Re: [ccp4bb] how to reduce protein solubility
Hi Francesca, It may be worth trying D2O (rather than H2O) in the final protein buffer. It sometimes causes problems in NMR experiments. One of my colleagues had success with this approach. Crystal screening will need to be done under oil (micro batch) to stop losing D2O by vapor diffusion. Alun Sent from my Samsung Galaxy smartphone. Original message From: Mattiroli,Francesca francesca.mattir...@colostate.edu Date:17/02/2015 04:34 (GMT+00:00) To: CCP4BB@JISCMAIL.AC.UK Cc: Subject: [ccp4bb] how to reduce protein solubility Hi all, I am struggling with a protein complex that is too soluble. I have reached about 20 mg/ml but I still observe very little precipitation (clear drops in 90-95% of the tested conditions). The proteins are expressed in insect cells and going to higher concentration is not easily achievable. I have tried different buffer conditions (salt concentration and pH) and I am testing temperatures. I am at a loss with what to try next. Do you think PTMs (phosphorylation, acetylation) might be causing this? Any input on how to decrease solubility? Thank you very much in advance, Francesca
Re: [ccp4bb] P3212--1's in Space Group Names?
the same as the 1 in P1 ;-) seriously, if you look at space group names: 143 P3 144 P31 145 P32 149 P312 151 P3112 153 P3212 150 P321 152 P3121 154 P3221 the 1s in 149 to 154 are necessary to differentiate them all. On 18 Feb 2015, at 04:51, Keller, Jacob wrote: Dear Crystallographers, I don't understand what the 1's are doing in space group names like P3212 or P3112--can someone fill me in? Not easy to google this one. JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.org *** Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij
[ccp4bb] how to reduce protein solubility
Hi Francesca, since your protein is expressed in eucarytic cells, glycosylation might be an issue. Glycosylation usually enhances the solubility and can influence the crystallisation negativly because of high flexibility. There are commercially available deglycosylation kits. You can try methylation of primary amines of your protein. For this purpose commercially available kits are existing too. My experience was a drastic reduction of solubility. Good Luck Max Original message From: Mattiroli,Francesca francesca.mattir...@colostate.edu Date:17/02/2015 04:34 (GMT+00:00) To: CCP4BB@JISCMAIL.AC.UK Cc: Subject: [ccp4bb] how to reduce protein solubility Hi all, I am struggling with a protein complex that is too soluble. I have reached about 20 mg/ml but I still observe very little precipitation (clear drops in 90-95% of the tested conditions). The proteins are expressed in insect cells and going to higher concentration is not easily achievable. I have tried different buffer conditions (salt concentration and pH) and I am testing temperatures. I am at a loss with what to try next. Do you think PTMs (phosphorylation, acetylation) might be causing this? Any input on how to decrease solubility? Thank you very much in advance, Francesca Forschungszentrum Juelich GmbH 52425 Juelich Sitz der Gesellschaft: Juelich Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498 Vorsitzender des Aufsichtsrats: MinDir Dr. Karl Eugen Huthmacher Geschaeftsfuehrung: Prof. Dr.-Ing. Wolfgang Marquardt (Vorsitzender), Karsten Beneke (stellv. Vorsitzender), Prof. Dr.-Ing. Harald Bolt, Prof. Dr. Sebastian M. Schmidt
[ccp4bb] PhD studentship at Queen Mary University of London (In silico study of the effects of pathogenic mutations on the structure and dynamics of heart muscle proteins)
Applications are invited for a PhD studentship in Dr. Arianna Fornili's group at Queen Mary University of London starting from October 2015. Using a combination of state-of-the-art Molecular Modelling, Molecular Dynamics and Bioinformatics techniques, the successful candidate will investigate how cardiomyopathy-related mutations affect the motion and stability of key components of the cardiac sarcomere. Applications are welcome from outstanding European/UK students with or expecting to obtain a degree in Chemistry, Biochemistry, Physics, Biophysics or related disciplines. Previous experience in molecular modelling/simulation and/or computer programming is desirable but not essential. For more information: http://www.sbcs.qmul.ac.uk/prospectivestudents/research/projects/147840.html http://www.findaphd.com/search/ProjectDetails.aspx?PJID=61492LID=1316 For informal enquires please contact Dr. Fornili (a.fornili at qmul.ac.ukhttp://qmul.ac.uk/) and include your CV, a covering letter explaining eligibility and interest in the project and the contact details of two academic referees. The deadline for formal applications is 5 pm (GMT) on 09 March 2015. Dr. Arianna Fornili BHF Research Fellow School of Biological and Chemical Sciences Joseph Priestley Building (room G.04) Queen Mary, University of London Mile End Road E1 4NS London UK email: a.fornili at qmul.ac.ukhttp://qmul.ac.uk Tel: +44 (0) 207 882 8446
Re: [ccp4bb] P3212--1's in Space Group Names?
