1. NMR vs. Crystallography for 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.  = 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

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, , then do
side chains
NOE: Wuthrich, (1989) Science 243, 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
NCBI PubMed
Homology modeling tutorial
Principles of Protein Structure, Comparative Protein Modeling and Visualization
( DeepView    -  download a free version of this viewer.  Its also for linux computers.  (
A tutorial for Deep View was made by Gale Rhodes, the author of CMCC. (