[ccp4bb] RES: [ccp4bb] Choosing test set for twin refinement, with multiple operators
Dear all, thank you for the valuable inputs. I realize that one important piece of information that must be added concerns the relative orientation of the domain for which the electron density becomes poor upon merging data towards higher symmetry, with respect to the rest of the protein. We have found that, at the monoclinic symmetry, this domain can be safely located for 6 of the 20 monomers in the asymmetric unit. By safely located, I mean that these 6 copies were automatically positioned by Phaser and refined well throughout. On the other hand, for the remaining 14 copies, not even manual positioning worked. At the tetragonal space group, neither of the 5 copies for this domain refine well, after borderline MR solutions for 2 copies. Therefore, based on your responses I understand that, while the indication of twinning in P21 may be misleading, P42212 can be a case is a pseudo symmetry. The intrinsic flexibility of this domain, as a rigid body, is however most properly resolved in the P21 symmetry. I will soon try refining again in P21 without imposing twinning. If the quality of the electron density map for the domains is reproduced, with and Rfree in the range of 30-ish%, this would be a more correct approach, right? I will make sure I incorporate all you suggestions in this subsequent analysis. As a matter of fact, xtriage concludes that twinning is not indicated or not possible for the P42212 sg. Thank you again and kind regards, Andre. -Mensagem original- De: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Em nome de Kay Diederichs Enviada em: terça-feira, 6 de dezembro de 2016 14:06 Para: CCP4BB@JISCMAIL.AC.UK Assunto: Re: [ccp4bb] Choosing test set for twin refinement, with multiple operators Hi Andre, you should first optimize your data processing as much as possible ... try different programs, and do test refinements against the data they give you. Unfortunately Rwork/Rfree is a poor indicator unless the Wilson B is the same; what is much more robust is the correlation coefficient CCwork/CCfree as a function of resolution. If you get 25% Rfree in twin refinement in a low-symmetry spacegroup with equal twin fractions, and 33% in the high-symmetry spacegroup without twin refinement, then the high-symmetry spacegroup may be the correct one. As Phil says, the intensity stats may be skewed due to close spots. To answer your question: for an unbiased free set, assign the free reflections in the high-symmetry space group and expand them (using sftools or similar) to the low symmetry space group. best, Kay
[ccp4bb] Choosing test set for twin refinement, with multiple operators
Dear all, We are currently refining a low resolution model (3.9 A max), obtained by MR. Dataset was collected from a single crystal with one long cell axis (~620 A) and high solvent content (74%). Best refinement results (by far!) are obtained in Refmac, by imposing P21 sg (20 multidomain monomers/AU) and allowing for amplitude-based twin refinement. Accordingly, Refmac identifies four twin operators, which I understand have considerable fractions, as follows: Twin operators with estimated twin fractions Twin operator: H, K, L: Fraction = 0.249; Equivalent operators: -H, K, -L Twin operator: -H, -K, L: Fraction = 0.260; Equivalent operators: H, -K, -L Twin operator: -K, -H, -L: Fraction = 0.235; Equivalent operators: K, -H, L Twin operator: K, H, -L: Fraction = 0.257; Equivalent operators: -K, H, L I suspect that the low Rfree (~25%) may be artificial and results from an inappropriate selection of the test due to the multiple operators. However, I cannot figure out how to properly select the test set when such a high number of operators must be considered. FYI, space group P42212 resulted in the best processing statistics (5 monomers/AU); however, model never improved from an Rfree of 33%, with the quality of the electron density distribution for some of the domains being heavily compromised. I honestly apologize if I am missing some obvious points here and will be happy to provide more info, including statistics on data processing. Since is a multidomain protein, is it also appropriate to include TLS refinement at this low resolution? I thank you all in advance. Best, Andre.
