[ccp4bb] Register Now! ACA 2018 - Small Angle Scattering Workshop
<https://gallery.mailchimp.com/6dbb03108468b01ea4f9d2ad2/images/84093648-968d-419e-a392-4b41d1ced9dc.jpg> “Applications of Small Angle Scattering to Structural Biology: An Introduction.” On behalf of the organizing committee, we are pleased to announce a workshop on “Applications of Small Angle Scattering to Structural Biology: An Introduction.” This workshop will take place on Friday, July 20th, 2018 at this year’s American Crystallographic Association (ACA) Meeting in Toronto, Canada. The meeting itself will run from July 20-24. Over the past two decades, SAS has become a mainstay technique for the study of structure and composition in solution for structural biologists around the world. In contrast to the more intensive SAS courses in Europe that extend across several days, this single-day workshop will be comprised of carefully constructed lectures and tutorials to serve as an introduction to investigators new to the technique. The workshop format will include lectures and a selection of hands-on practical exercises. Throughout the workshop the emphasis will be on practical application: knowing how to judge data quality, how to troubleshoot during data collection, and the expectations for a successful experiment and acceptable publication. Students will also learn about aspects of home laboratory data collection and will be introduced to experiments at national user facilities (synchrotrons and research reactors). Students will be expected to bring laptops with the appropriate pre-installed software. Extra laptops will be provided in case of unexpected hardware or software issues on-site, and network connectivity will be provided as part of the course, as some exercises and tutorials will reply on external computer-server resources.Preloaded portable disks and memory sticks will be provided to help reduce the need for large downloads over conference bandwidth. We thank Anton-Paar, Rigaku Corporation, SAXSLAB, and Wyatt Technology Corporation for their kind support of this event. Tentative Program Schedule Time Topic 8:00 AM Registration and Software Installation Help; Speaker Introduction 8:30 AM BioSAS: Overview Why is SAS helpful? 9:00 AM The Scattering Profile Guinier analysis, concentration, monodispersity, etc. 9:30 AM Tutorial 1: Basic Data Analysis I with RAW Is this good data? Radiation Damage? 10:00 AM Coffee Break 10:15 AM Data Analysis: Model-independent Features Molecular Weight Determination, Invariant Plots and Flexibility, The P(r) function 11:00 AM Tutorial 2: Basic Data Analysis II with RAW Calculate a P(r) function, determine Molecular Weight, Dmax, Rg, Volume, etc. 12:00 PM Working Lunch - Laboratory Source SAXS Discussion with Scientists, Experimental Advice 1:00 PM SEC-SAXS Application and Practical Considerations 1:30 PM Sample Preparation Sample purification and preparation, aggregation, buffer selection, etc 1:45 PM Complementary Biophysical Methods for Sample Assessment The importance of supporting biophysical methods including static and dynamic light scattering and analytical ultracentrifugation 2:00 PM Data Analysis: Model-dependent Analysis of SAS Data Calculated scattering profiles from atomic models, Shape reconstruction, Atomistic modeling 2:30 PM Tutorial 3: Three-dimensional modeling from SAS data 3:15 PM Coffee Break 3:30 PM Guest Research Lecture – Thomas Grant, Research Asst. Professor, University of Buffalo/Hauptman-Woodward Medical Research Institute 4:30 PM SANS and Contrast Variation 5:00 PM Publishing SAS data 5:30 PM Conclusion Pre-registration is mandatory. The fee to attend is: * Student (undergrad, grad or postdoctoral) - $185 (CAN) * Corporate - $355 (CAN) * Non-corporate or non-student - $185 (CAN) Lunch will be included. Contact: Kushol Gupta, Perelman School of Medicine, University of Pennsylvania. <mailto:kgu...@upenn.edu> kgu...@upenn.edu Confirmed Instructors: Angela Criswell, Rigaku Corporation Srinivas Chakravarthy, BioCAT Group, Advanced Photon Source Richard Gillilan, Macromolecular Diffraction Facility, Cornell High Energy Synchrotron Source Thomas Grant, University of Buffalo/Hauptman-Woodward Medical Research Institute Jesse Hopkins, BioCAT Group, Advanced Photon Source Maxim Petoukhov, BIOSAXS Group, EMBL Hamburg Soren Skou, XENOCS (SAXSLAB) More information is available at: <https://upenn.us16.list-manage.com/track/click?u=6dbb03108468b01ea4f9d2ad2=c23962f609=6031dddf6d> http://www.amercrystalassn.org/2018-workshops <https://upenn.us16.list-manage.com/track/click?u=6dbb03108468b01ea4f9d2ad2=a6594b9e23=6031dddf6d> http://www.amercrystalassn.org/2018-meeting-homepage
[ccp4bb] ACA 2018 - Small Angle Scattering Workshop
"Applications of Small Angle Scattering to Structural Biology: An Introduction." On behalf of the organizing committee, we are pleased to announce a workshop on "Applications of Small Angle Scattering to Structural Biology: An Introduction." This workshop will take place on Friday, July 20th, 2018 at this year's American Crystallographic Association (ACA) Meeting in Toronto, Canada. The meeting itself will run from July 20-24. Over the past two decades, SAS has become a mainstay technique for the study of structure and composition in solution for structural biologists around the world. In contrast to the more intensive SAS courses in Europe that extend across several days, this single-day workshop will be comprised of carefully constructed lectures and tutorials to serve as an introduction to investigators new to the technique. The workshop format will include lectures and a selection of hands-on practical exercises. Throughout the workshop the emphasis will be on practical application: knowing how to judge data quality, how to troubleshoot during data collection, and the expectations for a successful experiment and acceptable publication. Students will also learn about aspects of home laboratory data collection and will be introduced to experiments at national user facilities (synchrotrons and research reactors). Students will be expected to bring laptops with the appropriate pre-installed software. Extra laptops will be provided in case of unexpected hardware or software issues on-site, and network connectivity will be provided as part of the course, as some exercises and tutorials will reply on external computer-server resources. Preloaded portable disks and memory sticks will be provided to help reduce the need for large downloads over conference bandwidth. We thank Anton-Paar, Rigaku Corporation, SAXSLAB, and Wyatt Technology Corporation for their kind support of this event. Tentative Program Schedule Time Topic 8:00 AM Registration and Software Installation Help; Speaker Introduction 8:30 AM BioSAS: Overview Why is SAS helpful? 9:00 AM The Scattering Profile Guinier analysis, concentration, monodispersity, etc. 9:30 AM Tutorial 1: Basic Data Analysis I with RAW Is this good data? Radiation Damage? 10:00 AM Coffee Break 10:15 AM Data Analysis: Model-independent Features Molecular Weight Determination, Invariant Plots and Flexibility, The P(r) function 11:00 AM Tutorial 2: Basic Data Analysis II with RAW Calculate a P(r) function, determine Molecular Weight, Dmax, Rg, Volume, etc. 12:00 PM Working Lunch - Laboratory Source SAXS Discussion with Scientists, Experimental Advice 1:00 PM SEC-SAXS Application and Practical Considerations 1:30 PM Sample Preparation Sample purification and preparation, aggregation, buffer selection, etc 1:45 PM Complementary Biophysical Methods for Sample Assessment The importance of supporting biophysical methods including static and dynamic light scattering and analytical ultracentrifugation 2:00 PM Data Analysis: Model-dependent Analysis of SAS Data Calculated scattering profiles from atomic models, Shape reconstruction, Atomistic modeling 2:30 PM Tutorial 3: Three-dimensional modeling from SAS data 3:15 PM Coffee Break 3:30 PM Guest Research Lecture - Thomas Grant, Research Asst. Professor, University of Buffalo/Hauptman-Woodward Medical Research Institute 4:30 PM SANS and Contrast Variation 5:00 PM Publishing SAS data 5:30 PM Conclusion Pre-registration is mandatory. The fee to attend is: * Student (undergrad, grad or postdoctoral) - $185 (CAN) * Corporate - $355 (CAN) * Non-corporate or non-student - $185 (CAN) Lunch will be included. Contact: Kushol Gupta, Perelman School of Medicine, University of Pennsylvania. <mailto:kgu...