Transcript Comparison of NMR and X

NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
As we have seen to this point, that an NMR
structure is determined indirectly by combining
NMR experimental data as target functions
with traditional geometrical potential energy
functions.
Conversely, an X-ray structure is determined
by directly fitting the structure against the
electron density maps. This approach still uses
XPLOR to refine the structure and maintain
proper geometry (bond lengths, bond angles)
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
As a result, a single optimal structure can be
determined to represent the experimental X-ray
data where the r-factor indicates the quality of
the fit and the data indicates the resolution of
the structure
The EMBO Journal (2000) 19(13) 3179
Conversely, the NMR data can be equally
represented by an ensemble of structures and
there currently is no corresponding equivalent
to the r-factor or resolution
Biochemistry (2000) 39(31), 9146-9156
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
The correctness of a solution to a particular crystal is
usually measured by the R-factor:
compares the experimentally observed intensities of
reflection:
with the intensities of reflection calculated from the
structure:
typically ranging from 16% (high resolution
structure) to 28% (lower resolution).
X-ray Diffraction Pattern for a Protein
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Example of Ultra-High
Resolution X-ray Diffraction
Pattern
Resolution increases (d) as you
move out concentric circles in
the X-ray diffraction pattern
Bragg equation: 2dsinf =nl
Xfd
X
Note: diffraction intensity decreases
as you move to outer circle
Acta Cryst. (2000). D56, 1015–1016
The resolution of the structure is the minimum separation of two groups in the
electron-density plot that can be distinguished from one another.
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
NMR and X-ray structures generally exhibit the same
fold
Local differences may be attributed to:
1) dynamics
2) crystal-packing interactions
3) solid vs. solution state
- solvent is present in crystals
- lowest energy conformer in crystal?
4) Resolution/experimental error
Nevertheless, there are some examples where
distinct functional differences are observed
between the NMR and X-ray structures
Protein Science (1996). 5:2391-2398.
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Illustration of the large differences
between the NMR (blue) and X-ray
(red) structures of the Ca2+–calmodulin
complex
X-ray structure suggested a “dumb-bell”
structure with an extended a-helix
NMR structure indicated the central
helix was unstructured and dynamic.
“The difference between the crystal and solution structures of Ca 2+–calmodulin
indicates considerable backbone plasticity within the domains of calmodulin, which is
key to their ability to bind a wide range of targets.”
Nature Structural Biology (2001), 8(11), 990-997.
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Protein Dynamics Is Routinely Measured From
NMR Data
Dynamic Data Is Also Implied From the X-ray BFactor (temperature factor in the PDB).
Overall Poor Correlation Between NMR Dynamic
Data and B-factors
1) dynamic regions may have low B-factors
if stabilized by an interaction not present in
solution
2) low dynamic regions may have high Bfactors due to resolution issues not related to
dynamics – various crystal contacts, lack of
uniformity in crystals, etc.
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
large ribosomal subunit X-ray structure
There is no theoretical limit to the size of the structure
that can be determined by X-ray crystallography.
Requires a crystal that diffracts!
- requires highly pure samples
- requires high solubility (~mM)
- requires high stability (crystal may take
weeks to months to form)
- requires absence of aggregation/ppt
- may requires seleno-Met labeling for
phase determination
- usually need to test 100s to 1,000s of
crystal conditions
- requires a protein that will form a crystal
(may require site-directed mutant, N-,Cterminal truncation or using sequences from
different species)
Science (2000) 289, 905-920
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Conversely, there is a molecular-weight upper limit for NMR
structures.

molecular-weight of a protein is related to its radius
which in turn is related to the protein’s rotational
correlation time (c) :
3
4r
c 
3kT
where:
r = radius
k = Boltzman constant
 = viscosity coefficient
rotational correlation time (c) is the time it takes a
molecule to rotate one radian (360o/2).
 the larger the molecule the slower it moves
 c is related to the efficiency of T2 relaxation

NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
As a Result of the Relationship Between MW, c and T2
as the MW of a protein increases, the NMR line-widths broaden to the point
of being undetectable
 also, the efficiency of correlating NOE and coupling constant information
decreases with increasing line-widths (MW)

Can estimate c for a spherical protein:
c  MW/2400 (ns)
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Illustrations of the Relationship Between MW, c and T2
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Consider an INEPT Based NMR
Experiment Where Chemical Shifts
are Correlated by Coupling Constants
transfer magnetization via
heteronuclear coupling from the
sensitive 1H to the less sensitive
15N and back to 1H for detection
 this module is a basic
component of 2D, 3D and 4D
NMR experiments

