3J Scalar Couplings 3 J HN-H a
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Transcript of 3J Scalar Couplings 3 J HN-H a
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3J Scalar Couplings 3JHN-H
•The 3J coupling constants are related to the dihedral angles by the Karplus equation, which is an empirical relationship obtained from molecules for which the crystal structure is known.
• The equation is a sum of cosines, and depending on the type of topology (H-N-C-H or H-C-C-H) we have different parameters:
3JN = 9.4 cos2( - 60 ) - 1.1 cos( - 60 ) + 0.4
3J = 9.5 cos2( - 60 ) - 1.6 cos( - 60 ) + 1.8
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Series1
Sometimes 3J has no unique solution and extra information is required! CS, NOE, Ramachandran plot!
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Measurement of CouplingsProblem: large linewidth, no splitting
quantitative J experiments
J is calculated from an intensity ratio
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IPAP-HSQC
Measure 1JHN-N by combining an InPhase and an AntiPhase HSQC
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J-Correlation through H-Bonds
ubq.pdb
H-N-C’ and
H-N … O=C’
Correlations
e- density in the H-bond
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B0 Dependence of Splittings Indicates Dipolar Contributions
Incomplete averaging of the dipolar interaction due to partial alignment in the magnetic field
IS=h*IS/rIS3(3cos2IS-1)
Angular dependance allows the measurement of angles and relative orientations, which has not been possible in NMR
Contains information about angles!
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Field induced Alignment• Dipolar Contribution to J Splitings• Proportional to B0
2, but effects are very small
• Few Hz in molecules with a large magnetic anisotropy e.g. 2gat.pdb
• ‘Artificial’ Alignment required
DIS is measured as the different splitting between different B0 fields
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Induced Alignment
Phospholipid
‘Bicelles’
colloidal
Phage particles
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Surfaces may be additionally charged to modulate the alignment
sample stability can be a BIG problem
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NMR in LC Phases
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NMR in Liquid Crystals
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Dipolar couplings along a Protein Backbone
Measured as difference in splitting between aligned (left) and isotropic phase (right) IS=JIS+DIS
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Dipolar Coupling
The magnitude of the residual dipolar coupling depends on the alignment tensor:
5 parameters
Da/Dr: magnitude and rhombicity
+ 3 rotation angles: orientation relative to the .pdb frame
Knowing the alignment tensor (e.g. by least squares fitting) DC can be simulated and compared to experimental data
(in the principal axis frame)
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Motion along a Conedipolar couplings can be used as restraints in
NMR structure determination
The measurement of a residual dipolar coupling limits the the orientation of a bond vector (relative to the alignment tensor) to a narrow cone on a unit sphere
It restricts the orientaion relative to a ‘global’ alignment frame
not relative to other vectors
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Two Tensors!almost unique solution (intersection of cones)
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Dipolar Homology
Arbitrary fragments from the .pdb are fitted to the collected dipolar couplings
The dipolar agreement is used for the scoring
The best fragments are kept
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Dipolar Homology Mininguse measured DC to search for matching
overlapping peptide fragments
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Molecular Fragment Replacement
Fragments must share one common alignment frame
So the relative orientation can be inferred
Ambiguities:
0, 180x, 180y, 180z can be resolved by coordinate overlap
Use ‘Long Range Information in the Assembly Process
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Assemble a Protein Structure
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Protein Structure by MFR