@upenn.edu> kgu...@upenn.edu Confirmed Instructors: Angela Criswell, Rigaku Corporation Srinivas Chakravarthy, BioCAT Group, Advanced Photon Source Richard Gillilan, Macromolecular Diffraction Facility, Cornell High Energy Synchrotron Source Thomas Grant, University of Buffalo/Hauptman-Woodward Medical Research Institute Jesse Hopkins, BioCAT Group, Advanced Photon Source Maxim Petoukhov, BIOSAXS Group, EMBL Hamburg More information is available at: <https://upenn.us16.list-manage.com/track/click?u=6dbb03108468b01ea4f9d2ad2; id=e2acf7d04e=ca6eaea91e> http://www.amercrystalassn.org/2018-workshops <https://upenn.us16.list-manage.com/track/click?u=6dbb03108468b01ea4f9d2ad2; id=1a190253ae=ca6eaea91e> http://www.amercrystalassn.org/2018-meeting-homepage
Re: [ccp4bb] R: [ccp4bb] fit multiple structures into SAXS envelope
My two cent suggestion: if you have a good atomic inventory, perform the fitting directly against the experimental profile using your partial structures in programs like CORAL and SASSIE and EOM; use the bead model as a corroborating result. SAXS-derived ab initio bead models really aren’t the same thing as EM reconstructions, and can be horribly misleading if parts of your atomic inventory are flexible or disordered. (what are your NSD values for the bead calculations using what program? What do your Kratky and Porod-Debye plots look like?) Kushol Kushol Gupta, Ph.D. Research Assistant Professor Department of Biochemistry and Biophysics Perelman School of Medicine at The University of Pennsylvania <mailto:kgu...@upenn.edu> kgu...@upenn.edu / 215.573.7260 / 267.259.0082 / <http://www.stwing.upenn.edu/~kgupta> www.stwing.upenn.edu/~kgupta From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of dritan.siliqi Sent: Friday, November 4, 2016 4:51 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] R: [ccp4bb] fit multiple structures into SAXS envelope You can try with sculptor http://sculptor.biomachina.org In this pgm you can calculate first a map (a bĺured model) from saxs model (dammif) , then load your xray models and fit them simultaneously into the envelope. for the fitted model you can then calculate the saxs curve and compare with saxs data. D Dritan Siliqi Institute of Crystallography- CNR Via G. Amendola, 122/O 70126 Bari, Italy Phone: +39 0805929164 <tel:+39%200805929164> Fax: +39 0805929170 <tel:+39%200805929170> Email: dritan.sil...@ic.cnr.it skypeID: dritano Inviato da smartphone Samsung Galaxy. Messaggio originale Da: Natalia O <natalie.c...@gmail.com> Data: 04/11/16 21:32 (GMT+01:00) A: CCP4BB@JISCMAIL.AC.UK Oggetto: [ccp4bb] fit multiple structures into SAXS envelope Dear All, I have a question regarding SAXS, I collected SAXS data on the protein of interest and I can generate the molecular envelope, I also solved x-ray structures of its domains, and I would like to fit my x-ray structures into SAXS envelope, or alternatively find some program which could assemble my domains based on the original SAXS data (not even based on the processing product -envelope). I know that CRYSOL can produce a scattering curve based on some given X-ray structures, but to use it, I would have to fit my x-ray structures in the envelope manually, and then produce the scattering curve to compare with the experimental curve. However, I wonder if there is a program that could do the fitting itself? Thank you! Yours, Natalia
Re: [ccp4bb] How to fit BioSAXS shape to the Structure
I'd advise A LOT of caution here. If you're new to the technique, there are several considerations to make well before you go docking a structure into a reconstruction. Some suggestions: 1. Is your sample truly singular? BSA is a wonderful example of this conundrum: at high enough concentrations, BSA can be seen sampling a monomer-dimer-trimer equilibrium. (seen as plain as day for BSA in SEC-MALS, SV, SE..) Did you scatter a monomer, or a mixture? SAXS profiles are a volume weighted averages of the scatter of the individual components, so those trimers and dimers, even in small amounts, are contaminating your signal and hence the data upon which your reconstructions are based (hence volumes are skewed). Is mass by I(0) or Qr that of a monomer? Is Rg concentration dependent? 2. Is your particle compact or flexible? Flexible species and lack of compactness undermine the reliability of a shape reconstruction. (see Kratky and Porod Debye Plots) 3. What is the chi for an individual reconstructions in your calculations? 4. What is the NSD (Normalized Spatial Discrepancy) for the 10++ individual calculations that you performed using DAMMIF/N or GASBOR? If that number is too high for what the program prescribes (also seen visually), are you then justified in averaging those shapes together? 5. Is your atomic inventory complete (vs the construct you scattered) - very difficult to reconcile these volumes with partial structures without landmarks or contrast variation. The missing bits matter. 6. Be sure your bead file is properly rendered in Pymol with the bead radius prescribed in the header of the damfilt file. Wrong bead sizes or just rendering a surface over the beads are very misleading in assessing the volume intended by the protein. What you show below does not look proper. Use the 'set sphere_scale, X.X' command to set the bead radius. If you pass those many criteria, then you're ready to dock a structure into a reconstruction. A good reality check before this would be to start with CRYSOL and see what the discrepancy between the structure and the primary scattering and in what part of the data those discrepancies lie. I can suggest a number of citations on each of the bullet points above if you'd like. Good Luck, Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Group Department of Biochemistry and Biophysics Perelman School of Medicine at The University of Pennsylvania mailto:kgu...@upenn.edu kgu...@upenn.edu / 215.573.7260 / 267.259.0082 / http://www.stwing.upenn.edu/~kgupta www.stwing.upenn.edu/~kgupta From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ritika Sethi Sent: Friday, June 26, 2015 5:42 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] How to fit BioSAXS shape to the Structure I use SUPCOMB from the ATSAS package to fit the Xtal structure to the SAXS model. Then open this fitted file and your SAXS model again in pymol and you'll see the fit. From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Weifei Chen Sent: 26 June 2015 04:56 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] How to fit BioSAXS shape to the Structure Dear all, I am new to saxs and I get the model by saxs data and I have a structure. I want to fit the structure to the shape but I can just open them in PyMol. Can any one teach me to fit them? Best, Weifei
Re: [ccp4bb] [off topic] Fitting unknown model in SAXS envelope
