Varian 700 MHz NMR Magnet
Solving structures by NMR
PREMIUM SHIELDED PERFORMANCE
The Varian 700 MHz NM
Shielded design, is the re
of the Superscreened™ t
benefits for high resoluti
was pioneered by Magne
subsidiary of Varian, Inc.
Some slides adapted from Joanna Swain,
now at Adnexus Therapeutics
700 MHz, 16.4 T
Significant reduction
magnets minimizes sp
on surrounding areas
Minimized ceiling hei
easier siting
Concepts to be covered
 Why use NMR instead of crystallography?
 The basics of structure determination:
What is the data, and how is it used?
 How to evaluate the quality of an NMR structure.
How does it compare to a crystal structure?
Well focus on solving protein structures using
solution-state NMR, but many of the same
considerations apply to nucleic acids and/or solid
state techniques.
What do you already know about NMR?
•  Why the constant push to higher field?
•  Why so much alphabet soup ?
COSY, NOESY, TROSY, RDC
•  Why is NMR limited to small proteins – or is it?
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Comparing NMR and crystal structure determination
NMR structures
Crystal structures
Family or ensemble of
structures calculated
Single structure calculated
(more satisfying??)
Limited to smaller
proteins
No limit on protein size
Protein must be
soluble at 50 µM*
Protein must first be
crystallized
Can observe protein
dynamics
Static structure
1st structure 1984
1st structure 1959
•  Sample requirements were 0.5 mL x 1 mM  100µL x 50 µM (100-fold less)
“Macromolecular NMR Spectroscopy for the nonspectroscopist” Kwan et al (2011) FEBS J. 278, 687-703.
Current pdb is 87% x-ray, 12% solution NMR, 1% other
NMR has lots of uses beyond structure determination
- NMR is especially useful for studying protein complexes and protein dynamics
- applicable to minor species, in cell, …
Talk Overview
 Some basics: chemical shift, multi-dimensional methods
 Acquiring the data that go into the structure calculation
- introduction to the NOE and distance restraints
- the problem of sequential assignment
 Structure calculation
 Assessing structural quality
 Emerging methods/applications
Each hydrogen atom (proton) in a protein resonates at a characteristic frequency on
the NMR chemical shift scale, defined by its local structural environment.
Wüthrich, J. Biomol. NMR, 27: 13-39, 2003
Resonances in folded proteins are much better dispersed than in unfolded ones, due
to unique environments with shielding and/or enhancement of the externally applied
magnetic field by local structure.
http://u-of-o-nmr-facility.blogspot.com/2008/03/2d-nmr.html
Correlations:
Through-bond (J coupling)
Through-space (dipolar coupling)
“Macromolecular NMR Spectroscopy for the nonspectroscopist” Kwan et al (2011) FEBS J. 278, 687-703.
The Data
The nuclear Overhauser enhancement (NOE)
 Individual protons in the protein act as tiny dipoles, and two dipoles that are close in
space affect one another. The closer they are, the stronger the effect (dipolar coupling).
 The interaction can be monitored in a two-dimensional NOESY correlation spectrum
Off-diagonal peaks correspond to NOEs between two protons in the protein
The intensity of the peak is proportional to r-6 (r = distance between protons)
Limited to protons within about 5 Å of each other
Wüthrich, J. Biomol. NMR, 27: 13-39, 2003
The Data
The Sequential Assignment Problem!
 Each off-diagonal peak represents a
short-distance interaction between two
specific protons within the protein
sequence. Need to know resonant
frequency of each proton.
 Integration of peak intensity gives
interproton distance (strong, medium,
weak).
 Hundreds or thousands of inter-proton
distances are used to calculate threedimensional structures that are
consistent.
Wüthrich, J. Biomol. NMR, 27: 13-39, 2003
The Problem of Sequential Assignment
Solution: Use through-bond correlations (as opposed to the throughspace correlations that underlie the NOE) to discover which resonances in
the spectrum are connected through bonds in primary sequence.
Different strategies are utilized for small proteins versus larger proteins, where
peak overlap becomes more of a problem.
Wüthrich, J. Biomol. NMR, 27: 13-39, 2003
Sequential Assignment for proteins <15 kDa
 Two-dimensional COSY spectrum shows correlations between protons connected
through three or fewer bonds (indicated by ······, below left).
 Each residue is a closed system, called a spin system , isolated by the carbonyl.
Can usually identify a spin system as a particular amino acid type based on the
number of resonances and their chemical shifts.
 Spin systems are connected sequentially using short-range NOE correlations from a 2D
NOESY spectrum, usually dαN and dNN (indicated by ------, below left).
2D COSY
Wüthrich, J. Biomol. NMR, 27: 13-39, 2003
From NMR of Proteins & Nucleic Acids, by K. Wüthrich,
pp. 54-55
Sequential assignment for larger proteins (>15 kDa)
Two problems with larger proteins:
1. Many more protons lie in same spectral range, and peaks overlap.
2. Molecule tumbles more slowly as a whole, leading to broad peaks.
 Problem #1 can be overcome by
labeling protein with other NMRsensitive nuclei, such as 13C and 15N.
