made an exception in proximity to Cu++, Fe++ or Zn++ ions). Azide -N=N+=N- is also suspitious as a negative ion is a bad counterion for a carboxylate. Enrico. On Mon, 08 Jul 2013 11:19:46 +0200, herman.schreu...@sanofi.com wrote: Dear Ed, What is the pH of your crystallization buffer? If it is acidic, either the azide or the carboxylate may be protonated. Also the local environment of the carboxylate can make a hugh difference in PKa. You could also use some Bayesian logic: given the elongated linear density, what else of the available components of your crystallization drop would fit? Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward A. Berry Gesendet: Sonntag, 7. Juli 2013 22:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] help identifying ligand In a structure I'm refining, there are a couple of oblong blobs associated with carboxylates. (screenshots at http://sb20.lbl.gov/berry/ccp4/azide/) If I modeled with two waters, they refine too close together for normal H-bond, 2.3 to 2.5 A; and their density is connected. I considered one water with alternate locations, but the distal position wouldn't make much sense if the proximal water wasn't there. The density is the right size for azide, which was present in the medium, but I expect a chemist would find it unreasonable to have anionic azide (pKa of hydrazoic acid ~4.6) associating with a carboxylate. Would that make sense? or does anyone have other suggestions? (resolution is 2.2A, contour 0.25 e/A^3 or about 1.3 sigma) Thanks, Ed -- Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette, FRANCE http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100sortby=pubdate http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71
Re: [ccp4bb] AW: [ccp4bb] help identifying ligand
Thanks all, the pH is 6.7, azide is 3 mM, and there is no added ammonium. I could get away with modeling as two waters since the separation is well above the 2.2A that gets flagged as a clash in the PDB, still it's close enough to suggest that two waters is not really what's there. Enrico Stura wrote: Dear CCP4BB, The most likely components are those at the highest concentration in the crystallization or cryosolution. And a few wild ideas to continue the discussion that is very important as the ligands are always very difficult to identify. Example: If you have 1.5 M ammonium sulfate you should consider hydrated ammonium ions H3O+ + NH3 in equilib. H2O + NH4+ The pH will determine the equilibrium point and NH4+ would be a good ligand for a carboxylate. Assuming 200mM Li2SO4: A lithium ion (H20-Li-H20 with a Li-O distance of 2.14 Ang) Li+ is often associated with more that two H2O molecules with an angle of 105° not 180° but cannot be excluded in proximity of a carboxylate where the environ ment could be distorted (not very believable). (H2O, Na+ and Mg++ 10 electrons) water is always the most probable. 2 H2O in equilib. OH- + H3O+ Carboxylates are often destroyed by radiation damage. The most probable ligand will be at high concentration in the mother liquor the moment the crystal was flashcooled. This is rarely the case for typically 0.02% azide (I would made an exception in proximity to Cu++, Fe++ or Zn++ ions). Azide -N=N+=N- is also suspitious as a negative ion is a bad counterion for a carboxylate. Enrico. On Mon, 08 Jul 2013 11:19:46 +0200, herman.schreu...@sanofi.com wrote: Dear Ed, What is the pH of your crystallization buffer? If it is acidic, either the azide or the carboxylate may be protonated. Also the local environment of the carboxylate can make a hugh difference in PKa. You could also use some Bayesian logic: given the elongated linear density, what else of the available components of your crystallization drop would fit? Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward A. Berry Gesendet: Sonntag, 7. Juli 2013 22:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] help identifying ligand In a structure I'm refining, there are a couple of oblong blobs associated with carboxylates. (screenshots at http://sb20.lbl.gov/berry/ccp4/azide/) If I modeled with two waters, they refine too close together for normal H-bond, 2.3 to 2.5 A; and their density is connected. I considered one water with alternate locations, but the distal position wouldn't make much sense if the proximal water wasn't there. The density is the right size for azide, which was present in the medium, but I expect a chemist would find it unreasonable to have anionic azide (pKa of hydrazoic acid ~4.6) associating with a carboxylate. Would that make sense? or does anyone have other suggestions? (resolution is 2.2A, contour 0.25 e/A^3 or about 1.3 sigma) Thanks, Ed
