ARTICLES
PUBLISHED ONLINE: 18 MARCH 2012 | DOI: 10.1038/NCHEM.1299
Protein fold determined by paramagnetic magicangle spinning solid-state NMR spectroscopy
Ishita Sengupta1†, Philippe S. Nadaud1†, Jonathan J. Helmus1‡, Charles D. Schwieters2
and Christopher P. Jaroniec1 *
Biomacromolecules that are challenging for the usual structural techniques can be studied with atomic resolution by solidstate NMR spectroscopy. However, the paucity of distance restraints >5 Å, traditionally derived from measurements of
magnetic dipole–dipole couplings between protein nuclei, is a major bottleneck that hampers such structure elucidation
efforts. Here, we describe a general approach that enables the rapid determination of global protein fold in the solid phase
via measurements of nuclear paramagnetic relaxation enhancements (PREs) in several analogues of the protein of interest
containing covalently attached paramagnetic tags, without the use of conventional internuclear distance restraints. The
method is demonstrated using six cysteine–EDTA–Cu21 mutants of the 56-residue B1 immunoglobulin-binding domain of
protein G, for which ∼230 longitudinal backbone 15N PREs corresponding to distances of ∼10–20 Å were obtained. The
mean protein fold determined in this manner agrees with the X-ray structure with a backbone atom root-mean-square
deviation of 1.8 Å.
D
etermination of the three-dimensional structures of biological
macromolecules is critical to understanding their physiological function and is also the primary focus of structural genomics initiatives1. Magic-angle spinning (MAS) solid-state NMR
provides a set of spectroscopic tools for structural studies of biological systems that cannot be induced to form sufficiently diffracting
crystals and are also not amenable to solution NMR due to size
limitations, such as certain membrane-bound polypeptides2,3 and
amyloids4,5. However, in spite of the remarkable recent progress
in determining relatively high-resolution structural models for proteins with molecular weights up to 20 kDa by MAS solid-state
NMR5–19, the routine generation of such structures continues to
present considerable challenges.
Even for small- to medium-sized uniformly 13C,15N-labelled or
uniformly 15N- and extensively 13C-labelled proteins (the latter generally prepared by using complementary [1,3-13C] and
[2-13C]glycerol-based expression media6 to achieve improved spectral resolution and magnetization transfer efficiencies6,15 by suppressing many one-bond 13C–13C dipolar and J-couplings), the twodimensional 13C–13C and 15N–13C chemical shift correlation
spectra, which form the basis for the vast majority of current
solid-state NMR structure determination protocols, are typically
highly congested and contain many tens to hundreds (or even thousands) of resonances. These spectra rely on magnetization transfers
using radiofrequency pulse schemes such as transferred echo double
resonance (TEDOR)20,21, proton-driven spin diffusion (PDSD) or
dipolar-assisted rotational resonance (DARR)22, CHHC, NHHC
or NHHN23, or proton-assisted recoupling (PAR)12. They report
on the magnitudes of through-space 13C–13C, 15N–13C and
1
H–1H magnetic dipole–dipole couplings, and thereby distances,
among the protein nuclei. The use of ultrahigh-field (≥ 20 T)
NMR spectrometers, additional spectral dimensions, tailored
isotope labelling schemes (for example, perdeuterated proteins containing sparsely distributed proton nuclei at a limited number of
amide and methyl sites)18–20 and structure calculation algorithms
that are able to deal with ambiguous distance restraints8,9 has certainly helped to alleviate some of the problems related to NMR
signal overlap. However, the fundamental limitation in most
solid-state NMR structure determination endeavours has been the
relative scarceness of unambiguous long-range interatomic distance
restraints. This is primarily because dipolar couplings between 1H,
13
C and 15N spins, which scale with the inverse third power of the
internuclear distance, become vanishingly small for distances in
the 5–10 Å regime and beyond (typical coupling magnitudes are
on the order of a few hertz to tens of hertz). The detection and
reliable quantification of these couplings is often further complicated by the fact that they are part of tightly coupled multiple
spin networks. Consequently, correlations that encode information
about the most structurally relevant inter-residue dipolar couplings
typically comprise only a small fraction of the observable resonances
in multidimensional solid-state NMR data sets, and are also usually
associated with some of the weakest cross-peak intensities.
In this Article, we describe a general approach that has the potential to substantially accelerate the solid-state NMR protein structure
determination process. In broad terms, this methodology involves
the measurement of nuclear paramagnetic relaxation enhancements
(PREs)24,25 in several point mutants of the protein of interest modified with covalently attached paramagnetic tags, followed by the
application of these PREs as structural restraints to establish the
global protein backbone fold. The PRE phenomenon is well
known in magnetic resonance; the magnitude of the effect scales
with the inverse sixth power of the electron–nucleus distance24,25
and can be non-negligible for distances up to 20–25 Å or longer
depending on the observed nucleus and paramagnetic centre (that
is, distances that are at least a factor of three to four larger than
those obtained using traditional dipolar coupling-based solid-state
NMR techniques). Indeed, PRE-based methods of this kind, using
nitroxide spin labels and measurements of longitudinal or
1
Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA, 2 Division of Computational Bioscience, Center
for Information Technology, Building 12A, National Institutes of Health, Bethesda, Maryland 20892, USA, † These authors contributed equally to this work,
‡
Present address: Department of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington,
Connecticut 06030, USA. * e-mail: [email protected]
410
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transverse amide 1H relaxation enhancements, have been used previously with success to define the fold of soluble proteins with
limited nuclear Overhauser effect (NOE) data26–28.