Andre, some suggestions: 1. First be sure that shape reconstructions as an approach to interpreting your data is justified – the shape reconstruction approach can be quite misleading in some circumstances: i. Does your data show any evidence of flexibility and lack of compactness from Kratky and Porod-Debye analyses? Such aspects can exaggerate molecular volumes, undermining the ability to implement shape reconstruction algorithms to arrive at stable solutions. Extra floppy bits like extended linkers his-tags and mixtures of conformations can also affect apparent volumes. ii. Does your data agree with your understanding of molecular mass at those concentrations (e.g., Mass by Qr, Mass by I(0), Mass by empirical relationships with Porod Volume, etc.). Does Oligomer/Mixture analysis tell you that you have all tetramer, rather than say 95% tetramer/5% dimer…? Is there evidence of aggregation or concentration-dependence through multiple concentrations? As one can guess, mixtures and aggregation can undermine reliable shape reconstruction and interpretation. iii. How does the atomic inventory of your model compare to that of the reconstruction? Commonly, x-ray crystal structures are missing sequences that might otherwise be in a full-length/native construct. If your scattered sample has a composition not entirely represented in your atomic model, automated approaches might be misled, and doing it by eye might be difficult without obvious landmarks or constraints. It could be very helpful to scatter a few different truncations and then to employ simultaneous solution approach used in MONSA. Increasing your data-to-parameters always helps! iv. How does a CRYSOL/FOXS fit between your model and the primary data look, independent of the shape reconstruction calculations? v. Does a symmetry-free (P1) calculation using DAMMIF or GASBOR agree with a symmetry-imposed calculation?...What is the distribution of the Normalized Spatial Discrepancies (NSDs) and Chis like for 10+ calculations? Is averaging justified by the statistics? 2. With regards to software suggestions, in addition to SUPCOMB, I might suggest looking at the SITUS/SCULPTOR package. It uses a real-space approach to reconcile atomic models with volumetric representations, and does provide a real-space correlation coefficient for fits. Hope that helps, Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Group Department of Biochemistry and Biophysics Perelman School of Medicine at The University of Pennsylvania mailto:kgu...@upenn.edu kgu...@upenn.edu / 215.573.7260 / 267.259.0082 / http://www.stwing.upenn.edu/~kgupta www.stwing.upenn.edu/~kgupta From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andre Godoy Sent: Monday, February 2, 2015 5:53 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] [off topic] Fitting unknown model in SAXS envelope Dear users I'm having some troubles to fit my x-ray model in my SAXS envelope.. more about: 1) I have a SAXS model with enough room for 6 monomers. 2) I have the crystallographic structure, but AU or any generate symmetry related doesn't appears to be the biological unit (I mean, crystal packing is different from SAXS packing) Is there any piece of software that can take monomers and find the best (or least worst) RMSD between a SAXS envelope and a generated coordinate system? Or anyone have a good ideia for me to do so? All the best, Andre Godoy PhD Student IFSC - University of Sao Paulo - Brazil
[ccp4bb] Federal Post‐Doctoral Fellowship Opening: Joint project between MedImmune NIST
See attached and please contact Steven Hudson (steven.hud...@nist.gov) for more information. =-= Federal Post‐Doctoral Fellowship Opening: Joint project between MedImmune NIST There is an immediate opening for a post‐doctoral researcher at the National Institute of Standards and Technology (NIST) in Gaithersburg, MD (about 25 miles from downtown Washington DC). Project description: A major goal during formulation and fill/finish operations of biologics such as monoclonal antibody therapeutics is to minimize sub-visible particle (SVP) formation, yet the mechanisms responsible for SVP formation are not well understood. The objective of this project is to elucidate how flow encountered during manufacturing unit operations and the presence of different material surfaces affect antibody conformation and stability, viz., SVP formation. We will exploit in-situ small-angle neutron scattering (SANS) under flow (Rheo-SANS) for this purpose, using special fixtures to explore the effect of various surfaces commonly encountered by monoclonal antibodies during clinical and commercial manufacturing and fill/finish operations of drug products. Collaboration: This is a joint project as part of a comprehensive Cooperative Research Development Agreement (CRADA) between MedImmune, a member of the AstraZeneca group, and NIST for several joint projects. This project is led by Dr. Jai Pathak (Formulation Sciences Dept., MedImmune), Dr. Steven Hudson (Polymers and Complex Fluids Group, NIST), and Dr. Joseph Curtis (NIST Center for Neutron Research). The post-doc will be a US federal employee at NIST, and will have access to MedImmune scientific expertise as well as laboratories at the global RD headquarters of MedImmune, which are located adjacent to the NIST campus in Gaithersburg, MD. Suitable disciplines: Candidates who will have been awarded Ph.D. degrees in Physics, Bio- Physics, Applied Physics, Pharmaceutical Sciences, Chemistry, Biochemistry, Materials Science Engineering, Chemical Engineering, and Mechanical Engineering will be considered. Preference will be given to candidates who have demonstrated competence in neutron scattering methods and modeling. Previous background in rheology, computer simulation, protein biophysics, surface adsorption is preferred, but not required. A strong background in scattering methods and colloid and interface science will be a distinguishing asset. Salary and benefits: At a salary of $66,000 / year, the fellowship is funded for a maximum of three years; renewal will be granted at the end of each year based on satisfactory performance. For benefits information see http://www.nist.gov/ohrm/benefits/index.cfm Citizenship requirement: Interested applicants must have US citizenship, since the successful candidate will be a US federal govt. employee. Non US citizens will not be considered for this position. Application procedure: If interested, please send a cv and cover letter to Steven Hudson (steven.hud...@nist.gov). Kushol Gupta, Ph.D. Research Associate - Van Duyne Group Department of Biochemistry and Biophysics Perelman School of Medicine at The University of Pennsylvania mailto:kgu...@upenn.edu kgu...@upenn.edu / 215.573.7260 / 267.259.0082 / http://www.stwing.upenn.edu/~kgupta www.stwing.upenn.edu/~kgupta MedINIST Federal job opportunity Hudson Curtis Pathak.docx Description: MS-Word 2007 document
Re: [ccp4bb] asymmetric homotrimer in the asu
Hi everyone, Just wanted to second the SAXS suggestion: the approach should provide a very rigorous way of testing the candidate models apparent in the crystallographic lattice, and can assist with questions of mass. (among the most common applications) With regards to the mass question, test as many concentrations as you can to get a pulse on the strength of the interaction, and also use other complementary solution methods such as AUC or SEC-MALS. Note that the concentrations tested in SAXS are several fold higher than those examined in AUC or SEC/SEC-MALS, and may not be physiologically relevant in that regard. Indeed, a 12.5 kD monomer will have very weak scattering power (intensity varies as the square of molecular volume). You can compensate for that by increasing the concentration or using synchrotron radiation. The ‘rule of 100’ is a good guide – in your SAXS expt, start at a sample concentration where MW (kD) * concentration (mg/mL) ~100 and then adjust accordingly. (I’ve successfully studied molecules as small as a 8.5 kD Tudor domain and a ~7kD CHAPS micelle in this way.) Hope that helps, Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Group Department of Biochemistry and Biophysics Perelman School of Medicine at The University of Pennsylvania mailto:kgu...@upenn.edu kgu...