At the risk of muddying the waters further, the difference between 312 and 321 is that the 2-fold normal to the 3-fold is along the diagonal of the ab face in 312, while it is along the a (or b, by equivalence) in 321. The 1 is definitely no place holder. It has a very significant meaning. The increasing number of apparently redundant numbers in a space group name is not accidental or incremental. It is simply because in e-mail, people could not (in recent history) write the subscripts. So, P3112 used to be written as P 3(1) 1 2, which meant there is a 3sub1 3-fold screw, with a 2-fold normal to it along the diagonal in the ab plane. The round brackets used to save us from confusion, but their use seems to have dropped out from the lexicon. Who'd be a crystallographer today! Pierre Rizkallah -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: 18 February 2015 16:42 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P3212--1's in Space Group Names? Well, I meant no harm to the poor 1 by calling it a placeholder, but that in the case of P3212, the 1 is simply to tell you that there is no rotation about the second axis but is instead about the third. Saying okay, nothing here amounts to being a place-holder to avoid ambiguity in assigning the loci of the rotations. Place-holders are important too, e.g. the 0's in 1000, perhaps. Maybe to be rigorous we should start calling p1 p111? (not really...) JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kay Diederichs Sent: Wednesday, February 18, 2015 11:05 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P3212--1's in Space Group Names? Hmm, placeholder for me does not seem to emphasize enough the role that this number plays in the space group names. My understanding (but I fail to remember where I read this ...) is that the first number is the order of the rotation (i.e. 6,4,3,2 or 1) of the unique unit cell axis (often the one with the highest symmetry), the second number is the rotation order of a secondary axis, and the third number gives the rotation order of a tertiary axis - which is the third axis in the orthorhombic system, but a diagonal at least in the trigonal and tetragonal (and I think cubic) systems. This makes it clear that each (baseline) letter in the spacegroup name has its specific role, and tells you about the order of the rotation axis. On top of that comes the screw axis information which is much easier to read when using subscripts. But obviously the naming scheme was chosen such that even if screw axes are not indicated with subscripts, the resulting names are unambiguous. best, Kay
Re: [ccp4bb] P3212--1's in Space Group Names?
...The 1 is definitely no place holder. It has a very significant meaning. Not sure why place holders are inimical to significance. I think you are saying that the 1 tells you there is no rotation here, like the 0 in the number 201 tells you there are no tens in that number. I call that a place holder. To check myself I looked up its definition, and serendipitously found its mathematical definition a significant zero in the decimal representation of a number. The increasing number of apparently redundant numbers in a space group name is not accidental or incremental. It is simply because in e-mail, people could not (in recent history) write the subscripts. So, P3112 used to be written as P 3(1) 1 2, which meant there is a 3sub1 3-fold screw, with a 2-fold normal to it along the diagonal in the ab plane. The round brackets used to save us from confusion, but their use seems to have dropped out from the lexicon. Who'd be a crystallographer today! Pierre Rizkallah -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: 18 February 2015 16:42 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P3212--1's in Space Group Names? Well, I meant no harm to the poor 1 by calling it a placeholder, but that in the case of P3212, the 1 is simply to tell you that there is no rotation about the second axis but is instead about the third. Saying okay, nothing here amounts to being a place-holder to avoid ambiguity in assigning the loci of the rotations. Place-holders are important too, e.g. the 0's in 1000, perhaps. Maybe to be rigorous we should start calling p1 p111? (not really...) JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kay Diederichs Sent: Wednesday, February 18, 2015 11:05 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P3212--1's in Space Group Names? Hmm, placeholder for me does not seem to emphasize enough the role that this number plays in the space group names. My understanding (but I fail to remember where I read this ...) is that the first number is the order of the rotation (i.e. 6,4,3,2 or 1) of the unique unit cell axis (often the one with the highest symmetry), the second number is the rotation order of a secondary axis, and the third number gives the rotation order of a tertiary axis - which is the third axis in the orthorhombic system, but a diagonal at least in the trigonal and tetragonal (and I think cubic) systems. This makes it clear that each (baseline) letter in the spacegroup name has its specific role, and tells you about the order of the rotation axis. On top of that comes the screw axis information which is much easier to read when using subscripts. But obviously the naming scheme was chosen such that even if screw axes are not indicated with subscripts, the resulting names are unambiguous. best, Kay
Re: [ccp4bb] Poor experimental phases for a largish structure...