The latest advances in MAS NMR spectroscopy of paramagnetic
proteins29–36 have laid the foundation for the application of the PREbased approach to the structural analysis of biological macromolecules in the solid phase. Using several 13C,15N-labelled analogues
of a model 56-residue protein, the B1 immunoglobulin binding
domain of protein G (GB1), with EDTA–Cu2þ tags attached to
cysteine residues introduced into the protein especially for this
purpose37,38, we have recently demonstrated that residue-specific
longitudinal PREs can be rapidly determined for most backbone
amide 15N nuclei by multidimensional solid-state NMR
methods34,35. These 15N PREs, which correspond to 15N–Cu2þ distances of 10–20 Å, were found to be in good quantitative agreement with those predicted for protein structural models based on
the wild-type GB1 fold. Here, we show that a set of 230 such
PREs recorded for six Cys–EDTA–Cu2þ mutants of GB1 (that is,
4–5 restraints per residue), supplemented only by standard
chemical shift-based restraints on the secondary structure and,
importantly, in the absence of any dipolar coupling-based
internuclear distance restraints, is sufficient to derive the correct
global fold for GB1 in a de novo fashion. Notably, because PRE
restraints can be extracted easily with high sensitivity from
routine two- or three-dimensional solid-state NMR chemical shift
correlation spectra, this methodology is expected to become
widely applicable to larger proteins that are available in
limited quantities.
Results
Determination of 15N PREs in GB1 mutants by solid-state NMR
spectroscopy. Residue-specific backbone amide 15N longitudinal
PREs (GN
1 ) were determined from measurements of longitudinal
15
N relaxation rate constants using a series of two-dimensional
15
N–13CO (NCO) spectra recorded with different values of
relaxation delay, trelax (Fig. 1a, Supplementary Fig. S1), as described
in the Methods. Relaxation data were collected for six pairs of GB1
mutants containing non-native Cys–EDTA–Cu2þ/Zn2þ sidechains
incorporated at solvent exposed sites corresponding to residues N8,
E19, K28, E42, D46 or T53 in the wild-type protein (Fig. 1b). All of
these GB1 analogues exhibit the wild-type fold, as judged by the
similarity of solution and solid-state NMR backbone chemical shifts
to those of wild-type GB133–35. For brevity, the GB1 mutants used
in this study are labelled as 8EDTA–Cu2þ/Zn2þ, 19EDTA–
28EDTA–Cu2þ/Zn2þ,
42EDTA–Cu2þ/Zn2þ,
Cu2þ/Zn2þ,
46EDTA–Cu2þ/Zn2þ and 53EDTA–Cu2þ/Zn2þ, according to the
location of the Cys–EDTA–Cu2þ/Zn2þ tag.
Figure 1c,e presents representative small regions of two-dimensional NCO spectra for 8EDTA–Cu2þ/Zn2þ and 28EDTA–
Cu2þ/Zn2þ recorded with trelax values of 100 ms (reference
spectra) and 4 s. Although the reference spectra for the corresponding EDTA–Cu2þ and Zn2þ proteins are effectively indistinguishable
from one another and contain all of the expected intense one-bond
15
N–13CO correlations, a number of cross-peaks are severely attenuated or altogether missing in spectra recorded for the EDTA–Cu2þ
proteins with longer relaxation delays (trelax ≈ 2–4 s). The most
attenuated resonances (for example G9, G14 and T53 in 8EDTA–
Cu2þ and T25, C28 and Q32 in 28EDTA–Cu2þ) are invariably
associated with residues located in the spatial proximity of the paramagnetic tag34,35. This is to be expected based on the Solomon
dipolar relaxation mechanism24,25, and we have demonstrated previously that most experimental solid-state NMR 15N PREs and
15
N–Cu2þ distances determined in this fashion are in quantitative
agreement with the corresponding values predicted using protein
structural models based on the wild-type GB1 fold34,35. Figure 1d,f
shows the complete experimental relaxation trajectories and best fits
to decaying single exponentials (that is, I ¼ A exp{2R1 × trelax},
where I is the instantaneous cross-peak intensity, A is the peak intensity at trelax ¼ 0, and R1 is the 15N longitudinal relaxation rate constant;
the two fit parameters are A and R1) for representative residues in
8EDTA–Cu2þ/Zn2þ and 28EDTA–Cu2þ/Zn2þ, respectively.
Collectively, these experiments yield 231 amide 15N longitudinal
PRE restraints (Supplementary Table S1) for the six paramagnetic
GB1 mutants (that is, an average of 38 out of 55 possible restraints
per mutant or 4–5 restraints per residue; the remaining PREs
could not be determined reliably due to peak overlap in the twodimensional NCO spectra). Approximately half of the PRE
restraints (110 of 231) correspond to GN
1 values greater than
0.1 s21 and were used as is in the course of the protein structure calculations discussed below. The remaining 121 restraints were converted to purely repulsive ‘NOE-type’ distance restraints,
preventing the associated atoms from approaching closer than
15.1 Å. This was done to avoid potential complications with the
convergence of the calculated structures, associated with small but
detectable effects on the extracted GN
1 values stemming from the
presence of intermolecular 15N–Cu2þ couplings and secondary
protein Cu2þ binding sites36.
Validation of the utility of solid-state NMR 15N PRE restraints
for protein structure determination. To evaluate the ability of
the longitudinal solid-state NMR 15N PREs to determine the
correct protein fold in the absence of traditional dipolar couplingbased distance restraints, we performed a set of preliminary, ‘ideal
case’ scenario calculations for GB1 in Xplor-NIH39 using
protocols where all native sidechains as well as the backbone
atoms for regular secondary structure elements were frozen in
rigid bodies corresponding to their conformations in the 1.14 Å
GB1 X-ray structure40,41 (PDB entry 2GI9). The Cys–EDTA–Cu2þ
sidechain conformations (initially optimized using PRE data and
2GI9 atomic coordinates) were also fixed. The regular secondary
structure elements included the a-helix (residues 22–37) and
the four b-strands, b1 to b4, spanning amino acids 2–8, 13–19,
42–46 and 51–55, respectively. The backbone torsion angles for
the remaining residues were randomized. Moreover, to enable the
simultaneous use of all PRE restraints within the same structure
calculation protocol, the Cys–EDTA–Cu2þ tags were placed on all
six modified residues (even though separate NMR experiments
were used to record PRE data for each of the GB1 mutants) with
the concurrent disabling of interactions between atoms associated
with the different tags.