[ccp4bb] AW: [ccp4bb] AW: [ccp4bb] help identifying ligand
Dear Ed, For me, 3 mM is a significant concentration. If you have another crystal left, you could transfer it to a storage buffer without azide and collect a data set and see if the density disappears. A very small molecule, non-covalently bound on the outside of the protein should disappear in minutes if the compound is not present in solution. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward A. Berry Gesendet: Montag, 8. Juli 2013 15:31 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] AW: [ccp4bb] help identifying ligand Thanks all, the pH is 6.7, azide is 3 mM, and there is no added ammonium. I could get away with modeling as two waters since the separation is well above the 2.2A that gets flagged as a clash in the PDB, still it's close enough to suggest that two waters is not really what's there. Enrico Stura wrote: Dear CCP4BB, The most likely components are those at the highest concentration in the crystallization or cryosolution. And a few wild ideas to continue the discussion that is very important as the ligands are always very difficult to identify. Example: If you have 1.5 M ammonium sulfate you should consider hydrated ammonium ions H3O+ + NH3 in equilib. H2O + NH4+ The pH will determine the equilibrium point and NH4+ would be a good ligand for a carboxylate. Assuming 200mM Li2SO4: A lithium ion (H20-Li-H20 with a Li-O distance of 2.14 Ang) Li+ is often associated with more that two H2O molecules with an angle of 105° not 180° but cannot be excluded in proximity of a carboxylate where the environ ment could be distorted (not very believable). (H2O, Na+ and Mg++ 10 electrons) water is always the most probable. 2 H2O in equilib. OH- + H3O+ Carboxylates are often destroyed by radiation damage. The most probable ligand will be at high concentration in the mother liquor the moment the crystal was flashcooled. This is rarely the case for typically 0.02% azide (I would made an exception in proximity to Cu++, Fe++ or Zn++ ions). Azide -N=N+=N- is also suspitious as a negative ion is a bad counterion for a carboxylate. Enrico. On Mon, 08 Jul 2013 11:19:46 +0200, herman.schreu...@sanofi.com wrote: Dear Ed, What is the pH of your crystallization buffer? If it is acidic, either the azide or the carboxylate may be protonated. Also the local environment of the carboxylate can make a hugh difference in PKa. You could also use some Bayesian logic: given the elongated linear density, what else of the available components of your crystallization drop would fit? Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Edward A. Berry Gesendet: Sonntag, 7. Juli 2013 22:21 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] help identifying ligand In a structure I'm refining, there are a couple of oblong blobs associated with carboxylates. (screenshots at http://sb20.lbl.gov/berry/ccp4/azide/) If I modeled with two waters, they refine too close together for normal H-bond, 2.3 to 2.5 A; and their density is connected. I considered one water with alternate locations, but the distal position wouldn't make much sense if the proximal water wasn't there. The density is the right size for azide, which was present in the medium, but I expect a chemist would find it unreasonable to have anionic azide (pKa of hydrazoic acid ~4.6) associating with a carboxylate. Would that make sense? or does anyone have other suggestions? (resolution is 2.2A, contour 0.25 e/A^3 or about 1.3 sigma) Thanks, Ed
Re: [ccp4bb] ctruncate bug?