Dear William, In a similar circumstance, we cut out the density from one crystal, and used that to solve the structures of non-isomorphous crystals by molecular replacement. At this point, we had an envelope (needed to cut out the density), initial maps, and operators to place one copy of the density in the other crystals, which is all you need for multi-crystal averaging. The more non-isomorphous your crystals are, the more powerful this is. What’s important (Kevin Cowtan explained this in a talk at the IUCr meeting in Glasgow) is that the size of the difference in cell dimensions should preferably be comparable to the resolution of the data (dmin) or larger. Our case was a bit difficult because the non-isomorphism was marginal — enough to stop us from using MIR(AS) but barely enough to help the phasing. There are other cases of people using similar approaches in the literature. Best wishes, Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +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 On 18 Feb 2015, at 12:51, William Chao william.c...@cancer.org.uk wrote: Dear all, I am trying to phase a largish novel structure of 130 kDa with P21 (sometimes P222) space group. So far I have collected a few 3.5ish-Å 3-wavelength MAD datasets from SeMet derivatives as well as an anisomorphous 6Å Hg peak dataset (with detectable anomalous signal). As my crystal is rather anisotropic, CC1/2 of one direction of the Se data drops below 0.5 at 4Å. I can generate a map from a MAD dataset that gives a overall shape of the molecule with clear molecular boundary after solvent flattening, resembling the shape of some EM class averages that I obtained earlier. However, the density of this map is very discontinuous and is impossible to build any helix in by machine or by eye. As I use an insect-cell expression system and the occupancy of Se is expected to be low, the programmes that I used could only find a 2 reasonable sites out of 24 Se per molecule. I have about 10 Se crystals and 50 native crystals (which I shall use for derivatisation) left for one last trip before the synchrotron shuts down for two months. Could someone advise me on a reasonable data collection strategy that could maximise my chance on this upcoming trip? I am sure that many people have encountered difficult data like this one and have solved their structures successfully. Would anyone be able to advise me on how it'd be best to improve my phases/density given the limitations of the data? Also to mention that the best native crystal can diffract to 3A with CC1/2 of the worst direction dropping to 0.5 at 3.5A. Many thanks in advance! William --- NOTICE AND DISCLAIMER This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. Cancer Research UK Registered charity in England and Wales (1089464), Scotland (SC041666) and the Isle of Man (1103) A company limited by guarantee. Registered company in England and Wales (4325234) and the Isle of Man (5713F). Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
Re: [ccp4bb] P3212--1's in Space Group Names?
Laurent Maveyraud laurent.maveyr...@ipbs.fr writes: this is explained in details in table 2.2.4.1 of vol A of International Tables of Crystallography (p 18 in my edition). For trigonal/hexagonal, the primary direction is along c, along the 3-fold (6-fold axis). It's the same in tetragonal (obviously for the 4-fold axis !).. The secondary directions for trigonal/hexagonal are [100], the a vector, [010], the b vector, and [-1-10] the diagonal direction between -a and -b vectors (and therefore of between the a and b vectors). In tetragonal, the secondary directions are only [100], a, and [010], b. The tertiary direction for trigonal/hexagonal are [120] and [-2-10] which are directions perpendicular to a and to b, respectively, and [1-10]. None of these directions are the diagonal of a and b vectors. In tetragonal, the tertiary directions are [110], the diagonal of the a and b vectors, and [1-10], the diagonal of the a and -b vectors. I find it less confusing in these cases—not everyone might agree—to use three equivalent axes ⟂ to Z: X, Y and U, and hence four‐index denotations for directions, h k i l where h + k + i = 0. Someone has borrowed my copy of McKie and McKie, and not brought it back, but I’ve made the enclosed badly‐drawn diagram from memory. -- Ian ◎
Re: [ccp4bb] Odd diffraction
Dear Fang Lu, These spots are from a small molecule crystal. There is one very short axial length (large distance between reciprocal lattice rows) and one quite long axis (the spots are close together in the straight reciprocal lattice row near the bottom of your diffraction pattern) You can measure these distances and come up with an approximate unit cell for the 2 dimensions from this one pattern. It is certainly not a protein. Michael James On Wed, Feb 18, 2015 at 5:15 AM, Fang Lu fangluwork...@gmail.com wrote: Hi all, I got this odd diffraction. Not sure what it is, detergent or protein? Any ideas? The crystallisation condition is 2.4M Sodium Malonate. GF buffer contains HEGA-10,Tris, KAcetate, MgAcetate, EDTA and DTT. Protein size is around 100kDa. Thank you for your time. Fang [image: 内嵌图片 1]
Re: [ccp4bb] P3212--1's in Space Group Names?