Figure 2a,b presents representative sets of ten lowest-energy
structures (from a total of 1,000 structures) calculated without
and with the use of solid-state NMR longitudinal 15N PRE
restraints, respectively, in addition to the more conventional potential energy terms described in the Methods. Not surprisingly, when
no experimental NMR data were incorporated into the calculations,
the lowest-energy protein structures obtained in Xplor-NIH are
effectively random and show little resemblance to one another or
to the GB1 X-ray structure (Fig. 2a). Within this set of ten lowestenergy structures, no structure, even the one displaying the smallest
backbone atom (C′ , Ca, N and O) coordinate root-mean-square
deviation (RMSD) of 3.4 Å relative to 2GI9, correctly predicts the
protein fold. Although occasionally (three times out of 1,000 for
this data set) the calculations that included no experimental
restraints were able to yield protein structures with the correct
GB1 fold (backbone RMSDs versus 2GI9 of 1.5–2 Å), these
‘correct’ structures were associated with energies that were considerably higher than those obtained for numerous ‘incorrect’ structures
having backbone atom RMSDs in the 3–9 Å range. In stark
contrast, analogous calculations that also include the 231 experimental 15N PRE restraints yield lowest-energy protein structures
that cluster tightly together and, most importantly, correspond to
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ϕ1
a
b
y
19
CP
1H
DOI: 10.1038/NCHEM.1299
XiX
46
XiX
28
53
ψ
13C
t1
G9
G9 G41
G41
T44
G38
T51
G38
T51
112
τrelax = 100 μs
106
(ppm)
15N
110
G9
T44
G38
T51
τrelax = 100 μs
116
G38
T51
(ppm)
15N
120
τrelax = 4 s
T53
176
175
13C (ppm)
28EDTA–Zn2+
174
C28
E27
T25
118
178
(ppm)
118
15N
116
120
F30
28EDTA–Cu2+
Q32
Y33
F30
V21
A48
V29
A34
D36
τrelax = 4 s
Y33
E27
N35
Q32
V29
A34
D36
τrelax = 100 μs
181
180
K31 F30
179
178
(ppm)
13C
1.0
2.0
3.0
4.0
Relaxation delay, τrelax (s)
1.0
0.8
0.6
0.4
28EDTA–Zn2+
28EDTA–Cu2+
0.2
A48
0.0
T25
A24
0.2
1.0
C28
E27
N35
V21
122
A24 K31
A48
A34
D36
τrelax = 100 μs
0.4
0.0
f
C28
E27
T25
V29
0.6
174
N35
A24 K31
0.8
T53
176
175
13C (ppm)
V21
122
8EDTA–Zn2+
8EDTA–Cu2+
0.2
0.0
177
N35
Q32
0.4
0.0
112
e
0.6
1.0
T44
177
0.8
T51
G41
178
1.0
T53
8EDTA–Cu2+
G14
108
τrelax = 4 s
T53
Relative intensity
d
G14
T44
42
WALTZ-16
Relative intensity
(ppm)
15N
110
CP
8EDTA–Zn2+
G14
108
8
ϕ3
Relative intensity
106
t2
b
A48
Y33
177
V21
A24
180
A48
A34
D36
τrelax = 4 s
181
K31 F30
Y33
Relative intensity
c
τrelax
CP
15N
a
ϕ2
y
δ
δ
CP
0.8
0.6
0.4
0.2
Q32
0.0
179
178
(ppm)
177
0.0
13C
1.0
2.0
3.0
4.0
Relaxation delay, τrelax (s)
Figure 1 | Determination of longitudinal backbone amide 15N PREs in Cys–EDTA–Cu21 GB1 mutants by solid-state NMR spectroscopy. a, Pulse scheme
used to determine residue-specific longitudinal 15N rate constants (R1) from a series of two-dimensional 15N–13CO correlation spectra recorded with
different values of relaxation delay (trelax). See Supplementary Fig. S1 for a complete pulse scheme description. b, Ribbon diagram of GB1 (PDB entry
2GI9) with the locations of the non-native Cys–EDTA–Cu2þ/Zn2þ tags in point mutants investigated in this study (residues 8, 19, 28, 42, 46 or 53)
indicated by blue spheres. c, Small regions of two-dimensional 15N–13CO spectra recorded for 13C,15N-labelled 8EDTA–Zn2þ (green contours) and
8EDTA–Cu2þ (magenta contours) mutants with longitudinal 15N relaxation delays of 100 ms and 4 s, as indicated in the bottom left corner of each
spectrum. The resonance assignments were established previously35, and cross-peaks are labelled according to the amide 15N frequency of the residue
involved. d, Representative site-resolved 15N longitudinal relaxation trajectories for residues T51 and T53 in 8EDTA–Cu2þ and Zn2þ. Experimental
trajectories for 8EDTA–Zn2þ (green circles) and 8EDTA–Cu2þ (magenta circles) correspond to integrated cross-peak intensities in a series of twodimensional NCO spectra recorded as a function of the relaxation delay. Simulated best-fit trajectories to decaying single exponentials used to
extract the longitudinal 15N relaxation rate constants are shown as solid lines in the corresponding colour. The 15N PREs are calculated by taking
the difference between the 15N R1 values obtained for the EDTA–Cu2þ and EDTA–Zn2þ proteins. e, As in c but for 28EDTA–Zn2þ (blue contours) and
28EDTA–Cu2þ (red contours). f, As in d but for residues Q32 and A48 in 28EDTA–Zn2þ (blue circles/lines) and 28EDTA–Cu2þ (red circles/lines).
412
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a
b
β2
β1
β4
α
β3
d
Total energy (kcal mol–1)
Total energy (kcal mol–1)
c
3,000
2,000
1,000
0
6,000
5,000
4,000
3,000
0
2
4
6
8
10
RMSD from reference structure (Å)
0
2
4
6
8
10
RMSD from reference structure (Å)
Figure 2 | Results of idealized preliminary structure calculations for GB1 in the absence and presence of solid-state NMR longitudinal 15N PRE restraints.