 Overcrowded spectra can then be
spread out in additional dimensions.
 Accomplished by growing cells in a
minimal growth medium with single
carbon/nitrogen sources (e.g. 13Cglucose and 15NH4Cl for E. coli).
 Disadvantage is the cost of isotopic
labeling.
Wider, Biotechniques, 29: 1278-1294, 2000
Sequential assignment for larger proteins (>15 kDa)
With carbon and nitrogen labeling, spin systems no longer isolated by the carbonyl.
- Can utilize through-bond couplings to trace directly along backbone from
one amino acid to the next
(Slices removed from 3D spectrum)
Example: An HNCA experiment
yields a strong intra-residue
correlation between the amide
proton, nitrogen and alpha carbon
(peaks labeled i in figure), plus a
weak correlation from the amide
proton and nitrogen to the alpha
carbon of the i-1 (preceding)
residue (peaks labeled s in
figure).
Wider, Biotechniques, 29: 1278-1294, 2000
More tricks for even larger proteins (>25 kDa)
 Segmental isotopic labeling can solve problems with peak overlap:
♦ Two portions of protein are expressed separately, with only one
isotopically labeled.
♦ Two segments are then ligated in vitro to re-create the full-length
protein.
N
C
N
+
N
C
C
 Partial labeling with deuterium slows relaxation of NMR signals, and can
narrow peaks .
 New TROSY and CRINEPT experiments give sharper peaks for very
large proteins, especially with high-field spectrometers (900 MHz).
Some big proteins studied to date:
82 kDa malate synthase-protein global fold determined (L.E. Kay, 2005)
110 kDa dihydroneopterin aldolase octamer assigned (K. Wüthrich, 2000)
900 kDa GroEL tetradecamer partially assigned (K. Wüthrich, 2002)
720 kDa proteasome gates regulating proteolysis (L.E. Kay, 2010)
“Macromolecular NMR Spectroscopy for the nonspectroscopist” Kwan et al (2011) FEBS J. 278, 687-703.
Structure Calculation
 List of unambiguous structural restraints input into distance geometry or simulated
annealing protocol
- a set of 30-100 structures are calculated that are consistent with restraints
- structures are refined by restrained molecular dynamics or energy minimization
 Initial structures usually of poor quality due to inadequate numbers of NOEs or
incorrectly assigned NOEs.
-structures help to assign NOEs that were ambiguous, and fix incorrect ones.
 Repeat this process iteratively. 15-25 best structures are selected for NMR model.
Wider, Biotechniques, 29: 1278-1294, 2000
A typical representation of an NMR structure
20 structures
superimposed,
all consistent
with the
available data
Wüthrich, J. Biomol. NMR,
27: 13-39, 2003
 Backbone and core side chains usually better defined than the
solvent-exposed side chains and the chain termini.
 Ill-defined regions may indicate conformational dynamics in solution
or a lack of data in that region.
- Dynamics can be confirmed by relaxation measurements
 Remember, proteins are not static! Dynamics can be substantial
and functionally important.
1tvj: chicken cofilinbackbone rmsd = 0.25 Å ± 0.05 Å for residues 5-166
“Macromolecular NMR Spectroscopy for the nonspectroscopist” Kwan et al (2011) FEBS J. 278, 687-703.
Assessing Structural Quality
1998 IUPAC Task Force recommended the following structural statistics be
reported:
1. Number and type of NOEs used {intraresidue, sequential, medium range (≤5
residues apart), long range (>5 residues apart), intermolecular}
2. Number of torsion angle restraints
3. Number of hydrogen bond restraints
4. Maximum restraint violation and the average violation per constraint
5. Deviations from idealized geometry (I.e., unusual bond lengths or bond
angles)
6. Precision of structures: RMSD with respect to the mean structure (backbone
versus all heavy atoms)
7. Percentage of residues falling into allowed regions of φϕ space
1 and 6 are the best indicators of structural quality.
Goal:
1. >20 restraints per residue
2. 0.3-0.6Å rmsd for backbone atoms, 0.5-0.8Å rmsd for heavy atoms
derived structural restraints per structured residue.
examining the percentage of structures determined
Table 1. A guide for judging the ‘resolution’ of NMR-derived protein structures.