Well let me further muddy the waters by insisting that the 1 IS DEFINITELY a place-holder, telling you that there is no rotation (greater than 1-fold) along the a or b axes, and that the 2 therefore refers to 2-fold rotation along the next axis, the ab diagonal (no the -a,b diagonal which is perpendicular to a and c but is symmetry related to the a,b diagonal). just like in the decimal number 102 the zero tells you that the 0 signifies there is no multiple of 10^2 and the 1 must therefore be 1*10^3. I guess it comes down to whether you see a 1-fold rotation as being a rotation, in which case this is the order of the rotation about a, or as no rotation at all in which case it is a placeholder bumping the 2 on to the third axis. I guess the original question was why can't the 1's be omitted in the short form, as for p21 (P 1 21 1) or C2 (C 1 2 1). Calling it a placeholder to position the 2 then seems a reasonable description. C1 2 1 is like decimal 001, the leading zero's can be dispensed with without creating ambiguity. eab On 02/18/2015 12:05 PM, Pierre Rizkallah wrote: At the risk of muddying the waters further, the difference between 312 and 321 is that the 2-fold normal to the 3-fold is along the diagonal of the ab face in 312, while it is along the a (or b, by equivalence) in 321. The 1 is definitely no place holder. It has a very significant meaning. The increasing number of apparently redundant numbers in a space group name is not accidental or incremental. It is simply because in e-mail, people could not (in recent history) write the subscripts. So, P3112 used to be written as P 3(1) 1 2, which meant there is a 3sub1 3-fold screw, with a 2-fold normal to it along the diagonal in the ab plane. The round brackets used to save us from confusion, but their use seems to have dropped out from the lexicon. Who'd be a crystallographer today! Pierre Rizkallah -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Keller, Jacob Sent: 18 February 2015 16:42 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P3212--1's in Space Group Names? Well, I meant no harm to the poor 1 by calling it a placeholder, but that in the case of P3212, the 1 is simply to tell you that there is no rotation about the second axis but is instead about the third. Saying okay, nothing here amounts to being a place-holder to avoid ambiguity in assigning the loci of the rotations. Place-holders are important too, e.g. the 0's in 1000, perhaps. Maybe to be rigorous we should start calling p1 p111? (not really...) JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kay Diederichs Sent: Wednesday, February 18, 2015 11:05 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P3212--1's in Space Group Names? Hmm, placeholder for me does not seem to emphasize enough the role that this number plays in the space group names. My understanding (but I fail to remember where I read this ...) is that the first number is the order of the rotation (i.e. 6,4,3,2 or 1) of the unique unit cell axis (often the one with the highest symmetry), the second number is the rotation order of a secondary axis, and the third number gives the rotation order of a tertiary axis - which is the third axis in the orthorhombic system, but a diagonal at least in the trigonal and tetragonal (and I think cubic) systems. This makes it clear that each (baseline) letter in the spacegroup name has its specific role, and tells you about the order of the rotation axis. On top of that comes the screw axis information which is much easier to read when using subscripts. But obviously the naming scheme was chosen such that even if screw axes are not indicated with subscripts, the resulting names are unambiguous. best, Kay
[ccp4bb] Group Leader positions in Epigenetics and Integral Membrane Protein studies in Oxford
Dear all, The SGC and Target Discovery Institute at the University of Oxford are seeking applicants for two new group leader positions. To find out more about the positions and to apply go to the University's jobs and vacancies pages https://www.ox.ac.uk/about/jobs/index.html. Chemical Biology Group Leaderhttp://www.thesgc.org/careers/oxford/sgc-oxford-chemical-biology-group-leader We are seeking a Chemical Biology Group Leader to develop an independent research program in the area of epigenetics within the SGC. As a group leader, you will develop your own target area in epigenetic chemical biology, manage a small team of research staff and coach members of the group in specialist methodologies or procedures. You will be primarily physically based in the Target Discovery Institute but will report to the Chief Scientist in the Structural Genomics Consortium. Working closely with the medicinal chemistry and cell assay groups, your research program will support the effort by focusing on biochemistry and structural biology. You will regularly write research articles at a national level for peer-reviewed journals, book chapters, and reviews, and present papers at national conferences. You will submit applications and obtain grant funding for posts in the group either independently or in collaboration. Vacancy ID : 116825 This post is fixed-term to 30 June 2019. Closing Date: 1200 hours GMT on 23rd February 2015 Queries about the post should be sent to elena.mcphil...