@upenn.edu / 215.573.7260 / 267.259.0082 / http://www.stwing.upenn.edu/~kgupta www.stwing.upenn.edu/~kgupta From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David Briggs Sent: Friday, December 12, 2014 12:37 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] asymmetric homotrimer in the asu Hi Hay, I think SAXS should be more than capable of discriminating between a 12.5 kDa monomer vs ~37.5 kDa trimer. Lysozyme is a useful standard used in SAXS (as with most structural biology!), and Lysozyme is only slightly larger than your proteins. Cheers, Dave On Fri Dec 12 2014 at 5:13:26 PM Hay Dvir hd...@tx.technion.ac.il wrote: Tanner: Thanks, GREAT reference on asymmetric homo oligomers! SAXS sounds like a good idea for a bit larger particles. I'm afraid it might be very difficult to get enough resolution to resolve oligomerization of a rather small 12.5 kDa protein like ours, but will look into it more closely. Joes: Thanks, we are aware of the serious problem of instability of asymmetric homo-oligomers which could essentially polimerize as you nicely explain and cite. Indeed one of the hypothesis we aim to test if we get additional evidence about the the trimeric assembly concerns its known function to interact with another protein, which could potentially provide the complementary quaternary stability. Interface mutational analysis sounds like a good approach to take in such cases. Thanks again an very best, Hay On Dec 12, 2014, at 5:39 PM, Tanner, John J. wrote: Two thoughts on asymmetric oligomers. 1. Here is a recent survey of asymmetric homodimers in the PDB. I know you are looking for trimers, but at least this provides a precedent for asymmetric oligomers. Swapna LS, Srikeerthana K, Srinivasan N. Extent of structural asymmetry in homodimeric proteins: prevalence and relevance. PLoS One. 2012;7(5):e36688. doi: 10.1371/journal.pone.0036688. Epub 2012 May 22. PubMed PMID: 22629324; PubMed Central PMCID: PMC3358323. 2. SAXS is a very effective method for determining whether assemblies observed in crystals are stable in solution, since it provides not only the oligomeric state, but also the quaternary structure. The oligomeric state can be obtained from the volume of correlation (1) and Porod-Debye analysis (2). The quaternary structure can be deduced by comparing the experimental SAXS curve to theoretical curves calculated from oligomer models identified by PISA or from manual inspection. The FoXS server and CRYSOL are good tools for this. FoXS also allows ensembles of oligomers (MES) to be used in fitting the data (e.g. mixture of monomer + dimer). I believe ATSAS also has an ensemble program, but the name escapes me at this time. We have used this approach to show that assemblies that are predicted to be stable by PISA are not found in solution (3 and unpublished results). 1: Rambo RP, Tainer JA. Accurate assessment of mass, models and resolution by small-angle scattering. Nature. 2013 Apr 25;496(7446):477-81. doi: 10.1038/nature12070. PubMed PMID: 23619693; PubMed Central PMCID: PMC3714217. 2: Rambo RP, Tainer JA. Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers. 2011 Aug;95(8):559-71. doi: 10.1002/bip.21638. Epub 2011 Apr 20. PubMed PMID: 21509745; PubMed Central PMCID: PMC3103662. 3: Luo M, Singh RK, Tanner JJ. Structural determinants of oligomerization of δ(1)-pyrroline-5-carboxylate dehydrogenase: identification
[ccp4bb] FW: post-doc opening at NCNR/NIST
Hi all, See below and contact Joseph Curtis (joseph.cur...@nist.gov) for more information. Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Group Department of Biochemistry and Biophysics Perelman School of Medicine at The University of Pennsylvania mailto:kgu...@upenn.edu kgu...@upenn.edu / 215.573.7260 / 267.259.0082 / http://www.stwing.upenn.edu/~kgupta www.stwing.upenn.edu/~kgupta From: Curtis, Joseph [mailto:joseph.cur...@nist.gov] Sent: Tuesday, November 4, 2014 4:35 PM Subject: post-doc opening at NCNR/NIST Hi, We are looking to hire a new post-doc to work with us. While this position is year-to-year it has been renewed continuously since 2009. If you have any interested graduate students / post-docs, please pass along the following job-description. U.S. Citizenship is required. Cheers, Joseph Postdoctoral Fellowship Opening at the National Institute of Standards and Technology The NIST Center for Neutron Research in Gaithersburg, Maryland has an immediate opening for a postdoctoral fellow to work on scattering applications to biotechnological problems to support a joint NIST-Amgen collaborative research project. The initial appointment is for one calendar year with an option of a second year pending the availability of continued funding. The position requires a Ph.D. in chemistry, physics, biophysics, biology, or related engineering disciplines. Experience in either scattering (x-ray or neutron), or computational chemistry or biotechnology is required. This position requires full U.S. citizenship. Interested applicants should send the curriculum vitae to mailto:joseph.cur...@nist.gov joseph.cur...@nist.gov. *** Joseph E. Curtis, Ph. D. Research Chemist NIST Center for Neutron Research 100 Bureau Drive, Gaithersburg, MD 20899 (301) 975-3959 mailto:joseph.cur...@nist.gov joseph.cur...@nist.gov *** Joseph E. Curtis, Ph. D. Research Chemist NIST Center for Neutron Research 100 Bureau Drive, Gaithersburg, MD 20899 (301) 975-3959 joseph.cur...@nist.gov
Re: [ccp4bb] SAXS facility and Cryo EM facility
What part of the world are you in? Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Group Department of Biochemistry and Biophysics Perelman School of Medicine at The University of Pennsylvania mailto:kgu...@upenn.edu kgu...@upenn.edu / 215.573.7260 / 267.259.0082 / http://www.stwing.upenn.edu/~kgupta www.stwing.upenn.edu/~kgupta From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ruby Sharma Sent: Thursday, June 12, 2014 12:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] SAXS facility and Cryo EM facility Hello folks can u please help me by telling that where can i avail SAXS facility?? and Cryo EM too?? Regards Ruby
Re: [ccp4bb] Problems with SANS data analysis
I agree with Ed's suggestion - the folks on that forum are very helpful and very insightful with regards to small-angle scattering. Some thoughts to offer: Mark - it's hard to evaluate from the Primus screen shots, simply because Primus is not rendering the experimental noise. I'd suggesting plotting your data out on a log-log plot in Origin or via the Igor Pro macros if this data is from an American beamline (NIST, ORNL) and using those renderings to evaluate. Rendering the experimental noise is important, as in those middle D2O concentrations you're going to have a considerable amount of incoherent scatter contributing to your profiles. In your first 30% data shown, the discrepancy is concerning. Check the reduction parameters and make sure the correct correction files were used for that particular sample to detector distance. The rendering of the experimental noise would be helpful in evaluating. In your 50% and 70% data, the experimental noise would be helpful to evaluate, but on the whole they seem pretty good. The discrepancy seen at middle Q in this profile (http://postimg.org/image/m358pazb7/) is a little concerning though. I'm concerned with the first set of 90% data you show, assuming the incoherent scatter is very low at that concentration of D20 and signal-to-noise is high. There seems to be a discrepancy in the middle Q regime (~0.15 Q) Again, might be worth double-checking the correction files used for reduction. HTH, Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Laboratory Perelman School of Medicine University of Pennsylvania BLOCKED::mailto:kgu...