[ccp4bb] Postdoc Position on Structural Biology of Membrane Proteins in Brazil
Dear colleagues, I intend to implement work on structural biology (by X-ray crystallography) of membrane proteins in my lab, more specifically on the subset of inner mitochondrial membrane carriers. For this, I would like to take this opportunity and invite anybody who is prospecting a postdoc in the field, and for the second semester of 2014, to consider coming to Brazil. My laboratory is located at the Brazilian National Laboratory for Biosciences (LNBio), which is hosted in the same campus as the Brazilian Synchrotron Light Source (LNLS) as well as the third-generation synchrotron source Sirius, currently under construction. LNBio has an exceptional infrastructure dedicated to all aspects of structural biology. The campus is located in Campinas, about 1 hour driving, inland from the city of São Paulo. Brazilian research grants to principal investigators seldom allow the payment of salaries to postdocs or students, so as a rule fellowships or scholarships have to be applied on demand to the funding agencies, as in this case. Specifically, the PD fellowship proposal I advertise will be presented - in the form of a research project - to the funding agency which has been funding my lab since its establishment (about 4 years ago), as a joint application by myself and the chosen candidate. The time frame for analysis is around 100 days from submission. If approved, the PD fellowship is granted for 24 months and can be renewed for an extra 12 months. The stipend is currently R$ 5.908,80 or approximately USD 2,500.00, tax-free. According to the agency rules, on demand PD fellowship applications that receive a favorable assessment from ad hoc reviewers will be put forward for a monthly comparative analysis session, which in my experience, takes especially into consideration the previous experience of the candidate in the field; thus, a publication record in structural biology and/or membrane proteins may grant considerable advantage to the candidate. So, if yourself or anybody you know would like to consider this opportunity, please, do not hesitate to contact me, sending a copy of your CV and the name of a reference scientist to whom I could ask for recommendation. I will be more than happy to provide further details on every aspect of this position (including the research project, the fellowship, my institution or myself) to serious, motivated and talented candidates. The funding agency requires that the candidate should have concluded a doctorate less than seven years before the beginning of the PD fellowship, which would be around August or September 2014. Thank you for your attention, Andre LB Ambrosio LNBio, CNPEM - Brazil
[ccp4bb] Summary: Identity of a Bacterial lipid
Dear all, I would to acknowledge everyone that replied to my post, both privately and to the BB, for the very useful comments and suggestions. Here is my conclusion so far: Taking into consideration especially the planarity resulting from the presence of double bonds, it seems that indeed there is only one unsaturation - at position 11 - which suggests the fatty acid identity as the vaccenic acid or (11-cis)-octadecanoic acid, very frequent in E. coli. Cis conformations at positions 5, 7, 9 and 13 remain, but all modeled as single bonds now. Plus, the extra group is well explained by a glycerol molecule. If it is actually a glycerol esterified at more than one position, or even a phospholipid, we cannot tell from the map. I need to read a little more about it to learn what is plausible to propose, especially in regards to an actual function for this protein. Mass spec and NMR are due soon, anyways. Thanks once again and kind regards, -Andre. De: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Em nome de Uma Katre Enviada em: sexta-feira, 4 de outubro de 2013 18:12 Para: CCP4BB@JISCMAIL.AC.UK Assunto: Re: [ccp4bb] Identity of a Bacterial lipid Perhaps this will help- these people used mass spectrometry to identify the lipid. The expression system was E. coli and the lipid was 18-carbon cis-vaccenic acid. Detection and removal of an artefact fatty acid from the binding site of recombinant Bombyx mori pheromone-binding protein. Oldham NJhttp://www.ncbi.nlm.nih.gov/pubmed?term=Oldham%20NJ%5BAuthor%5Dcauthor=truecauthor_uid=11418499, Krieger Jhttp://www.ncbi.nlm.nih.gov/pubmed?term=Krieger%20J%5BAuthor%5Dcauthor=truecauthor_uid=11418499, Breer Hhttp://www.ncbi.nlm.nih.gov/pubmed?term=Breer%20H%5BAuthor%5Dcauthor=truecauthor_uid=11418499, Svatos Ahttp://www.ncbi.nlm.nih.gov/pubmed?term=Svatos%20A%5BAuthor%5Dcauthor=truecauthor_uid=11418499. Chem Senses.http://www.ncbi.nlm.nih.gov/pubmed/11418499?dopt=Abstract 2001 Jun;26(5):529-31. Uma. -- Uma Katre Columbus, IN 47201 From: Andre Luis Berteli Ambrosio andre.ambro...@lnbio.cnpem.brmailto:andre.ambro...@lnbio.cnpem.br To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Sent: Thursday, October 3, 2013 8:42 AM Subject: [ccp4bb] Identity of a Bacterial lipid Dear colleagues, We have just determined the crystal structure (at 1.1 A max resolution) of a recombinant protein that crystallized in complex with a leftover bacterial lipid, the full identity of which we are currently struggling to identify. Please see attached (3 views of the molecule). The map strongly suggests an 18-carbon long polyunsaturated fatty acid, with 5 conjugated unsaturations (at positions 5, 7, 9, 11 and 13, all cis), covalently bound to some extra chemical group at is polar head. This extra group seems to be comprised of 4 (5?) atoms, though I am afraid cannot tell if it extends further into not-so-well-structured atoms. Myself and a student have spent the last two days searching on the web for possible matches for this ligand without any success. For instance, we have generated a SMILES formula for the aliphatic tail comprising the unsaturations and browsed for similar compounds at PubChem with different similarity cutoffs, but nothing seemed to resemble the complete molecule. We would appreciate if you could make any comments on the nature of this ligand or perhaps suggest your favorite computational tools. We will perform mass spec on it soon. Thank you beforehand. Andre LB Ambrosio, DSc Laboratório Nacional de Biociências - LNBio CNPEM, Brazil