a, Backbone traces (blue) for the ten lowest-energy GB1 structures (from 1,000 total structures), calculated using Xplor-NIH39 in the absence of experimental
15
N PRE restraints. Shown for comparison is the backbone trace (red) corresponding to the 1.14 Å GB1 X-ray structure40,41 (PDB entry 2GI9). Native sidechain
torsion angles and backbone torsion angles for residues in regular secondary structure elements (b1, aa 2–8; b2, aa 13–19; a, aa 22–37; b3, aa 42–46; b4,
aa 51–55) were fixed to their X-ray structure values during the calculations, and the remaining backbone torsion angles were randomized (a detailed
description of the structure calculations is provided in the Methods). The backbone coordinates of the ten lowest-energy structures show RMSDs ranging
from 3.4 to 9.1 Å relative to the X-ray structure. b, As in a except that 231 15N PRE restraints recorded for the six paramagnetic GB1 mutants were also
included in the structure calculations. The backbone coordinates of the ten lowest-energy structures show RMSDs relative to 2GI9 ranging from 0.9 to 1.8 Å.
c,d, Plots of total energy versus backbone atom coordinate RMSD from the 2GI9 reference X-ray structure for each of the 1,000 structures calculated
without (c) and with (d) the use of 15N PRE restraints. In each plot, the ten lowest-energy structures are indicated by red circles.
the correct GB1 fold with backbone RMSDs of 0.9–1.8 Å relative to
2GI9 (Fig. 2b).
The complete results for both sets of calculations (using large
ensembles of 1,000 random starting protein structures) are summarized in Fig. 2c,d. These data clearly illustrate that in the absence of
15
N PRE restraints there is effectively no correlation between the
total energy of a particular protein structure obtained in XplorNIH and that structure displaying the correct GB1 fold (Fig. 2c).
Indeed, nearly all structures obtained in this manner bear little
resemblance to the reference GB1 X-ray structure. On the other
hand, the addition of 15N PRE restraints to the structure calculation
protocol leads to a funnel-like effect where a relatively strong correlation emerges between the total energy of a calculated structure and
its RMSD relative to the 2GI9 backbone atom coordinates (Fig. 2d).
Although the convergence properties of these calculations may not
be ideal and the calculation procedure generates multiple incorrect
protein structures, it is important to note that all of these incorrect
structures have high energies and can be readily identified. Most significantly, however, the lowest-energy structures (associated with
total energies of 3,000 kcal mol21 or less in this case; the ten
lowest-energy structures are indicated by red circles in Fig. 2c,d)
all have similar backbone folds that also closely match the GB1
X-ray structure (Fig. 2b).
De novo determination of the GB1 backbone fold. Having
established the utility of solid-state NMR 15N PRE restraints for
protein structure calculations, we proceeded to investigate whether
the fold of GB1 could be determined in a de novo manner using
only 15N PRE data (Supplementary Table S1) and standard solidstate NMR chemical shift-based backbone dihedral angle
restraints for wild-type GB1 (Supplementary Table S2) to define
the local secondary structure. As described in detail in the
Methods, these realistic de novo calculations consisted of a twostage protocol. In the initial stage, backbone atoms for residues
comprising regular secondary structure elements (identified within
the torsion angle likelihood obtained from shifts and sequence similarity (TALOS+) software42 as having a or b conformation with
greater than 85% confidence) were frozen in rigid bodies with
their dihedral angles fixed to the ideal TALOSþ predicted values,
while all other backbone and sidechain torsion angles (including
the Cys–EDTA–Cu2þ tags) were randomized. We found that
fixing the TALOSþ-identified secondary structure regions during
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a
b
8,000
Total energy (kcal mol–1)
DOI: 10.1038/NCHEM.1299
β2
6,000
4,000
β4
α
2,000
β1
0
c
2
4
6
8
RMSD from reference structure (Å)
β3
10
N
N
90º
C
C
Figure 3 | De novo calculation of the GB1 fold using solid-state NMR longitudinal 15N PRE restraints. a, Plot of total energy versus backbone atom RMSD
from the reference X-ray structure obtained during the course of the de novo calculations. The ten lowest-energy structures from the initial calculations are
indicated by red circles. These ten structures were further refined as described in the text to yield a set of 100 structures. From this final set, the 20 lowestenergy structures indicated by yellow circles were used to obtain the regularized mean structure (magenta circle). b, Backbone traces for the 20 lowestenergy GB1 structures (blue), corresponding to the yellow circles in a, and the reference X-ray structure (red). c, Comparison of the GB1 X-ray structure with
the regularized mean solid-state NMR backbone fold determined using realistic, de novo calculations. The coordinates of the mean NMR structure (blue)
show a backbone atom RMSD of 1.8 Å and an all-heavy-atom RMSD of 2.7 Å relative to the X-ray structure (red). The grey cloud is a reweighted atomic
probability map50 of the final ensemble of 20 lowest-energy NMR structures shown in b plotted at 15% of maximum value, representing the conformational
space occupied by the associated backbone atoms. Structure calculations were performed as described in the Methods using 231 longitudinal 15N PRE
restraints recorded for six paramagnetic GB1 analogues and solid-state NMR chemical shift-based dihedral angle restraints for wild-type GB1 obtained using
TALOSþ (ref. 42).
this initial stage resulted in significantly improved convergence
compared to the standard protocols (which allow more degrees of
freedom), as well as improved accuracy in the final calculated structures. The initial calculation stage was followed by a second stage, in
which the ten lowest-energy structures from the initial calculations
(indicated by red circles in Fig. 3a) were further refined. Ten different sets of randomized velocities were used in a protocol that
allowed all sidechains to move as well as all backbone dihedral
degrees of freedom constrained only by the TALOSþ dihedral
angle potential with minimum dihedral angle uncertainties of
+208. This led to a final set of 100 structures. From this set, the
20 lowest-energy structures, shown in Fig. 3b and indicated by
yellow circles in Fig. 3a, were used to obtain a regularized
mean structure.
Remarkably, using these largely unrestricted de novo calculations,
which did not make use of any conventional dipolar coupling-based
interatomic distance restraints or structural information derived
414
from X-ray diffraction data, we were able to determine a backbone
fold for GB1 that is in good agreement with the 1.14 Å X-ray structure (Fig. 3c; structure indicated by the magenta circle in Fig. 3a).
The mean solid-state NMR structure obtained in this manner
(blue) displays a backbone atom RMSD of 1.8 Å and an all-heavyatom RMSD (excluding the sidechains of residues 8, 19, 28, 42, 46
and 53) of 2.7 Å relative to the 2GI9 reference structure (red).