@stwing.upenn.edu kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed Pozharski Sent: Wednesday, August 07, 2013 11:54 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Problems with SANS data analysis This question may be better suited for more small-angle-oriented forum, e.g. http://www.saxier.org/forum/ On 08/07/2013 11:22 AM, Remec, Mark wrote: Dear CCP4bb, I have a few questions concerning SANS data recently collected that I'm having trouble analyzing. The data was collected at 2 different detector distances (4m, 2.5m) to achieve higher q-range, but I worry that the curves don't overlap enough at intermediate q, which might indicate a problem with the data. The links below are pictures of the corresponding datasets, before truncating the 4m high-q data and merging them into one. Is there a problem evident with the data, or am I imagining a problem? http://postimg.org/image/qb00y20qr/ http://postimg.org/image/8trbp7akj/ http://postimg.org/image/hni86axj7/ http://postimg.org/image/3sjxnu343/ http://postimg.org/image/4ysj0dgsj/ http://postimg.org/image/9ypz8bmf7/ http://postimg.org/image/m358pazb7/ http://postimg.org/image/jzuthmzib/ My second question concerns the values obtained in the analysis of the final scattering curves. The second sample in my experiment shows serious deviation in the values obtained for I(0) and Rg by Guinier analysis compared to the values obtained by the P(r) analysis. In other words, either the P(r) values match the Guinier and the P(r) fit is terrible, or else the P(r) fit is good but doesn't match the Guinier at all (5-10 difference in Rg, 2x difference in I(0)). I've checked to make sure the buffer subtraction algorithm was OK, and I'm pretty certain that the buffers were exact matches, so I don't know how to explain this variation. There's no evidence of aggregation or polydispersity to throw off the values, either. Does anyone know how this can happen? -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs
Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Mea culpa - I'm thinking minutes at 0.5 ml/min, not mLs! (clearly I'm overdue for my afternoon caffeine...) Kushol -Original Message- From: Zhang, Zhen [mailto:zhen_zh...@dfci.harvard.edu] Sent: Thursday, June 20, 2013 4:18 PM To: 'Kushol Gupta'; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Puzzling observation about size exclusion chromatography Hi Kushol, No. The void for the column is 8ml and the whole volume of the column is 24ml. You must be talking about a different column. Zhen -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kushol Gupta Sent: Thursday, June 20, 2013 4:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography Isn't 18 mLs into a Superdex 200 10/300 column run out near where the 670kD marker is, just after the void at ~15 mLs? Zhen, did you mean ~500kD rather than 5kD?. Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Laboratory Perelman School of Medicine University of Pennsylvania kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 Hi Zhen, I'm not sure that binding to a monoclonal antibody is good evidence that the protein is in a natively folded state. I would be suspicious of such a result as the protein could be improperly, which is causing it to interact with the column matrix. It could be useful to use some other techniques (Activity Assay, Circular Dichroism, DSC, Native Page etc. to validate the refolding). Best, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Loll [pat.l...@drexel.edu] Sent: 20 June 2013 20:39 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography If your protein elutes very late, that means it's binding to the column matrix (so all estimates of size go into the trash). Check to see that the ionic strength of buffer is reasonable (equivalent to, say, 150 mM NaCl). If so, then the only solution is to go to a different matrix type. Pat On 20 Jun 2013, at 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.=
Re: [ccp4bb] Do my SAXS data agree with the crystal structure?
Two cents - A good deal of caution must be exercised when working with composite particles such as a protein-DNA complex in SAXS because of the contrast problem. Simply, protein and DNA scatter differently in x-rays, with a bias towards the DNA component. As a result, experimental Rgs could be slightly deflated versus what their true values would be at infinite contrast. Mass estimation by I(0) analysis with a protein standard of known mass and concentration is not really valid because the contrast terms are different. Because the particle is heterogeneous in composition and distribution, shape reconstruction from SAXS alone, which assumes homogeneity, can also be misleading (although in practice it is still reasonably instructive). It is for these reasons that SANS and the contrast variation approach can be extremely useful. With those caveats, the strategy you describe - comparison of experimental and theoretical profiles from an experimental structure using CRYSOL or FoxS is definitely the best way to go in the case of a protein-DNA complex with SAXS alone. Showing comparisons of the experimental with the calculated should make the point. Test other possible models inferred from lattice packing to further your point (if applicable). Regarding populations of monomer and dimer - . it is generally good to constrain your interpretation of scattering data with other orthogonal solution measures which demonstrates the homogeneity of your complex in comparable experimental conditions, such as sedimentation velocity or gel filtration. . Have some determination of affinity of the complex in the same solution conditions (including temperature!). This will allow you to argue that your sample concentrations are well in excess of any monomer-dimer association behavior (eg, mixtures!). Scattering of mixtures can undermine your ability to accurately assess the structural properties of your complex. . Collect a concentration series and extrapolate to infinite dilution, if possible, to ensure elimination of the S(q) term from your data. Interparticle interactions can be an issue with complexes containing DNA if the buffers aren't quite right. (I've seen this a lot) Lastly, remember that the scattering profile represents the solution average of the particle, not just a single snapshot. Some discrepancies like those you note should be expected. Hope that helps, Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Laboratory HHMI / Perelman School of Medicine University of Pennsylvania kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xun Lu Sent: Saturday, June 16, 2012 2:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Do my SAXS data agree with the crystal structure? Dear all, I have solved a protein-DNA structure, and I also did SAXS to get some ideas of the solution structure. The SAXS data were good, no aggregation at all three tested concentrations. I tried to use Crysol to see if my crystal structure fits the SAXS. The fitting to the scattering profile seems good to me and the Chi2 is 1~1.4. Then I wanted to see how the P(r) looked like (wanted to make a figure for my paper:). I calculated the theoretical scattering profile of the crystal structure from an online server (FOXS). I then run GNOM to make P(r). To my surprise, this theoretical P(r) looks a little different from the P(r) of SAXS data. There's a very small bump that was peaked at 70A (Dmax is 108A, which seems reasonable from the crystal structure). The major peak was at 25A. As some people said, P(r) is indeed quite sensitive to subtle differences. The protein is a dimer in the crystal, although it can also bind DNA as a monomer (much more loosely). The estimated MW from SAXS indicates it's a dimer in solution as well. It seems that I got the information I wanted from the SAXS experiment, but maybe not. Due to the low resolution of SAXS, maybe I can only say that the majority is a dimer?? Would it be possible to see the monomer if there's only 10% of them in the solution? How to interpret the discrepancy between the P(r) from crystal and the P(r) from SAXS? Any comments are welcome! Xun Sent from my iPad=
Re: [ccp4bb] {**SPAM**} [ccp4bb] screen kit
The screen described here might be worth checking out as well:
Crystallization of bFGF-DNA aptamer complexes using a Sparse Matrix designed
for protein-nucleic acid complexes
Jamie J. Cannone, , Cindy L. Barnes, , Aniruddha Achari, and Craig E.