@ndm.ox.ac.ukmailto:elena.mcphil...@ndm.ox.ac.uk?subject=Chemical%20Biology%20Group%20Leader Group Leader - Integral Membrane Protein Studieshttp://www.thesgc.org/careers/oxford/sgc-oxford-group-leader-integral-membrane-protein-studies We are seeking a Group Leader to lead a new IMP group. The Group Leader for this group will pursue a research programme in the area of Integral Membrane Proteins structure and/or function studies, using technologies and studying families of proteins that are complementary to existing strengths within the SGC. You will report to and directly support the Chief Scientist of the SGC. Key performance indicators for this position are the publication of research discoveries in first rate peer-reviewed journals, presentation of work at national and international conferences and the application for and award of external funding to augment your research programme. Vacancy ID : 116561 This post is fixed-term to 30 June 2019. Closing Date: 1200 hours GMT on 5 March 2015 Queries about the post should be sent to orcrb.person...@ndm.ox.ac.ukmailto:orcrb.person...@ndm.ox.ac.uk?subject=Group%20Leader%20-%20Integral%20Membrane%20Protein%20Studies
[ccp4bb] Position for a person experienced in structure based drug design.
Dear All, I'd like to call your attention to a second position announcement for a PhD level structural biologist to join a new biotechnology company called HarkerBIO. I am posting this announcement for any members of the CCP4 board that may be looking for positions but please note that I am not involved with the company. Again, all enquiries should be directed to the i...@harkerbio.com email address. Thanks, Eddie --- HarkerBIO Accelerating Drug Discovery with Structural Biology 700 Ellicott Street, Buffalo, NY 14203 www.harkerbio.com HarkerBIO is a newly established biotechnology company backed by the world-renowned Hauptman-Woodward Medical Research Institute, namesake of Nobel Laureate Dr. Herbert Hauptman. Expanding on six decades of scientific research in structural biology and a proven high throughput protein crystallization technology, HarkerBIO provides unique drug discovery and development services to the pharmaceutical and biotechnology industries. Using three-dimensional structures of drug target molecules, HarkerBIO improves the early stages of drug discovery by creating visual scaffolds upon which new drugs can be built. HarkerBIO structurally guides the optimization of therapeutic candidates. By partnering with pharmaceutical and biotechnology companies, HarkerBIO is discovering and designing the next generation of drug therapies. HarkerBIO is located on the Buffalo Niagara Medical Campus, a consortium of the region's premier health care, life sciences research, and medical education institutions, all located on 120 acres in downtown Buffalo, New York. Buffalo offers cosmopolitan opportunities in an easily navigable city with a reasonable cost of living. Now with revenues established, an early and expanding customer base, HarkerBIO is seeking an experienced PhD-level structural biologist/x-ray crystallographer with expertise and proven experience in structural biology gained both academically and through working in industry, including structure-based design, protein expression, production (including high-throughput), purification and biochemical assays, fragment-based screening, molecular modelling, computer-assisted docking and structure-determination. Experience working in pharmaceutical and biotechnology R D is highly desirable, as is key therapeutic area specialization. Required Qualifications * PhD in structural biology, x-ray crystallography or structure-based drug design. * Proven experience including published work in structural biology and work on de novo structures. * Working knowledge of current best-in-class approaches to drug discovery using structural biology and adjacent technologies. * Experience and proven capability working with structural biology within pharmaceutical and biotechnology segments in key therapeutic areas, e.g neurological disorders, cardiovascular disease, oncology, diabetes, infectious disease. * Strong formal academic qualifications in structural biology to PhD-level, including post-doctoral work. * Knowledge of and access to key research contacts within pharmaceutical and biotechnology markets globally relevant to structure-based approaches. * Prior experience working in a CRO or drug discovery services provider. * The ability to work as a collaborative team member, and share responsibilities in a fast-paced and entrepreneurial environment. * Must be willing and able to travel both domestically and internationally. Compensation The selected candidate will be offered a compensation package with a base salary, incentive rewards tied to performance. A full fringe package will also include health benefits and a 401K plan. Some remote work may be an option with the ideal candidate and circumstances. To Apply Please send a CV and cover letter to: i...@harkerbio.com --- Edward Snell Ph.D. CEO Hauptman-Woodward Medical Research Institute Assistant Prof. Department of Structural Biology, SUNY Buffalo 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu Heisenberg was probably here!