Also, the backbone atoms for the vast majority of residues in the
X-ray structure (with the exception of several amino acids located
in the loop regions or near the N-terminus) fall within the conformational space occupied by the ensemble of the 20 lowest-energy
conformers (grey cloud). Finally, in the course of these calculations
we also considered the possible influence of Cys–EDTA–Cu2þ sidechain disorder on the quality of the derived structures. The PRE
refinement facilities within Xplor-NIH allow such structural heterogeneity to be modelled using ensembles of several sidechains, by
either representing the PRE as a sum of contributions from each
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ensemble member (corresponding to slow interconversion between
ensemble members) or 1/r 6 averaging of the different ensemble
members (for fast interconversion). Both approaches were
attempted using ensembles of two and three Cys–EDTA–Cu2þ
tags. We found that the use of such ensembles did not improve
the results over using a single Cys–EDTA–Cu2þ conformer, presumably due to the presence of additional degrees of freedom for
which there are insufficient data in this study.
Discussion
Notwithstanding the tremendous current progress5–19, the routine
determination of protein structures by solid-state NMR remains a
formidable task, largely due to the challenges associated with the
detection of large numbers of correlations that report on internuclear distances of .5 Å. Measurements of such long-distance
restraints in paramagnetic proteins, including PREs33,34 and pseudocontact shifts (PCS)30,31, have the potential to significantly accelerate
and enhance solid-state NMR protein structure elucidation efforts.
In fact, the utility of paramagnetic restraints of this type was recently
illustrated by Bertini and co-workers for a 159-residue (17.6 kDa)
native metalloprotein, cobalt(II)-substituted matrix metalloproteinase 12 (refs 31,32). In that work, over 300 13C PCS restraints
were used in conjunction with 300 (ref. 31) and 800 (ref. 32)
internuclear distances obtained from PDSD/DARR, CHHC and
PAR experiments to arrive at three-dimensional protein structures.
In the approach to global protein fold determination described
here, we use PRE restraints recorded for structural analogues of
the protein of interest containing covalently linked paramagnetic
tags. This is readily applicable to a wide variety of protein molecules
in the solid phase, including natively diamagnetic proteins. Nuclear
PREs yield almost exclusively structurally relevant inter-residue
restraints generally corresponding to distances of .10 Å, but this
approach also has another major advantage. These PRE restraints
are very straightforward to extract by monitoring cross-peak intensities (as a function of relaxation delay) in the simplest two- or
three-dimensional solid-state NMR chemical shift correlation
spectra, which contain a minimum number of signals arising
from adjacent, strongly dipolar coupled nuclei in the protein. As
such, this methodology is pertinent to larger protein systems.
Although the implementation of this method requires the preparation of several point mutants of the protein under study, such
biochemical manipulations are routine. It is also important to
note that although the known three-dimensional structure of GB1
was helpful in identifying suitable sites for introducing the
Cys–EDTA–Cu2þ tags in this study, the incorporation of such
tags into proteins of unknown structure, with minimal influence
on the backbone fold38, should be readily implementable using
the approach discussed by Battiste and Wagner27. This uses secondary structure information obtained from chemical shifts for the
wild-type protein and the analysis of hydrophilicity patterns to
identify the most probable solvent-accessible residues43.
The current study demonstrates that 4–5 longitudinal amide
15
N PRE restraints per residue, collected using a condensed NMR
data acquisition approach35 for a set of six Cys–EDTA–Cu2þ
mutants available in small quantities (150–200 nmol of 13C,15Nlabelled protein per sample), are sufficient to obtain a protein backbone fold for GB1 that is in close agreement with the X-ray structure, without the requirement for any conventional internuclear
distance restraints. Although the global protein fold determined in
this manner does not correspond to the structure with the highest
achievable resolution, it can be derived very rapidly and provides
a reasonable structural model that is likely to be sufficient for
many applications. Moreover, such a PRE-based model can serve
as a major aid for resolving assignment ambiguities in conventional
dipolar coupling-based solid-state NMR spectra. It can be refined
further to higher resolution, if desired, by incorporating additional
interatomic distance restraints or by combining the PRE restraints
with more advanced fragment-based chemical shift/molecular
mechanics (CSMM)-based structure calculation protocols such as
CHESHIRE13 or CS-ROSETTA14. It should be noted that the structure of GB1 has, in fact, been successfully solved (and to higher resolution) using the CHESHIRE approach with chemical shifts as the
sole experimental input13, whereas in this work we only extracted
broad backbone torsion angle ranges from chemical shift data.
Such CSMM methods (which make use of molecular mechanics
force fields to obtain the proper protein fold) have chalked up an
impressive record of correctly determining protein structures and
we strongly advocate their use. However, they are also known to
fail at times due to inadequacies of the MM force fields and the
fact that chemical shift data contain essentially no long-distance
information. In contrast, the PRE-based approach reported here
uses direct distance restraints simply related to experimental observables, and thus serves as an independent means to solve or validate
a protein fold.
Importantly, the number of paramagnetic-based structural
restraints in these studies can be dramatically increased (by at
least two to threefold) with minimal effort. This is achieved by
measuring other types of PREs, including longitudinal backbone
and sidechain 13C PREs and transverse PREs for the different
nuclei, in the same protein samples, as well as PCS restraints for
13
C, 15N and potentially 1H nuclei in analogous Cys–EDTA–Co2þ
samples. This is expected to substantially enhance the resolution
of the derived structures, and bodes well for the extension of this
paramagnetic solid-state NMR methodology to larger proteins for
which a significant fraction of resonances can be assigned. Indeed,
such sequential resonance assignments in larger systems may be
facilitated by using spectral editing approaches that take advantage
of the presence of the paramagnetic tags.
