NMR vs. Crystallography for CHEM 645

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Presentation transcript:

NMR vs. Crystallography for CHEM 645 Brian Bahnson Department of Chemistry & Biochemistry University of Delaware

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

Tortional Restraints through bond J-coupling

 = wF  whkl (|Fo| - |Fc|)2hkl + wideal Etotal 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

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)

NMR Structures of closed form calmodulin

X-ray Crystal Structures of calmodulin

Bundle of 20 NMR models of calmodulin Cases of Bundle Spread Missing restraints dynamics

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

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

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)