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Introduction into
solid-state NMR
Barth van Rossum, 21.02.2011
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Barth-Jan van Rossum: Solid-State NMR
X-ray crystallography requires high-quality single crystal
some systems notoriously difficult to crystallize
typical solid-state NMR systems:
membrane-integrated receptors, aggregates, fibrils, intact
organelles, (bio-)polymers...
Solid-state NMRno need for large well-ordered crystals or
highly-purified proteins
works for immobilised proteins, no inherent limitation on
complex size
Solid-state MAS NMR very well-suited to study protein complexes
that are difficult
to crystallize, insoluble or have tendency to aggregate.
Methods for structure determination (with atomic resolution)
rapid isotropic tumbling of the molecules
Solution-state NMRrequires rapid reorientation of soluble
biomolecules
Difficult to study proteins that either are large (> 50 kDa)
or form
large complexes (aggregates, fibrils, membrane proteins)
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Barth-Jan van Rossum: Solid-State NMR
proton chemical shift (ppm)
solid-state NMRno magic angle spinning no 1H-1H decoupling
liquid-state NMR
solids-state NMR vs. liquid-state NMR
100 80 60 40 20 0 -20 -40 -60 -80
5,000 Hz
40,000 Hz
dipolar coupling between protons:
~ 120 kHz @ 1.0 ~ 40 kHz @ 1.5
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Barth-Jan van Rossum: Solid-State NMR
solids-state NMR vs. liquid-state NMR
1 KW
720,844 mm3(1 mW / mm3)
67 mm3(15 W / mm3)
Solid-state NMR uses high-power RF pulses (1000 W) to manipulate
the spins
5,000 Hz
15 mm3(70 W / mm3)
Solid-state NMR is brute force
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Barth-Jan van Rossum: Solid-State NMR
tumbling rate
spectralcrowding
complex size versus protein size
solids and liquids:
protein size determines spectral crowding
liquids:
complex size determines tumbling rate
no inherent limitation on complex size : What does it mean?
Methods for structure determination (with atomic resolution)
SH3 domain62 residues (~7 kDa)
OmpG281 residues (~34 kDa)
solids:
no upper limit for complex size
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Barth-Jan van Rossum: Solid-State NMR
MAS mimics orientation averaging in liquids
by imposing a collective reorientation of all
molecules around a special axis
tumbling rate not dependent on size
Magic-Angle Spinning (MAS)
whats more:
- without MAS, only single crystals give a
high solid-state NMR resolution
- with MAS, you do not need crystals
However, it helps to have some sort of local
order
tumbling rate
spectralcrowding
complex size versus protein size
drive
bearing
Magic-Angle Spinning (MAS)
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Barth-Jan van Rossum: Solid-State NMR
What are good MAS NMR samples?
For MAS NMR, you do not need crystals
However, it helps to have some sort of local order
non-ordered solid: each molecule (protein) in a different
chemical environment
(identical spins experience different local field)
broadening of the NMR lines
NAV(micro-crystalline)
QIY(lyophilized)
non-ordered
ordered
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Barth-Jan van Rossum: Solid-State NMR
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ppm
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Barth-Jan van Rossum: Solid-State NMR
ppm
X-ray: long-range order required,
single-crystals required
MAS NMR: no long-range order required,
- short-range order sufficient
- non-crystallographic symmetries
Ordered system provides better NMR
resolution. But: no need for crystals in
the classical sense
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Barth-Jan van Rossum: Solid-State NMR
systems with high native
symmetry not good enough
for X-ray crystallography
- non-crystallographic symmetry
- rotation symmetry
- combination of rotation and
translation: helical symmetry
X-ray crystallography
requires unit-cell that
repeats in all directions
and is related by
translation symmetry only
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Barth-Jan van Rossum: Solid-State NMR
175180 ppm
70
65
60
55
50
45
40
35
30
25
20
15
10
5
70 65 60 55 50 45 40 35 30 25 20 15 10 ppm
70
65
60
55
50
45
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30
25
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LH2-complex: ~150 kDa9 protein units (in total 846 AA)+ pigments
(BChl a and carotenoid)
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Barth-Jan van Rossum: Solid-State NMR
unshielded
shielded
Electrons shield the nuclear spins from the external magnetic
field
Chemical shift anisotropy (CSA)
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Barth-Jan van Rossum: Solid-State NMR
solids: interactions depend on orientation of molecule
these interactions are called anisotropic
limit resolution in NMR spectra of biological macromolecules
liquids: rapid random tumbling averages
anisotropic chemical shifts and couplings
Chemical shift anisotropy (CSA)
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Barth-Jan van Rossum: Solid-State NMR
Chemical shift anisotropy (CSA)
axial symmetry
molecule perpendicular to B0 maximum deshielding
molecule parallel to B0 maximum shielding
non-axial symmetry
shielding is different in all three dimensions
spherical symmetry
shielding is similar in all three dimensions, CSA ~ 0
HCCH
H C C H
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Barth-Jan van Rossum: Solid-State NMR
Chemical shift anisotropy (CSA)spherical symmetry
11 = 22 = 33
axial symmetry
11 = 22 (or)
22 = 33
non-axial symmetry
11 = 22 = 33
11 = 2211,22 < 33
22 = 3322,33 > 11
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Barth-Jan van Rossum: Solid-State NMR
Chemical shift anisotropy (CSA)
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Barth-Jan van Rossum: Solid-State NMR
drive
bearing
Magic-Angle Spinning (MAS)
Magic-Angle Spinning (MAS)
isoAnisotropic interactions can be suppressed using a
technique called magic-angle spinning
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Barth-Jan van Rossum: Solid-State NMR
Magic-Angle Spinning (MAS)
magic angle : the angle between the body diagonal of a cube and
the z-axis
by rotation around this axis, a vector along z will cross the x
and y-axes
54.7
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Barth-Jan van Rossum: Solid-State NMR
Magic-Angle Spinning (MAS)
note : for any rotation, one could construct a 1x1x1 cube
around
however, the z-axis is special (B0 direction!) and has to be one
of the axes