Methods
Sample preparation. Plasmid cDNAs encoding for N8C, E19C, K28C, E42C, D46C
or T53C mutants were constructed as described previously33 using T2Q-GB1 cDNA
(referred to as GB1) and the Stratagene QuikChange II site-directed mutagenesis
protocol. Proteins were expressed in Escherichia coli BL21(DE3) with Luria-Bertani
medium or M9 minimal medium containing 1 g l21 15NH4Cl and 3 g l21
13
C-glucose (Cambridge Isotope Laboratories) and purified by gel filtration
chromatography33,34. Cysteines were reacted with the sulfhydryl-specific reagent
N-[S-(2-pyridylthio)cysteaminyl]EDTA37 (Toronto Research Chemicals), preloaded with 1.1 equiv. Cu2þ or Zn2þ (diamagnetic control). Protein microcrystals
for solid-state NMR were prepared by co-precipitating the 13C,15N–EDTA–Cu2þ/
Zn2þ proteins with natural abundance GB1 in a 1:3 molar ratio using microdialysis
at 4 8C and a 2:1:1 (v/v) 2-methylpentane-2,4-diol/isopropanol/deionized water
precipitant solution34,36. Samples, each containing 1.0–1.2 mg (150–200 nmol)
of 13C,15N-labelled protein, were packed into 1.6 mm zirconia rotors (Agilent
Technologies) by centrifugation.
NMR spectroscopy. NMR spectra were recorded on a 500 MHz Varian
spectrometer equipped with a 1.6 mm FastMAS probe. The MAS rate and sample
temperature were regulated at 40,000+20 Hz and 5 8C, respectively. Residuespecific amide 15N longitudinal relaxation rate constants (R1) for the EDTA–
Cu2þ/Zn2þ GB1 mutants were determined from series of two-dimensional NCO
spectra recorded using the pulse scheme in Fig. 1a with relaxation delays (trelax) of
100 ms to 4 s. Spectra were collected with acquisition times of 25.6 ms (t1) and 30 ms
(t2) and recycle delays of 0.4 s and 1.3 s for EDTA–Cu2þ and EDTA–Zn2þ proteins,
respectively. The total experiment time required to record 15N R1 data for all EDTA–
Cu2þ/Zn2þ GB1 mutants was 18 days. Spectra were processed using NMRPipe44
and analysed with home-built software (available at http://code.google.com/p/
2þ
2þ
15
N
nmrglue). 15N PREs (GN
1 ) were calculated as G1 ¼ R1(Cu ) – R1(Zn ), where N
R1 values for Cu2þ and Zn2þ proteins were obtained by fitting residue-specific
relaxation trajectories to decaying single exponentials.
Structure calculations. Structure calculations were carried out in Xplor-NIH39 with
a protocol in which backbone atoms of regular secondary structure elements were
frozen in rigid bodies corresponding to their X-ray (during initial calculations in
Fig. 2) or ideal target conformations as predicted from TALOSþ42 (during the
realistic de novo calculations in Fig. 3). Regions of secondary structure were
identified by inspection of the GB1 X-ray structure40,41 (PDB entry 2GI9) for the
initial calculations and from TALOSþ hits predicted with .85% confidence in the a
or b Ramachandran regions for the realistic calculations. The remaining backbone
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415
ARTICLES
NATURE CHEMISTRY
torsion angles were randomized. Sidechain torsion angles were fixed in their X-ray
conformation for the initial calculations and randomized for the realistic
calculations. The EDTA–Cu2þ tags were present on all six residues that were
modified, but the inter-residue interactions of the associated atoms were disabled. In
the initial calculations, conformations of the EDTA–Cu2þ sidechains were first
optimized using PRE data and the 2GI9 coordinates, and were held fixed during
structure calculations. For the realistic calculations, EDTA–Cu2þ sidechains were
treated as other sidechains, with torsion angles randomized.
PRE restraints consisted of explicit restraints using the PREPot term45 with an
21
electron correlation time of 3 ns (ref. 25) for GN
1 values above a cutoff of 0.1 s
(Supplementary Table S1). Smaller PREs were included using a purely repulsive
‘NOE-type’ term restraining the associated 15N–Cu2þ distances to values .15.1 Å.
TALOSþ was used to derive 102 (of 110 possible) backbone torsion angle restraints
from GB1 13C and 15N solid-state NMR chemical shifts (Supplementary Table S2).
The radius of gyration term46 was used to achieve an appropriate protein packing
density. Additional knowledge-based energy terms included the torsion angle
potential of mean force47, the hydrogen bond potential of mean force48 and the lowresolution residue contact term49. Standard bond, bond angle and improper torsion
angle restraints were used to maintain proper covalent geometry, and a repulsive
quartic van der Waals term was used to prevent atomic overlap. During calculations
in which EDTA–Cu2þ sidechains were allowed to move, the associated dihedral
angles were restrained by a dihedral energy term.
The protocol consisted of initial conjugate gradient minimization without the
PRE and repulsive distance terms, followed by minimization including all energy
terms. This was followed by variable-step-size dynamics for the smaller of 500 ps or
20,000 steps at 3,000 K.
Simulated annealing was performed from 3,000 K to 25 K in 12.5 K increments,
and at each temperature, dynamics for the smaller of 1.6 ps or 400 steps was run.
During simulated annealing, the force constants of various energy terms were
ramped to their final values as specified in Supplementary Table S3. For the realistic
calculations, the initial structure calculation was followed by an additional
calculation stage in which the ten lowest-energy structures from the initial
calculation were refined using ten different sets of randomized velocities (for a total
of 100 structures) in a protocol that allowed all backbone dihedral degrees of
freedom and omitted the initial minimization step. A regularized mean structure was
calculated from the 20 lowest-energy refined structures.
Received 24 October 2011; accepted 8 February 2012;
published online 18 March 2012
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Acknowledgements
This research was supported by the National Science Foundation (CAREER award
MCB-0745754 to C.P.J.). C.D.S. was supported by the National Institutes of Health
Intramural Research Program of the Center for Information Technology. The GB1 plasmid
was kindly provided by A.M. Gronenborn.
Author contributions
C.P.J. designed the research. I.S. and P.S.N. prepared the samples. I.S., P.S.N. and J.J.H.
recorded and analysed the NMR data. C.D.S. and C.P.J. performed the structure
calculations. C.D.S. and C.P.J. wrote the paper.
Additional information
The authors declare no competing financial interests. Supplementary information
accompanies this paper at www.nature.com/naturechemistry. Reprints and permission
information is available online at http://www.nature.com/reprints. Correspondence and
requests for materials should be addressed to C.P.J.