Kundrot ,
Journal of Crystal Growth
Volume 232, Issues 1-4, November 2001, Pages 409-417
Kushol
Kushol Gupta, Ph.D.
Research Associate
Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine
kgu...@mail.med.upenn.edu
215-573-7260 / 267-259-0082
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Vellieux Frederic
Sent: Thursday, May 26, 2011 2:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] {**SPAM**} [ccp4bb] screen kit
Hi,
Hampton research claims that their Natrix series of screening kits is
designed for nucleid acid and nucleic acid/protein complexes.
http://hamptonresearch.com/product_detail.aspx?cid=1sid=27pid=8
Fred.
dengzq1987 wrote:
hello all,
is there any screen kit that is highly effective for the
crystallization of protein-nucleic acids complexes?
deng.
Re: [ccp4bb] Histogram/Plot of Buried Surface Areas
Hi Jacob - These references might(?) be helpful: Proteins. 1995 Dec;23(4):580-7. Protein-protein interaction at crystal contacts. Janin J, Rodier F. J Mol Biol. 2004 Feb 27;336(4):943-55. A dissection of specific and non-specific protein-protein interfaces. Bahadur RP, Chakrabarti P, Rodier F, Janin J. Proteins. 2005 Jul 1;60(1):36-45. Hydration of protein-protein interfaces. Rodier F, Bahadur RP, Chakrabarti P, Janin J. The problem you describe is a tricky one - crystal packing can be deceiving. Complementing your structural studies with some biophysical measurements (ie: comparing computed hydrodynamic properties to those observed in gel filtration or centrifugation or SAXS or the like) could help flesh out these types of questions. Hope this helps, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller Sent: Wednesday, May 25, 2011 10:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Histogram/Plot of Buried Surface Areas Dear Crystallographers, is anyone aware of a reference or plot addressing buried surface area (or PISA output values) versus veracity of a complex? I am trying to determine the physiological relevance of a crystallographically-observed assembly, and would love to put my PISA output in the context of verified complexes versus crystal contacts. Thanks, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] DNA oligonucleotide purification
Hi Jamie - We've had good success with these columns from Dionex (the DNAPac PA-100 and the DNAPac PA200 (Analytical 4x250mm and Semi-Prep 9x250mm,)). We use them precisely for the application you describe. http://www.dionex.com/en-us/products/columns/bio/nucleic-acid/dnapac-pa100/l p-73370.html http://www.dionex.com/en-us/products/columns/bio/nucleic-acid/dnapac-pa200/l p-73371.html cheers, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine BLOCKED::mailto:kgu...@stwing.upenn.edu kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Wallen, Jamie Sent: Thursday, April 28, 2011 9:32 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DNA oligonucleotide purification All, Our laboratory is looking for a new column to purify short (40mers or less) DNA oligonucleotides that will be used for crystallization setups. I wanted to ask if anyone has suggestions of a column that will give excellent separation of N and N-1 products. We have a Shimadzu HPLC system. We are considering Dionex columns as well as Oligonucleotide Separation C18 columns from Waters, but if there are others that are better we would like to know. Any suggestions will be greatly appreciated. Thank you! Jamie Wallen
Re: [ccp4bb] DNA oligonucleotide purification
We're using an older Dynamax system that is outfitted with PEEK throughout. Cheers, Kushol From: Wallen, Jamie [mailto:jwal...@biochem.wustl.edu] Sent: Thursday, April 28, 2011 10:17 AM To: Kushol Gupta; CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] DNA oligonucleotide purification Kushol, Thank you for the reply. Might I ask what kind of HPLC system you have? Our HPLC is old and has stainless steel pumps, and to our knowledge this is not compatible with the Dionex columns (due to metal leaching). Dionex has recommended either a titanium or peek system. As our HPLC is still running well, we were hoping to find a column that can tolerate the stainless steel. Jamie On 4/28/11 9:11 AM, Kushol Gupta kushol.gu...@gmail.com wrote: Hi Jamie - We've had good success with these columns from Dionex (the DNAPac PA-100 and the DNAPac PA200 (Analytical 4x250mm and Semi-Prep 9x250mm,)). We use them precisely for the application you describe. http://www.dionex.com/en-us/products/columns/bio/nucleic-acid/dnapac-pa100/l p-73370.html http://www.dionex.com/en-us/products/columns/bio/nucleic-acid/dnapac-pa200/l p-73371.html cheers, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu BLOCKED::mailto:kgu...@stwing.upenn.edu 215-573-7260 / 267-259-0082 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Wallen, Jamie Sent: Thursday, April 28, 2011 9:32 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] DNA oligonucleotide purification All, Our laboratory is looking for a new column to purify short (40mers or less) DNA oligonucleotides that will be used for crystallization setups. I wanted to ask if anyone has suggestions of a column that will give excellent separation of N and N-1 products. We have a Shimadzu HPLC system. We are considering Dionex columns as well as Oligonucleotide Separation C18 columns from Waters, but if there are others that are better we would like to know. Any suggestions will be greatly appreciated. Thank you! Jamie Wallen
Re: [ccp4bb] 250 kDa standard
The Catalase tetramer is around there (232 kD according to the Table 1 in Mylonas and Svergun, J. Applied Cryst. 2007). The shape of the tetramer in the PDB (4BLC) seems reasonably globular. Cheers, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Alexandra Deaconescu Sent: Saturday, February 19, 2011 7:47 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] 250 kDa standard Dear ccp4bb enthusiasts: A question unrelated to ccp4: can anyone recommend a good 250 kDa standard for gel filtration that is commercially available? It could be a single polypeptide or an oligomer too... Thanks a lot! Bests, Alex
Re: [ccp4bb] MALLS analysis question
Hi Tommi - I find it to be very stable day-to-day. However, the alignment between UV, RI and light scattering can be very misleading depending on the size of the particle you're analyzing (and of course, that makes all the difference in the world in getting an accurate result). I find that very large things will have a scattering profile that doesn't align well at all with the UV and RI. What I usually do is use the alignment parameters from a small, more isotropic scatterer like cytochrome C or RNase A at high concentration for my experimental runs. Hope this helps, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of tommi kajander Sent: Friday, February 18, 2011 9:08 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] MALLS analysis question Hi, if you happen to have Wyatt's light scattering detector + their RI detector rEX, together with analytical HPLC/SEC system what are your experiences on the stability of the aligment? the digital connections dont seem to work for us. ie. apparently synchronizing the signals from different detectors (computers) to the analysis software computer dont work, even if the alignment (ie delay volumes between detectors) has been done. Should work with analog cables to one detector and then LAN connection from there on to the analysis PC. testing it.quit certain it will. but i would be happy to hear if anyone had similar problems Thanks! tommi Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] monomer-dimer