[ccp4bb] RES: [ccp4bb] Identity of a Bacterial lipid
Dear Michael, thank you for the prompt reply. The host was indeed E. coli. From what I have been reading I completely agree on the lack of biological support for such a molecule but still the map seems very convincing of the presence of cis bonds at such positions... Could such a conformation arise due to something other than unsaturations? We are working on isolating the lipid from the purified protein I will sure follow your suggestions on the additional characterization of this molecule, especially adding NMR analysis to it. Thanks also for referencing the literature. With kind regards, -Andre. De: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Em nome de R. M. Garavito Enviada em: quinta-feira, 3 de outubro de 2013 10:16 Para: CCP4BB@JISCMAIL.AC.UK Assunto: Re: [ccp4bb] Identity of a Bacterial lipid Dear Andre, It always impressive to see a near atomic resolution structure with a bound lipid. Congratulations. However, to identify what lipid you have requires a bit more analysis than just looking in databases. First, what is the bacterium you are using as the host? If it is E. coli, then the known lipids are very well characterized. Also, VERY FEW E. coli lipids have sites of unsaturation, and virtually polyunsaturated fatty acids (PUFAs) have one sp3 carbon in between the double bonds (arising from the mechanisms of biosynthesis). So your proposed structure doesn't seem right from a biological sense, which makes looking into databases unproductive. However, since you solved the structure, do your produce enough protein to isolate the putative lipid by simple TLC? With the help of a good lipid lab you should be able to tell what it with greater certainty. Try to get a copy of Techniques of Lipidology by Morris Kates from Elsevier. Although it is old school stuff, it will help you isolate enough for mass spec and NMR analysis. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.commailto:garav...@gmail.com On Oct 3, 2013, at 8:42 AM, Andre Luis Berteli Ambrosio wrote: Dear colleagues, We have just determined the crystal structure (at 1.1 A max resolution) of a recombinant protein that crystallized in complex with a leftover bacterial lipid, the full identity of which we are currently struggling to identify. Please see attached (3 views of the molecule). The map strongly suggests an 18-carbon long polyunsaturated fatty acid, with 5 conjugated unsaturations (at positions 5, 7, 9, 11 and 13, all cis), covalently bound to some extra chemical group at is polar head. This extra group seems to be comprised of 4 (5?) atoms, though I am afraid cannot tell if it extends further into not-so-well-structured atoms. Myself and a student have spent the last two days searching on the web for possible matches for this ligand without any success. For instance, we have generated a SMILES formula for the aliphatic tail comprising the unsaturations and browsed for similar compounds at PubChem with different similarity cutoffs, but nothing seemed to resemble the complete molecule. We would appreciate if you could make any comments on the nature of this ligand or perhaps suggest your favorite computational tools. We will perform mass spec on it soon. Thank you beforehand. Andre LB Ambrosio, DSc Laboratório Nacional de Biociências - LNBio CNPEM, Brazil FA-density.png
[ccp4bb] Off-topic: heterologous co-expression in yeast
Hi, We have been thinking of implementing recombinant protein expression in S. cerevisiae in our lab, and I would like to ask if there is a choice of vector designed for co-expression of any two intracellular binding partners, similarly to the pETduet-1 vector for E. coli. If you feel this is not of broad interest, please reply directly to me. I thank you for your input, Andre LB Ambrosio Laboratório Nacional de Biociências - LNBio CNPEM, Brazil
Re: [ccp4bb] Desalting columns
Hi, We have a case where a 260 KDa protein crystallized at 3 mg/ml, that was the highest concentration we could achieve, without playing with the protein buffer. Protein-to-well-solution ratio was 3:1. When screening, we tested all ratios between 4:1 and 1:1. Crystals only appeared at 3:1 ratio. That is another important variable to test, I guess. Regards, -Andre. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp (Hofkristallrat a.D.) [hofkristall...@gmail.com] Sent: Monday, February 27, 2012 1:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Desalting columns Why, in the first place, do you feel an urge to concentrate your protein above 3 mg/ml ? For crystallization, the concentration needs to be a) high enough to achieve supersaturation, meaning close enough to the maximum solubility in a given buffer so that the precipitant can drive the system in to supersaturation, preferably of a level where homogenous nucleation can occur (or you micro-seed, if necessary) b) high enough that sufficient material for crystals of acceptable size to grow is in the drop, which is generally the case, lest micro-crystal showers happen. There is ample evidence for proteins crystallizing below 3 mg/ml. The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased towards highly soluble, smaller (lower hanging fruit) proteins. Sometimes the shape of a distribution matters ;-) BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sangeetha Vedula Sent: Monday, February 27, 2012 8:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Desalting columns Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
[ccp4bb] Why 0.1% bandwidth?
Dear ccp4bb, I sometimes find the flux of x-ray sources reported in units of photons/s/0.1% bandwidth instead of simply photons/s. Where does the 1/0.1% bandwidth unit come from? I have also seen other percentages like 0.01% bw or 0.02% bw... Is it simply defining some degree of acceptance in energy (for example, the flux between 8 KeV +/- 8 eV for a given stored current)? Does it somehow have to do with energy resolution? Thank you in advance for your answers, -Andre Ambrosio
