NMR and Xray Structures

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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)
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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 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
The EMBO Journal (2000) 19(13) 3179
Biochemistry (2000) 39(31), 9146-9156
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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
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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, 10151016
The resolution of the structure is the minimum
separation of two groups in the electron-density
plot that can be distinguished from one another.
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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). 52391-2398.
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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
Ca2calmodulin 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 Ca2calmodulin 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.
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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
B-Factor (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 B-factors due to
resolution issues not related to dynamics
various crystal contacts, lack of uniformity in
crystals, etc.
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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-,C-
terminal truncation or using sequences from
different species)
Science (2000) 289, 905-920
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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 proteins
  rotational
• correlation time (tc)

where r radius k Boltzman constant h
viscosity coefficient

• rotational correlation time (tc) is the time it
  takes a
• molecule to rotate one radian (360o/2p).
• the larger the molecule the slower it moves
• tc is related to the efficiency of T2 relaxation

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NMR and X-ray Structures
Comparison of NMR and X-ray Structures

• As a Result of the Relationship Between MW, tc
  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 tc for a spherical protein tc
MW/2400 (ns)
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NMR and X-ray Structures
Comparison of NMR and X-ray Structures
Illustrations of the Relationship Between MW, tc
and T2
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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

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NMR and X-ray Structures
Comparison of NMR and X-ray Structures
Spin-Vector diagram of the INEPT sequence
illustrates the evolution of magnetization as a
function of J by waiting a delay (D) 1/4J

• During This Same Time Period T2 relaxation also
  is Occurring
• - as MW? T2? the signal
• decays significantly during D
• - the peak is weak or
• unobserved in the spectra

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NMR and X-ray Structures
Comparison of NMR and X-ray Structures
Transfer amplitudes for antiphase/in-phase convers
ion in CH, CH2 and CH3 spin systems
Again, we can see the efficiency of magnetization
transfer as a function of time and J
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NMR and X-ray Structures
Approximate Molecular Weight Limits for
Structure Determination by NMR
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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

Resolved 2D NMR Spectra of a Protein
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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,
  g2H/g1H 0.15, so
• replacement of 1H with 2H reduces line-widths
  
• by removing contributions from proton-proton
  dipolar
• relaxation
• 1H-1H 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 NHs that rapidly exchange with
  water.
• decrease in spin-diffusion pathways

13C-1H
13C-2H
Annu. Rev. Biophys. Biomol. Struct. 1998.
27357406
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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
15N, 2H labeled
only 15N labeled
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NMR and X-ray Structures
Comparison of NMR and X-ray Structures
Effects of Deuterium Labeling
3D HNCA spectrum of the 23 kDa Shc PTB
domain/phosphotyrosine peptide complex.
deuterium labeled
no deuterium labeling
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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
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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
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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)
• normally decouple 1H-15N 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, 1236612371
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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,
9594601
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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.