Hi Maia, this review and website might be a good place to start: http://analyticalultracentrifugation.com/images/AUCinProteinScience.pdf http://analyticalultracentrifugation.com/default.htm Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 Hi ccp4bb Could you please send me some references with the sedimentation equilibrium calculations of Kd, monomer/dimer ratio etc. Maia Maia Cherney wrote: Thank you. Now I understand the difference. I thought there was separation. Maia Xuewu Zhang wrote: Hi Maia, I have seen your post regarding this before and I just want to point out that you may have confused AUC (analytical ultracentrifugation) with gradient-based ultra-centrifugation methods for separating macromolecules. AUC does not involve separation of different species in the sample. There are two types of AUCs: sedimentation velocity and sedimentation equilibrium. In sedimentation equilibrium experiments, the system reaches the equilibrium at the end, and the monomer/dimer ratio, Kd, etc parameters can be worked out by fitting the data to a model globally. The shape of the molecule does not matter. For starters: http://en.wikipedia.org/wiki/Ultracentrifuge Xuewu Zhang On Wed, Aug 11, 2010 at 10:37 AM, chern ch...@ualberta.ca mailto:ch...@ualberta.ca wrote: Hi Anastassis, We are back to the same argument that AUC is not a good method. As everyone knows, it's a dynamic equilibrium between monomers and dimers that exists before separation. Once you started separation in any method, the equilibrium is disturbed now in each separated band. That will cause re-equilibration and constant migration of newly formed dimers from the monomer band and newly formed monomers from the dimer band. The t(eq) is the re-equilibration time. Your method of separation of monomers and dimers should be quick enough before any re-equilibration occurs (t(sep)t(eq)). Otherwise, you get a mess and smearing of bands. Also, most conventional methods depend on shape etc. I find SEC is most convenient. Maia - Original Message - *From:* Anastassis Perrakis mailto:a.perra...@nki.nl *To:* chern mailto:ch...@ualberta.ca *Sent:* Monday, July 05, 2010 2:38 PM *Subject:* Re: [ccp4bb] monomeric coiled coil--updated On 5 Jul 2010, at 22:04, chern wrote: Hi, Anastassis If you had just a monomer at the start time then t(eq) is the time to get to equilibrium with the dimer and vice versa. sorry to say but the definition of that time in a biophysical sense, is in my opinion equal to infinity and cannot be defined. I am being a bit pedantic here, but I am just saying that t(eq) cannot be defined, it can be approximated, and thus t(eq) is wrong to define. Why not talk about kD and kON and kOFF that have robust definitions based on kinetic properties and a physical meaning? When you separated the two bands (monomers and dimers) in AUC, and then the equilibrium is quickly established in each band again what's the point? So, to be successful in this method, you need to have t(eq) much lower than the separation run. Ideally, if you could separate monomers and dimers instantly and freeze them in the separated state, then you can have good estimate of the both fractions. I think this is clear. But, I disagree and I think what you say is wrong. The equilibrium is dynamic. Why do you insist there is a point in 'separation'? The monomer changes to a dimer and vise versa in a continuous fashion. All you can say is that in a given concentration the equilibrium is shifted towards one or the other form. But its a dynamic one. Even at a concentration which is 50-50 between two states, the molecules that are in one state or another are changing according to kinetic parameters that are characteristic for the complex. Even at 100% - lets say of a dimer - by your definition, (100% cannot exist since its reached asymptotically by any derivation about equilibriums) molecules will fall to monomer and will reassemble to a dimer rapidly. To be honest I think that talking about t(eq) is largely wrong in biophysical terms, since it does not exist. A. That's what I meant
Re: [ccp4bb] non-symmetric tetramer ? 2nd round
Fred, Two cents - I think the P1 SAXS solution should strongly guide your choice of symmetry constraint above all else in this case: do any of the symmetry-restrained shape reconstructions *improve* the statistics (chi) and stability of the shape (NSD) when compared to the P1 result? Also, it sounds like you have other data - do the theoretical Rs, f/fo, etc of the shapes generated agree well with your other measurements? Cheers, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 Of course, 222 has not a 4 axis, otherwise it would be a 4-fold axis. But that's the output of the program. P4 exp. model has a 4-fold axis along the longest axis, while the P222 MODEL has a 4-fold axis along the smallest, which doesn't make any sense. Can you imagine something build up with 4 identical subunits and 222 symmtry, but without a 4-fold axis at the molecular level (I mean at the envelop resolution level)? Em 29-07-2010 12:32, Vellieux Frederic escreveu: Hi, To quote you: even my P222 experimental envelop does have a 4-fold axis - this is not suprising, a particle with 222 symmetry does not have 4-fold symmetry. There are 3 mutually perpendicular 2-fold axes that intersect at the origin (of the particle, of the molecule) [and for the nomenclature, these axes are named the P Q and R axes]. Fred. Fred wrote: Thanks all of you who promptly replied my question. I should have been more precise. I was referring to the symmetry of the tetrameric particle (point symmetry) at the molecular level not at the atomic level. This question has arisen because I have collected some SAXS data of my protein in solution and I don't have a molecular model to superpose to the experimental envelop. Others experimental data, gel filtration and NAT-PAGE, suggest a tetrameric particle. On the other side, P1, P2, P222 and P4 experimental envelops are quite different. So, I am not sure which symmetry to take. Considering the native state (no ligands at all), 4 identical subunits and that the interface of oligomarization have to be conserved, I would take P222 or P4. However, I can be able to imagine such spacial arrangement without a 4-fold axis at the molecular level. Indeed, even my P222 experimental envelop does have a 4-fold axis. I appreciate if you could add some more comments on this. Thanks in advance, Fred
Re: [ccp4bb] SAXS EM comparison
Hi Andreas, this is the way I do it: I use the program SITUS to convert the volume (in some SITUS-friendly EM format) to a pdb bead model (vol2pdb). Then I'll input that PDB into CRYSOL to generate the theoretical scatter. I vaguely recall that Chimera has a SAXS function (not sure), but that might be worth googling as well. Cheers, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 Dear all, the other day I obtained SAXS data from which a low-resolution structural model was calculated. The model is simpler/less complex than one of the same protein that we obtained with cryo-EM. Is there a way to estimate theoretical SAXS data from a cryo-EM reconstruction to compare with the obtained raw data? Is there a program that does for a reconstruction what CRYSOL does for pdbs? I understand that there would be a huge amount of handwaving involved, but it might help us reconcile our models. Thanks. Andreas -- Andreas Förster, Research Associate Paul Freemont Xiaodong Zhang Labs Department of Biochemistry, Imperial College London http://www.msf.bio.ic.ac.uk