[ccp4bb] Biostruct-X transnational user access for the production and crystallization of bacterial and eukaryotic membrane proteins
Dear All, We have transnational user access slots for the production and crystallization of bacterial and eukaryotic membrane proteins from Biostruct-X available. If you are interested (or if know someone at your institute who might be interested) to produce together with us at PSI membrane proteins of interest, then please apply directly via Biostruct-X (http://www.biostruct-x.eu/) or forward this e-mail to people who might be interested. The application seems not a lot of work. If an application is successful, travel and accommodation costs will be reimbursed. The offer is valid only for European users and excludes users from Switzerland. Best wishes, Richard Paul Scherrer Institute Dr. Richard A. Kammerer Group Leader Department of Biology and Chemistry Laboratory of Biomolecular Research OFLC/106 CH-5232 Villigen PSI Switzerland Phone +41 56 310 47 65 richard.kamme...@psi.ch www.psi.ch
Re: [ccp4bb] Odd diffraction
The spots at the very edge is around 2.5A. 2015-02-18 12:15 GMT+00:00 Fang Lu fangluwork...@gmail.com: Hi all, I got this odd diffraction. Not sure what it is, detergent or protein? Any ideas? The crystallisation condition is 2.4M Sodium Malonate. GF buffer contains HEGA-10,Tris, KAcetate, MgAcetate, EDTA and DTT. Protein size is around 100kDa. Thank you for your time. Fang [image: 内嵌图片 1]
Re: [ccp4bb] P3212--1's in Space Group Names?
Dear Dear Crystallographers, Thanks very much to all who replied on and off the list—I feel I have to express my continuous admiration and gratitude to the folks who respond to these questions. I am not sure where there is a listserve like CCP4BB! Anyway, in the current issue I have gotten to complete lucidity, thanks to all who responded. I guess the summary is that the non-screw 1’s are essentially place-holders. All the best, Jacob Keller From: Ian Tickle [mailto:ianj...@gmail.com] Sent: Wednesday, February 18, 2015 1:45 AM To: Keller, Jacob Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] P3212--1's in Space Group Names? Hi Jacob If you look at SGs P312 and P321in ITC-A you'll see that they are quite different in terms of the arrangement of the a.u.s relative to the symmetry axes. So essentially the 1's are there to distinguish these. The extra 1 or 2 after the 3 of course signifies a screw axis. Note that for a rhombohedral cell SGs R312 and R321 would be identical so there is only R32. Note that the original SG names in ITC without intervening spaces were carefully designed to be unambiguous. The extra spacing that was added by the PDB is redundant (or maybe they thought that it improves readability). Hope this helps. -- Ian On Wednesday, 18 February 2015, Keller, Jacob kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org wrote: Dear Crystallographers, I don't understand what the 1's are doing in space group names like P3212 or P3112--can someone fill me in? Not easy to google this one. JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org ***
Re: [ccp4bb] P3212--1's in Space Group Names?
Hmm, placeholder for me does not seem to emphasize enough the role that this number plays in the space group names. My understanding (but I fail to remember where I read this ...) is that the first number is the order of the rotation (i.e. 6,4,3,2 or 1) of the unique unit cell axis (often the one with the highest symmetry), the second number is the rotation order of a secondary axis, and the third number gives the rotation order of a tertiary axis - which is the third axis in the orthorhombic system, but a diagonal at least in the trigonal and tetragonal (and I think cubic) systems. This makes it clear that each (baseline) letter in the spacegroup name has its specific role, and tells you about the order of the rotation axis. On top of that comes the screw axis information which is much easier to read when using subscripts. But obviously the naming scheme was chosen such that even if screw axes are not indicated with subscripts, the resulting names are unambiguous. best, Kay
[ccp4bb] Odd diffraction
Hi all, I got this odd diffraction. Not sure what it is, detergent or protein? Any ideas? The crystallisation condition is 2.4M Sodium Malonate. GF buffer contains HEGA-10,Tris, KAcetate, MgAcetate, EDTA and DTT. Protein size is around 100kDa. Thank you for your time. Fang [image: 内嵌图片 1]
Re: [ccp4bb] P3212--1's in Space Group Names?