Assessment criterion
Very high resolution
High resolution
Medium resolution
Low resolution
Restraints per residuea
Backbone rmsd (Å)b
Heavy-atom rmsd (Å)b
Ramachandran
Plot quality (%)c
Example PDB file
> 18
< 0.3
< 0.75
14–18
0.3–0.5
0.75–1.0
10–15
0.5–0.8
1.0–1.5
< 10
> 0.8
> 1.5
> 95
1TVJ [63]
85–95
2IL8 [65]
75–85
2FE0 [66]
< 75
1LMM [67]
a
Total number of interproton-distance, dihedral-angle and hydrogen-bond restraints per residue. Disordered regions should be excluded from
this calculation, and it is important that only structurally relevant restraints are included in the count. Unfortunately, many NMR studies give
a misleading indication of the true number of structural restraints by including interproton distances that do not restrain the protein conformation. For example, an upper-limit distance restraint of 4.5 Å between the Ha of residue i and HN of residue i + 1 is not a structural
restraint because this distance is always less than 3.5 Å, regardless of the conformation of the protein [68]. Note that interproton distance
restraints are often divided into categories of ‘intraresidue’, ‘sequential’ (NOEs between protons on adjacent residues), ‘medium range’
(NOEs between protons separated by two to five residues) and ‘long range’ (NOEs between protons separated by more than residues). The
number of medium-range and long-range restraints is the most important factor when determining the global fold of the protein. b rmsd calculated versus mean coordinate structure, with disordered regions excluded. c Percentage of residues in most favoured region of the Ramachandran plot as judged by MOLPROBITY. Note that these numbers will be slightly lower if PROCHECK is used for stereochemical analysis
because of the slightly different way in which the most favoured regions of the Ramachandran plot are defined.
FEBS Journal 278 (2011) 687–703 ª 2011 The Authors Journal compilation ª 2011 FEBS
Goal:
1. >20 restraints per residue
2. 0.3-0.6Å rmsd for backbone atoms, 0.5-0.8Å rmsd for heavy atoms
Very rough rule of thumb: an NMR structure calculated with ≥20 restraints
per residue is equivalent to a 2-2.5Å crystal structure
But… long range restraints are much more important than medium range,
sequential or intraresidue ones for making a high quality NMR structure
699
Wikipedia: residual dipolar coupling
“The blue arrows represent the orientation of the N - H bond of selected peptide bonds. By
determining the orientation of a sufficient amount of bonds relative to the external magnetic
field, the structure of the protein can be determined. From PDB 1KBH.”
Banci et al., Prog Nucl Magn Reson Spectrosc.,
56: 247-66, 2010
Fig. 3. Family of 30 conformers of Ca/Ce Calbindin obtained with (A) diamagnetic
and (B) paramagnetism-based restraints. (C) Different types of restraints used in the
structure calculation and their effect on the final resolution of the structure [76].
NMR of protein complexes
Even transient/weak ones
•  Free vs bound ∆chemical shift identifies interface
•  Isotope-filtered NMR experiments -> protein-protein NOE s
•  One case:
Combine x-ray of individual proteins + minimal NMR of complex
 efficient structure of complex
Pre-dock: x-ray structures
Red = NMR structure of complex
Green = Docked via 9 interprotein NOE s, 231
rdc s
Rmsd = 1.3 Å
rdc s to orient, NOE s to dock
Efficient!
Clore, PNAS 97: 9021-9025, 2000.
Solution NMR structures
of membrane proteins
NOW: 95 total, 65 unique.
Sanders & Sonnichsen (2006)
Magnetic Resonance in Chemistry!
44, S24 - S40.
2008
Human VDAC-1
32 kDa β barrel
19 strand
(mitochondria)
2K4T
2JK4
Bayrhuber et al., PNAS 105: 5370-5, 2008 .
2009
Diacylglycerol Kinase (DAGK)
43 kDa alpha helical (trimer)
2KDC
Van Horn Science 324:1726-9, 2009 .
Solid-state NMR
Two approaches
Magic Angle Spinning (MAS)
Oriented samples (membrane proteins)
Complete structures of small proteins
Local structure in large systems
Oriented samples
σ33
σ11, σ22
Concepts in
Magnetic
Resonance 14,
212.
Concepts in
Magnetic
Resonance 23A, 89.
Magic Angle Spinning
static: HCS = σisoγH0Iz + 1/2(3cos2θ -1)(σzz-σiso) γH0
H0
MAS: 3cos2θ -1 = 0 when θ = 54.7˚= magic angle
54.7˚
!r
Protein structures by solid-state NMR
25 unique as of 10/12/11
www.drorlist.com/nmr.html
*
Emerging methods 
Local structure in larger systems
Influenza M2 proton channel (Cady et al, J Mol Bio 385:1127-41, 2009)
a. MAS ssNMR
b. Static ssNMR
c. sol n NMR d. crystal structure
2kad
2h95
2rlf
3c9j
2009
2007, 2001
2008
2008
lipid
lipid
detergent
detergent
NMR (especially combined with other methods)
 next era of understanding proteins in action
Role of dynamics in mechanisms
Structures of noncrystallizable systems or states
-- complete or local
minor states: invisible (5-10%)
protein complexes
membrane proteins
proteins in cells
Dynamics Measured by NMR
Markwick et al (2008). PLoS
Comput Biol 4(9): e1000168