On Jul 7, 2013, at 1:44 PM, Ian Tickle ianj...@gmail.com wrote: On 29 June 2013 01:13, Douglas Theobald dtheob...@brandeis.edu wrote: I admittedly don't understand TDS well. But I thought it was generally assumed that TDS contributes rather little to the conventional background measurement outside of the spot (so Stout and Jensen tells me :). So I was not even really considering TDS, which I see as a different problem from measuring background (am I mistaken here?). I thought the background we measure (in the area surrounding the spot) mostly came from diffuse solvent scatter, air scatter, loop scatter, etc. If so, then we can just consider Itrue = Ibragg + Itds, and worry about modeling the different components of Itrue at a different stage. And then it would make sense to think about blocking a reflection (say, with a minuscule, precisely positioned beam stop very near the crystal) and measuring the background in the spot where the reflection would hit. That background should be approximated pretty well by Iback, the background around the spot (especially if we move far enough away from the spot so that TDS is negligible there). Stout Jensen would not be my first choice to learn about TDS! It's a textbook of small-molecule crystallography (I know, it was my main textbook during my doctorate on small-molecule structures), and small molecules are generally more highly ordered than macromolecules and therefore exhibit TDS on a much smaller scale (there are exceptions of course). I think what you are talking about is acoustic mode TDS (so-called because of its relationship with sound transmission through a crystal), which peaks under the Bragg spots and is therefore very hard to distinguish from it. The other two contributors to TDS that are often observed in MX are optic mode and Einstein model. TDS arises from correlated motions within the crystal, for acoustic mode it's correlated motions of whole unit cells within the lattice, for optic mode it's correlations of different parts of a unit cell (e.g. correlated domain motions in a protein), and for Einstein model it's correlations of the movement of electrons as they are carried along by vibrating atoms (an Einstein solid is a simple model of a crystal proposed by A. Einstein consisting of a collection of independent quantised harmonic-isotropic oscillators; I doubt he was aware of its relevance to TDS, that came later). Here's an example of TDS: http://people.cryst.bbk.ac.uk/~tickle/iucr99/tds2f.gif . The acoustic mode gives the haloes around the Bragg spots (but as I said mainly coincides with the spots), the optic mode gives the nebulous blobs, wisps and streaks that are uncorrelated with the Bragg spots (you can make out an inner ring of 14 blobs due to the 7-fold NCS), and the Einstein model gives the isotropic uniform greying increasing towards the outer edge (makes it look like the diffraction pattern has been projected onto a sphere). So I leave you to decide whether TDS contributes to the background! That's all very interesting --- do you have a good ref for TDS where I can read up on the theory/practice? My protein xtallography books say even less than SJ about TDS. Anyway, this appears to be a problem beyond the scope of this present discussion --- in an ideal world we'd be modeling all the forms of TDS, and Bragg diffraction, and comparing those predictions to the intensity pattern over the entire detector --- not just integrating near the reciprocal lattice points. Going on what you said above, it seems the acoustic component can't really be measured independently of the Bragg peak, while the optic and Einstein components can, or least can be estimated pretty well from the intensity around the Bragg peak (which means we can treat it as background). In any case, I'm going to ignore the TDS complications for now. :) As for the blocking beam stop, every part of the crystal (or at least every part that's in the beam) contributes to every part of the diffraction pattern (i.e. Fourier transform). This means that your beam stop would have to mask the whole crystal - any small bit of the crystal left unmasked and exposed to the beam would give a complete diffraction pattern! That means you wouldn't see anything, not even the background! That's all true, but you can detect peaks independently of one another on a detector, so obviously there is some minimal distance away from a crystal where you could completely block any given reflection and nothing else. Clearly the reflection stop would have to be the size of the crystal (or at least the beam). You could leave a small hole in the centre for the direct beam and that would give you the air scatter contribution, but usually the air path is minimal anyway so that's only a very small contribution to the total background. But let's say by some magic you were able to measure only the background, say
[ccp4bb] Heterogeneity during purification
Dear all I am working on a 30 kDa membrane protein which forms a functional dimer. The protein is His-tagged at N-terminal. In small scale expression screening from whole cells, there is only a single band on Western blot at 30 kDa. But, after purification, additional bands appear at 60 and 120 kDa on SDS-PAGE and Western blot. On size exclusion with Superdex 200, a large proportion elute near the void volume (8 ml). Detail purification For small scale screening, I lysed cells in 20 mM Tris pH 8, 100 mM NaCl, 1 mg/ml lysozyme, 1 % DDM and DNAse for 2 hours and then centrifuged at 16000 g. I then checked the supernatant on SDS-PAGE and scale it up for purification. For purification, I use the buffer 50 mM Tris pH 8, 300 mM NaCl, 20 mM imidazole, 0.05 % DDM (two times CMC of DDM). Is there suggestion to get homogeneous protein? Thank you. Theresa
[ccp4bb] crystallographic association
Hi everyone, I just wanted to ask if there is a professional association for crystallographers in the US. When I googled it I came across The American Crystallographic Association. Is that the only one? Thank you, Maher