Yes, the 1 in these space group names is the same as the 1 in P1. 1 is the identity symmetry operator. For a trigonal space group (and hexagonal) the symmetry operators are given in an axis order of (z) (a,b) and (i) where i is defined by the ab vector (the ab diagonal) So, for example in space group 149, P312 The z axis has 3-fold symmetry The a and b axes have identity symmetry The i axis (ab diagonal) has 2-fold symmetry HTH, Chris -- Christopher L. Colbert, Ph.D. Assistant Professor Department of Chemistry and Biochemistry North Dakota State University From: Mark van Raaij mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es Reply-To: Mark van Raaij mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es Date: Wednesday, February 18, 2015 4:10 AM To: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P3212--1's in Space Group Names? the same as the 1 in P1 ;-) seriously, if you look at space group names: 143 P3 144 P31 145 P32 149 P312 151 P3112 153 P3212 150 P321 152 P3121 154 P3221 the 1s in 149 to 154 are necessary to differentiate them all. On 18 Feb 2015, at 04:51, Keller, Jacob wrote: Dear Crystallographers, I don't understand what the 1's are doing in space group names like P3212 or P3112--can someone fill me in? Not easy to google this one. JPK *** Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: kell...@janelia.hhmi.orgmailto:kell...@janelia.hhmi.org *** Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij
Re: [ccp4bb] P3212--1's in Space Group Names?
Hi, this is explained in details in table 2.2.4.1 of vol A of International Tables of Crystallography (p 18 in my edition). For trigonal/hexagonal, the primary direction is along c, along the 3-fold (6-fold axis). It's the same in tetragonal (obviously for the 4-fold axis !).. The secondary directions for trigonal/hexagonal are [100], the a vector, [010], the b vector, and [-1-10] the diagonal direction between -a and -b vectors (and therefore of between the a and b vectors). In tetragonal, the secondary directions are only [100], a, and [010], b. The tertiary direction for trigonal/hexagonal are [120] and [-2-10] which are directions perpendicular to a and to b, respectively, and [1-10]. None of these directions are the diagonal of a and b vectors. In tetragonal, the tertiary directions are [110], the diagonal of the a and b vectors, and [1-10], the diagonal of the a and -b vectors. laurent Le 18/02/2015 17:04, Kay Diederichs a écrit : Hmm, placeholder for me does not seem to emphasize enough the role that this number plays in the space group names. My understanding (but I fail to remember where I read this ...) is that the first number is the order of the rotation (i.e. 6,4,3,2 or 1) of the unique unit cell axis (often the one with the highest symmetry), the second number is the rotation order of a secondary axis, and the third number gives the rotation order of a tertiary axis - which is the third axis in the orthorhombic system, but a diagonal at least in the trigonal and tetragonal (and I think cubic) systems. This makes it clear that each (baseline) letter in the spacegroup name has its specific role, and tells you about the order of the rotation axis. On top of that comes the screw axis information which is much easier to read when using subscripts. But obviously the naming scheme was chosen such that even if screw axes are not indicated with subscripts, the resulting names are unambiguous. best, Kay -- -- Laurent Maveyraud laurent.maveyraud AT ipbs DOT fr P I C T --- Plateforme Intégrée de Criblage de Toulouse Université Paul Sabatier / CNRS / I.P.B.S. UMR 5089 Département BiologieStructurale et Biophysique http://cribligand.ipbs.fr http://www.ipbs.fr 205 route de Narbonne 31077 TOULOUSE Cedex FRANCE Tél: +33 (0)561 175 435 Fax : +33 (0)561 175 994 --
Re: [ccp4bb] P3212--1's in Space Group Names?
