1 / 12

NMR vs. Crystallography for CHEM 645

NMR vs. Crystallography for CHEM 645. Brian Bahnson Department of Chemistry & Biochemistry University of Delaware. Distance Restraints. Through space - NOE. NOE  1/r 6 . f ( t c ). Tortional Restraints through bond J-coupling. NMR Refinement. ideal geometry. NMR term.

KeelyKia
Download Presentation

NMR vs. Crystallography for CHEM 645

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. NMR vs. Crystallographyfor CHEM 645 Brian Bahnson Department of Chemistry & Biochemistry University of Delaware

  2. Distance Restraints Through space - NOE NOE  1/r6.f (tc)

  3. Tortional Restraints through bond J-coupling

  4. NMR Refinement ideal geometry NMR term  = wNMR distance restraint +  tortional restraint + wideal Etotal violations violations X-ray Refinement ideal geometry X-ray term  = wFwhkl (|Fo| - |Fc|)2hkl + wideal Etotal hkl calculated observed

  5. 13C, 15N labeling, homogeneity Bigger magnet is better – 600, 750 or 900 MHz 2-D and 3-D homonuclear and heteronuclear pulse sequences Ikura et al., (1989) Biochemistry 29, 4659-4667., then do side chains NOE: Wuthrich, (1989) Science 243, 45-50. also: J-coupling ~ tortion Pattern recognition, build 100 models, select 20 best Minimize restraint violations, keep “good” geometry  = WN(distance restraint violation + WI (Etotal)

  6. NMR Structures of closed form calmodulin

  7. X-ray Crystal Structures of calmodulin

  8. Bundle of 20 NMR models of calmodulin • Cases of Bundle Spread • Missing restraints • dynamics

  9. Crystallography vs. NMR – advantage/disadvantages • Experimental difficulties • need for homogeneity in common • need good crystals for crystallography • need 13C and 15N label for NMR • size limits of NMR technique • solubility an issue for each technique • Reported structure(s) look different – i.e. bundle • crystal vs. solution structure • Complementary information • high resolution vs. dynamics • positional amplitude, • certainty time domains

  10. Molecular Replacement – homology modeling Molecular Replacement (MR) – another method to estimate phases – use a structurally homologous protein >25% sequence identity is sometimes possible >50% sequence identity is a safe bet • Make search model • - find structural model of sequence homolog • - from sequence alignment and homolog structure, create model • - mutate or trim down to what the two proteins have in common • - energy minimize to eliminate bad geometry (intro to refinement) Suppose you wanted to make a model of BSIDH

  11. Homology Modeling Links Swiss Model http://swissmodel.expasy.org//SWISS-MODEL.html NCBI PubMed http://www.ncbi.nlm.nih.gov/sites/entrez/ Homology modeling tutorial http://molvis.sdsc.edu/protexpl/homolmod.htm Principles of Protein Structure, Comparative Protein Modeling and Visualization (http://swissmodel.expasy.org//course/course-index.htm)DeepView    -  download a free version of this viewer.  Its also for linux computers.  (http://au.expasy.org/spdbv/) A tutorial for Deep View was made by Gale Rhodes, the author of CMCC. (http://www.usm.maine.edu/~rhodes/SPVTut/index.html)

More Related