NMR vs. Crystallographyfor CHEM 645

Brian Bahnson

Department of Chemistry & Biochemistry

University of Delaware

Through space - NOE

Distance Restraints

NOE 1/r6. f (c)

Tortional Restraintsthrough bond J-coupling

observed calculated

X-ray Refinement

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

X-ray term ideal geometry

hkl

NMR Refinement

= wNMR distance restraint + tortional restraint + wideal Etotal

NMR term ideal geometry

violations violations

13C, 15N labeling, homogeneity

Ikura et al., (1989) Biochemistry 29, 4659-4667., then do

side chains

NOE: Wuthrich, (1989) Science 243, 45-50. also: J-coupling ~ tortion

Bigger magnet is better – 600, 750 or 900 MHz

2-D and 3-D homonuclear and heteronuclear pulse sequences

Pattern recognition, build 100 models, select 20 best

Minimize restraint violations, keep “good” geometry = WN(distance restraint violation + WI (Etotal)

NMR Structuresof closed form calmodulin

X-ray Crystal Structuresof calmodulin

Bundle of 20 NMR models of calmodulin

Cases of Bundle Spread• Missing restraints • dynamics

Crystallography vs. NMR – advantage/disadvantages

1) Experimental difficultiesneed for homogeneity in commonneed good crystals for crystallographyneed 13C and 15N label for NMRsize limits of NMR techniquesolubility an issue for each technique

2) Reported structure(s) look different – i.e. bundlecrystal vs. solution structure

3) Complementary informationhigh resolution vs. dynamicspositional 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

Swiss Modelhttp://swissmodel.expasy.org//SWISS-MODEL.html

NCBI PubMedhttp://www.ncbi.nlm.nih.gov/sites/entrez/

Homology modeling tutorialhttp://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) 

Homology Modeling Links