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417
SUPPLEMENTARY INFORMATION
DOI: 10.1038/NCHEM.1299
Supplementary Information
Protein fold determined by paramagnetic magic-angle spinning
solid-state NMR spectroscopy
Ishita Sengupta1, Philippe S. Nadaud1, Jonathan J. Helmus1,
Charles D. Schwieters2 and Christopher P. Jaroniec1*
1
Department of Chemistry, The Ohio State University, 100 West 18th Avenue,
Columbus, Ohio 43210, USA
2
Division of Computational Bioscience, Center for Information Technology, Building 12A,
National Institutes of Health, Bethesda, Maryland 20892, USA
*E-mail: [email protected]
1
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Figure S1. The pulse scheme used to determine residue-specific longitudinal 15N relaxation rate
constants, R1, in the GB1 Cys-EDTA-Cu2+/Zn2+ mutants from series of two-dimensional 15N13
CO (2D NCO) correlation spectra recorded with different values of τrelax in the 100 μs to 4 s
range.1 The experiments were performed at 11.7 T (500 MHz 1H Larmor frequency) and 40 kHz
MAS rate. Narrow and wide black rectangles correspond to 90o and 180o pulses, respectively,
and all pulses have phase x unless indicated otherwise. The 1H, 13C and 15N carriers were placed
at ~4.7, 175, and 120 ppm, respectively. 1H-15N cross-polarization (CP)2 was achieved with a
~60 kHz 15N field applied with a tangent ramp,3 a ~100 kHz 1H field, and a contact time of 1.0
ms. SPECIFIC CP4 with a ~15 kHz 15N field, a ~25 kHz 13C field with a tangent ramp, and a 7
ms contact time was used to establish the 15N-13CO magnetization transfer. Proton XiX5 and
nitrogen WALTZ-166 decoupling was applied at field strengths of ~12 kHz and ~3.3 kHz,
respectively, during periods indicated in the figure. The spin-state-selective excitation (S3E)
13
CO-13Cα J-decoupling scheme,7,8 with the delay δ = 2.25 ms, consisted of two separate spectra
recorded with selective rSNOB 180o refocusing pulses9 of 250 μs duration applied to the 13CO
spins at the frequency of 175 ppm (filled black shapes) and 13Cα spins at the frequency of 55
ppm (filled gray shapes) in either position a or b. The spectra were processed as described by
Laage et al.8 The following phase cycles were employed to record spectra a and b. Spectrum a:
φ1 = 2(x), 2(-x); φ2 = x, -x; φ3 = y; ψ = x; receiver = x, -x, -x, x. Spectrum b: φ1 = 2(x), 2(-x); φ2 = x,
-x; φ3 = y; ψ = -y; receiver = x, -x, -x, x. Quadrature in the 15N dimension was achieved by
alternating phase φ2 according to the States method.10
2
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Table S1. Summary of longitudinal amide 15N PRE restraints and repulsive ‘NOE-type’ distance
restraints determined for the six GB1 mutants modified with paramagnetic EDTA-Cu2+ tags
Residue
EDTA-Cu2+
Residue
NOEb
PRE, G1N (s-1)a
tag position
number
number
8
4
0.14 ± 0.01
2
*
5
0.26 ± 0.02
17
*
9
0.34 ± 0.05
18
*
14
0.52 ± 0.05
19
*
15
0.54 ± 0.11
20
*
39
0.15 ± 0.01
21
*
40
0.24 ± 0.03
24
*
41
0.14 ± 0.03
25
*
43
0.27 ± 0.02
26
*
44
0.22 ± 0.03
27
*
45
0.14 ± 0.01
28
*
46
0.14 ± 0.01
29
*
47
0.11 ± 0.01
30
*
50
0.11 ± 0.02
31
*
51
0.14 ± 0.01
32
*
52
0.18 ± 0.01
33
*
53
0.29 ± 0.02
34
*
55
0.54 ± 0.11
35
*
56
0.20 ± 0.05
36
*
37
*
38
*
48
*
49
*
19
5
0.19 ± 0.02
7
*
9
0.18 ± 0.01
14
*
10
0.24 ± 0.04
33
*
11
0.19 ± 0.02
34
*
12
0.12 ± 0.02
35
*
15
0.14 ± 0.01
37
*
16
0.21 ± 0.01
43
*
20
0.74 ± 0.09
45
*
21
0.11 ± 0.01
46
*
23
0.19 ± 0.03
53
*
25
0.11 ± 0.02
54
*
26
0.17 ± 0.01
28
0.14 ± 0.01
38
0.12 ± 0.01
39
0.13 ± 0.02
40
0.22 ± 0.03
41
0.18 ± 0.04
42
0.22 ± 0.03
47
0.12 ± 0.01
3
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DOI: 10.1038/NCHEM.1299
Table S1. continued
EDTA-Cu2+
tag position
28
Residue
number
49
51
55
21
24
25
26
27
28
29
30
31
32
33
34
35
36
40
41
42
43
PRE, G1N (s-1)a
9
10
11
15
32
33
34
0.20 ± 0.01
0.33 ± 0.09
0.25 ± 0.06
0.12 ± 0.01
0.21 ± 0.03
0.15 ± 0.01
0.24 ± 0.03
42
0.12 ± 0.01
0.15 ± 0.01
0.15 ± 0.02
0.13 ± 0.01
0.33 ± 0.02
0.81 ± 0.05
0.21 ± 0.01
0.21 ± 0.01
0.56 ± 0.07
0.48 ± 0.03
0.22 ± 0.01
0.34 ± 0.03
0.51 ± 0.02
0.18 ± 0.01
0.13 ± 0.01
0.19 ± 0.01
0.12 ± 0.01
0.16 ± 0.01
0.23 ± 0.02
0.14 ± 0.01
0.17 ± 0.01
Residue
number
NOEb
2
4
5
6
7
8
9
10
11
12
13
14
15
17
18
19
20
37
38
39
44
45
46
47
48
49
50
51
52
53
54
55
56
7
14
18
19
20
21
24
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
4
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DOI: 10.1038/NCHEM.1299