NMR and X-ray Structures
Comparison of NMR
and X-ray Structures

consider a two spin system, I & S:
z
A
Iz
Sz
z
B
Sz
 = 1/4JIS
free precession
(90x)I
Iy
Spin-Vector diagram of the
INEPT sequence illustrates the
evolution of magnetization as a
function of J by waiting a delay
() =1/4J
x
C
y
x
z
D
(180x)I
(180x)S
z
 = 1/4J IS
free precession
1/ 2 [-Iy - 2IxSz]
During This Same Time Period T2
relaxation also is Occurring
- as MW↑ & T2↓ the signal
decays significantly during 
- the peak is weak or
unobserved in the spectra
y
y
x
E
1/ 2 [Iy + 2IxSz](I +_
JIS) t1
x
z
G
(90y)I
(90x)S
x
y
-2IxSz
(Ix antiphase
magnetization)
z
acquire on N
or C channel
y
x
-2IzSy
(Sy antiphase
magnetization)
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Transfer amplitudes for antiphase/in-phase
conversion in CH, CH2 and CH3 spin systems
Again, we can see the efficiency
of magnetization transfer as a
function of time and J
NMR and X-ray Structures
Approximate Molecular Weight Limits for Structure Determination by NMR
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Crowded 1D NMR
Spectra of a Protein
How Has the Molecular Weight Limits
for NMR Been Increased?
By 13C and 15N Isotope Labeling and 2D,3D
& 4D NMR
- increase information content
- spread information out into
nD eliminates overlap
- generally impractical for natural
abundance isotopes  too low
Isotope
Natural
Abundance (%)
1H
99.98
13C
1.11
15N
0.37
Resolved 2D NMR
Spectra of a Protein
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
How Has the Molecular Weight Limits for NMR Been Increased?
By 2H Isotope Labeling and Deuterium Decoupling

lower gyromagnetic ratio of 2H to 1H, g[2H]/g[1H] = 0.15, so
replacement of 1H with 2H reduces line-widths
 by removing contributions from proton-proton dipolar
relaxation
1
1
 H- H scalar couplings
 eliminates an efficient relaxation pathway
• decreases N-H T2 by 2-fold,
• decreases 13C-H dipolar interactions by a factor of 15
 eliminates most of the sources of distance constraints
(hydrogens)
• only observe NH’s that rapidly exchange with water.
• decrease in spin-diffusion pathways
Annu. Rev. Biophys. Biomol. Struct. 1998. 27:357–406
13C-1H
13C-2H
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Effects of Deuterium Labeling
2D 15N-NH HSQC spectrum of the
30 kDa N-terminal domain of
Enzyme I from the E. coli
only 15N labeled
15N, 2H
labeled
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Effects of Deuterium Labeling
no deuterium labeling
3D HNCA spectrum of the 23 kDa
Shc PTB domain/phosphotyrosine
peptide complex.
deuterium labeled
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Effects of Deuterium Labeling
Labeling a protein with deuterium severely decreases the density and distribution of observable
1H that are required to observe distance constraints and calculate a protein structure
fully protonated
PLCC SH2 domain.
deuterated PLCC
SH2 domain.
hydrogens are depicted as gray spheres
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Effects of Deuterium Labeling
Can re-introduce back some distance
constraints with 1H-methyl labeling of Leu,
Ile and Val in a fully deuterated protein
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
How Has the Molecular Weight Limits for NMR Been Increased?
By The TROSY Experiment
 select spin from multiplet pattern with “best” relaxation
characteristics
 eliminate dipole-dipole coupling (DD) and chemical shift
anisotropy (CSA)
1
15
 normally decouple H- N coupling (A)
 multiplet is observed with different line-widths if
1H-15N coupling is present (separation in peaks is J)
relaxation rates of different components of the multiplet. Note
different factors contribute to the individual relaxation rates
Proc. Natl. Acad. Sci. USA (1997) 94, 12366–12371
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Effects of TROSY Experiment
15N, 2H
labeled gyrase (45 kDa)
Current Opinion in Structural Biology 1999, 9:594–601
NMR and X-ray Structures
Comparison of NMR
and X-ray Structures
Effects of TROSY Experiment
The TROSY effect is field dependent
with a maximum at ~ 1GHz
The TROSY experiment also requires
deuterium labeling.
The TROSY effect is MW dependent,
more pronouncement for larger MW
proteins.