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NMR vs. Crystallography for CHEM 645 PowerPoint PPT Presentation


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.

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NMR vs. Crystallography for CHEM 645

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Nmr vs crystallography for chem 645 l.jpg

NMR vs. Crystallographyfor CHEM 645

Brian Bahnson

Department of Chemistry & Biochemistry

University of Delaware


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Distance Restraints

Through space - NOE

NOE  1/r6.f (tc)


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Tortional Restraints

through bond J-coupling


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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


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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)


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NMR Structures

of closed form calmodulin


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X-ray Crystal Structures

of calmodulin


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Bundle of 20 NMR models of calmodulin

  • Cases of Bundle Spread

  • Missing restraints

  • dynamics


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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


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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


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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)


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