Re: [ccp4bb] SAXS on a coiled coil protein
Rongjin, With regards to the SAXS part of post: I'm guessing your collaborators are making this determination from the SAXS data based on a Kratky plot analysis? Given the inherently low resolution of this technique, it may be difficult to assign the profile observed to a specific secondary structure element without more analysis or other data. Is the profile concentration dependent? How well does the profile correlate with the theoretical scattering from your crystal structure? It could be simply that your two helices are not crossing in solution in the conditions tested. Using bead model approach like GASBOR or the EOM approach with your two atomic models of the helical portions linked by beads and modeled against the SAXS data might be very informative (ie: generating an ensemble of shapes and seeing what type of shapes best agree with the data). There's a very active community of small-angle scattering geeks on the forum at www.saxier.org - might be worth posting this question there as well. Cheers, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine BLOCKED::mailto:kgu...@stwing.upenn.edu kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Rongjin Guan Sent: Thursday, July 15, 2010 11:24 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] SAXS on a coiled coil protein Sorry for a non-ccp4 question. We have determined a structure which is mainly a coiled coil motif. The two helices are from the same protein chain linked by a short turn. However, the SAXS data indicates that this protein is probably natively unfolded or may have very flexible domains and linkers as commented by our collaborators who did the SAXS experiments. Could this be due to the shifting of the turn connecting the two helices? Has this kind of flexibility in the turn position in a coiled coil motif been observed in other coiled coil structures? Thank you Rongjin Guan
Re: [ccp4bb] Postdoc wanted - NIST-ARRA Fellowship Program
Please respond to Joseph Curtis (joseph.cur...@nist.gov) and Susan Krueger (susan.krue...@nist.gov ) if you are interested. =-=- Here is the link. NIST-ARRA Fellowship Program http://www.nistfellows.umd.edu/index.htm If there is someone that is interested then they should contact us so that we can help write a paragraph to support the application. Briefly, we are looking for a post-doc to help develop algorithms and software for the analysis of neutron scattering data using atomistic models. The applicant should have programming experience. Fluency in Python, C, Fortran, OpenGl, and/or experience in molecular simulations is a plus. Cheers, Joseph
Re: [ccp4bb] LS / RI detector systems
Hi Tommy, While I cannot comment on the Viscotek or Varian/Agilent products, I can say a few things about the Wyatt product: We have a Dawn Heleos II 18-angle instrument with inline QELS here in the lab along with one of their older Optilab RI instruments. We use TSK columns for protein characterizations and GE S200-like columns for protein-nucleic acid complexes. We've had the set-up for about 18 months now, and we're very happy with the product. It's become a routine-use instrument in the lab for the characterization of protein and protein-nucleic acid preparations that are about to go into crystal trials and SAXS experiments. If used properly, the accuracy and reproducibility of the mass determinations for protein samples are very good - usually within a few percent of actual mass and about as good as what I would get from an AUC sedimentation equilibrium experiment. (This becomes a trickier issue with composite particles like protein-DNA, as you have to implement a mass-averaged dn/dc figure and so on) We've used it successfully on systems where the particles range from 12 kD to upwards of 660 kD. The customer technical support is good, too. Hope this helps, Kushol Kushol Gupta, Ph.D. Research Associate Van Duyne Laboratory - HHMI/Univ. of Pennsylvania School of Medicine kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Tommi Kajander Sent: Sunday, June 20, 2010 7:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] LS / RI detector systems Dear All, I would like to ask people's comments on usage/preferences on different providers MALLS/RALS detectors and RI detectors (to combine with HPLC SEC) Mainly we are looking at Wyatt vs Viscotek (or perhaps Varian/Agilent now also) tetradetektors(or triple) detectors at the moment, i am not aware of many other options.. In particular what is your take on Wyatt MALSS accuracy *(why bother with several angles although i kind of like the software and instrument...) vs RALS for proteins (which end up being angle independent in scattering anyhow) --or what do you think overall of the different manufacturers equipments accuracy, etc... e.g. viscometer seems rather unnessary to me, as does online DLs.. Thanks for comments, Tommi -- Tommi Kajander, Ph.D., Docent Macromolecular X-ray Crystallography Research Program in Structural Biology and Biophysics Institute of Biotechnology P.O. Box 65 (Street address: Viikinkaari 1, 4th floor) University of Helsinki FIN-00014 Helsinki, Finland Tel. +358-9-191 58903 Fax +358-9-191 59940
Re: [ccp4bb] tips on crystallizing a Protein-DNA complex
Hi Clare, Two papers that might be worth checking out that address some of your questions - 1. Tan et al, Crystallization of the Yeast MATa2/MCM1/DNA Ternary Complex: General Methods and Principles for Protein/DNA Cocrystallization J. Mol. Biol. (2000) 297, 947±959 2. Cannone et al, Crystallization of bFGF-DNA aptamer complexes using a Sparse Matrix designed for proteinnucleic acid complexes Journal of Crystal Growth, 2001 232 (2001) 409417 cheers, Kushol Kushol Gupta, Ph.D. Mathilde Krim Fellow in Basic Biomedical Research Van Duyne Laboratory - Univ. of Pennsylvania School of Medicine BLOCKED::mailto:kgu...@stwing.upenn.edu kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 I was hoping people could give some tips on the best way to go about crystallizing a protein-DNA complex. I have a large amount of experience in protein crystallisation but have never tried co-crystallisation with DNA until I started this project. If you want to reply to me personally I will then post a summary. My protein is a dimer and has been shown by several methods to bind to DNA with high affinity (KD ~ nM) with a footprint of ~26 bp. I have several questions: 1.Do people routinely try different lengths of DNA? 2.Do you start with blunt or sticky ends? 3.Would purification of the resultant complex by gel filtration be a good idea as the interaction is so tight? 4.Which screens would you try first? 5.Where do you order the DNA from as there is a large difference in price depending on supplier. What scale do you go for and what purification? 6.We expect 1:1 binding. What ratios of DNA to protein are generally used (bearing in mind the inaccuracies of protein estimation)? 7.Any other useful tips? Thanks in advance for the suggestions and advice Clare Dr. Clare E. M. Stevenson John Innes Centre, Department Biological Chemistry Colney Lane Norwich Norfolk NR4 7UH