Well, I meant no harm to the poor 1 by calling it a placeholder, but that in the case of P3212, the 1 is simply to tell you that there is no rotation about the second axis but is instead about the third. Saying okay, nothing here amounts to being a place-holder to avoid ambiguity in assigning the loci of the rotations. Place-holders are important too, e.g. the 0's in 1000, perhaps. Maybe to be rigorous we should start calling p1 p111? (not really...) JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kay Diederichs Sent: Wednesday, February 18, 2015 11:05 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] P3212--1's in Space Group Names? Hmm, placeholder for me does not seem to emphasize enough the role that this number plays in the space group names. My understanding (but I fail to remember where I read this ...) is that the first number is the order of the rotation (i.e. 6,4,3,2 or 1) of the unique unit cell axis (often the one with the highest symmetry), the second number is the rotation order of a secondary axis, and the third number gives the rotation order of a tertiary axis - which is the third axis in the orthorhombic system, but a diagonal at least in the trigonal and tetragonal (and I think cubic) systems. This makes it clear that each (baseline) letter in the spacegroup name has its specific role, and tells you about the order of the rotation axis. On top of that comes the screw axis information which is much easier to read when using subscripts. But obviously the naming scheme was chosen such that even if screw axes are not indicated with subscripts, the resulting names are unambiguous. best, Kay
[ccp4bb] The discrepancy for determination of Ramachandran outliers by Coot and MolProbity
Dear All, It often finds for the Ramachandran favored determined by Coot, MolProbity regards as Ramachandran outliers. There are earlier posts regards Coot and MolProbity has different database for the determination of the Ramachandran plots. Then will you please let me know the correct way to correct the Ramachandran outliers by Coot in order to meet the MolProbity Standards? I am looking forward to getting your reply. Smith
Re: [ccp4bb] Poor experimental phases for a largish structure...
Hi, If your Hg dataset is not isomorphous with the SeMet you can cut out some density and use cross-crystal averaging (molecular replacement using electron density) to transfer the Hg phases to the Se dataset. You can then try using an anomalous difference fourier map to find more or weaker Se sites. If that manages to get density good enough to even partially build a model, you can put it through multiple rounds of MR-SAD with phaser. Basically you use your model phases to help find more weak Se sites, which improves your phases and allows you to build a better model, and you go around again. Jason. — Dr Jason Busby Laboratory of Structural Biology School of Biological Sciences The University of Auckland Private Bag 92019 Auckland Mail Centre Auckland New Zealand ph: +64 9 3737599 ext 88958 On 19/02/2015, at 1:51 am, William Chao william.c...@cancer.org.ukmailto:william.c...@cancer.org.uk wrote: Dear all, I am trying to phase a largish novel structure of 130 kDa with P21 (sometimes P222) space group. So far I have collected a few 3.5ish-Å 3-wavelength MAD datasets from SeMet derivatives as well as an anisomorphous 6Å Hg peak dataset (with detectable anomalous signal). As my crystal is rather anisotropic, CC1/2 of one direction of the Se data drops below 0.5 at 4Å. I can generate a map from a MAD dataset that gives a overall shape of the molecule with clear molecular boundary after solvent flattening, resembling the shape of some EM class averages that I obtained earlier. However, the density of this map is very discontinuous and is impossible to build any helix in by machine or by eye. As I use an insect-cell expression system and the occupancy of Se is expected to be low, the programmes that I used could only find a 2 reasonable sites out of 24 Se per molecule. I have about 10 Se crystals and 50 native crystals (which I shall use for derivatisation) left for one last trip before the synchrotron shuts down for two months. Could someone advise me on a reasonable data collection strategy that could maximise my chance on this upcoming trip? I am sure that many people have encountered difficult data like this one and have solved their structures successfully. Would anyone be able to advise me on how it'd be best to improve my phases/density given the limitations of the data? Also to mention that the best native crystal can diffract to 3A with CC1/2 of the worst direction dropping to 0.5 at 3.5A. Many thanks in advance! William --- NOTICE AND DISCLAIMER This e-mail (including any attachments) is intended for the above-named person(s). If you are not the intended recipient, notify the sender immediately, delete this email from your system and do not disclose or use for any purpose. We may monitor all incoming and outgoing emails in line with current legislation. We have taken steps to ensure that this email and attachments are free from any virus, but it remains your responsibility to ensure that viruses do not adversely affect you. Cancer Research UK Registered charity in England and Wales (1089464), Scotland (SC041666) and the Isle of Man (1103) A company limited by guarantee. Registered company in England and Wales (4325234) and the Isle of Man (5713F). Registered Office Address: Angel Building, 407 St John Street, London EC1V 4AD.
