NMR vs Crystallography for CHEM 645 Brian Bahnson

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

Distance Restraints Through space - NOE 1/r 6. f (tc)

Tortional Restraints through bond J-coupling

NMR Refinement NMR term ideal geometry = w. NMR distance restraint + tortional restraint + wideal Etotal violations violations X-ray Refinement ideal geometry X-ray term = w. F whkl (|Fo| - |Fc|)2 hkl + wideal Etotal hkl observed calculated

13 C, 15 N 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 1) Experimental difficulties need for homogeneity in common need good crystals for crystallography need 13 C and 15 N label for NMR size limits of NMR technique solubility an issue for each technique 2) Reported structure(s) look different – i. e. bundle crystal vs. solution structure 3) 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 1) 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 Pub. Med 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-index. htm) Deep. View - 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)