Table S1. continued
EDTA-Cu2+
tag position
Residue
number
35
36
37
38
39
43
44
55
56
PRE, G1N (s-1)a
6
7
8
17
19
21
23
24
25
32
33
34
35
36
37
38
39
41
43
44
45
51
52
56
2
5
6
7
12
14
15
0.33 ± 0.03
0.38 ± 0.03
0.15 ± 0.03
0.17 ± 0.02
0.13 ± 0.03
0.12 ± 0.01
0.18 ± 0.02
0.16 ± 0.01
0.12 ± 0.02
0.11 ± 0.02
0.11 ± 0.02
0.12 ± 0.03
0.15 ± 0.03
0.14 ± 0.03
0.12 ± 0.02
0.13 ± 0.03
0.12 ± 0.02
0.25 ± 0.02
0.11 ± 0.02
0.21 ± 0.03
0.36 ± 0.02
1.11 ± 0.15
0.37 ± 0.04
0.13 ± 0.01
0.15 ± 0.02
0.15 ± 0.01
0.13 ± 0.02
0.18 ± 0.05
0.14 ± 0.03
0.14 ± 0.03
0.15 ± 0.03
46
53
0.33 ± 0.03
0.21 ± 0.01
0.19 ± 0.01
0.38 ± 0.02
0.18 ± 0.02
0.56 ± 0.03
0.15 ± 0.01
0.39 ± 0.02
0.48 ± 0.13
Residue
number
25
26
27
29
30
45
47
48
49
50
51
52
9
10
11
13
14
15
18
29
30
31
40
42
55
NOEb
9
10
11
13
16
17
19
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
5
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Table S1. continued
EDTA-Cu2+
tag position
Residue
number
21
43
49
51
PRE, G1N (s-1)a
0.12 ± 0.01
0.12 ± 0.01
0.17 ± 0.04
0.21 ± 0.03
Residue
number
24
25
27
29
31
32
33
34
36
37
38
39
41
42
46
NOEb
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
PREs were calculated as Γ1N = R1(Cu2+) – R1(Zn2+), where 15N R1 values for the corresponding pair of EDTA-Cu2+
and EDTA-Zn2+ tagged proteins were obtained by fitting residue-specific relaxation trajectories to decaying single
exponentials. A total of 110 quantitative PRE restraints (77 for residues located in regular secondary structure
elements and 33 for loop residues) were determined for the six GB1 analogs and used in the structure calculations
via the PREPot potential term (see main text and Supplementary Table S3 for additional details).
b
For residues where the PRE measurements yielded values of Γ1N £ 0.10 s-1 (indicated by asterisks) the PRE
restraints were converted into purely repulsive ‘NOE-type’ distance restraints with a lower-bound cutoff of 15.1 Å
and used in the structure calculations via the NOEPot potential term. A total of 121 residues for the six GB1 analogs
fell into this category (89 located in regular secondary structure elements and 32 loop residues).
a
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Table S2. TALOS+ dihedral angle restraints for GB1a
Residue number
 (degrees)
2
-115 ± 36
3
-129 ± 26
4
-133 ± 22
5
-110 ± 24
6
-105 ± 30
7
-101 ± 30
8
-108 ± 54
9
-109 ± 70
10
-67 ± 30
11
-102 ± 34
12
-117 ± 62
13
-125 ± 28
14
-150 ± 62
15
-135 ± 34
16
-121 ± 34
17
-132 ± 20
18
-128 ± 40
19
-121 ± 20
20
-114 ± 54
21
-84 ± 36
22

23
-62 ± 20
24
-63 ± 20
25
-68 ± 20
26
-63 ± 20
27
-63 ± 20
28
-61 ± 20
29
-64 ± 20
30
-67 ± 20
31
-65 ± 20
32
-67 ± 20
33
-64 ± 20
34
-63 ± 20
35
-60 ± 20
36
-61 ± 20
37
-97 ± 26
38
86 ± 20
39
-84 ± 32
40
-115 ± 48
41

42
-113 ± 32
43
-111 ± 38
44
-129 ± 44
45
-145 ± 32
 (degrees)
140 ± 36
149 ± 22
132 ± 20
122 ± 20
117 ± 30
124 ± 30
135 ± 54
170 ± 40
-32 ± 22
-4 ± 32
128 ± 42
161 ± 28
161 ± 34
151 ± 26
151 ± 32
156 ± 20
138 ± 26
129 ± 22
163 ± 30
-18 ± 32

-37 ± 20
-40 ± 20
-38 ± 20
-42 ± 20
-40 ± 24
-41 ± 20
-46 ± 20
-37 ± 20
-42 ± 20
-41 ± 20
-44 ± 20
-44 ± 20
-43 ± 20
-27 ± 20
7 ± 20
13 ± 20
134 ± 20
138 ± 44

128 ± 36
151 ± 28
158 ± 22
134 ± 34
7
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Table S2. continued
Residue number
46
47
48
49
50
51
52
53
54
55
a
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 (degrees)
-112 ± 32

-74 ± 22
-104 ± 20
61 ± 22
-102 ± 48
-116 ± 26
-129 ± 30
-129 ± 26
-107 ± 42
 (degrees)
133 ± 32

-25 ± 38
6 ± 34
34 ± 32
133 ± 20
153 ± 32
147 ± 20
132 ± 20
127 ± 28
Dihedral restraints were obtained using the 13C and 15N solid-state NMR chemical shifts reported for
microcrystalline GB1 (BMRB entry 15156) and the TALOS+ program11 in the ‘no-proton’ mode
(http://spin.niddk.nih.gov/bax/nmrserver/talos/). For predicted dihedral angles having uncertainties of less than
±20o, the uncertainties were set to ±20o for the structure calculations.
8
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Table S3. Potential term parameters during structure calculations
Force constant schedulea
Potential name
Description
Initial minimization
(force constant units)
Simulated annealing
and dynamics
PrePot
SSNMR PRE restraints
0.05
ramped: 0.05 Æ 1.0
(kcal◊sec/mol)
NOEPot
repulsive distance
2
ramped: 2 Æ 30
2
(kcal/mol/Å )
restraints
CDIH
TALOS+ based dihedral
1000
1000
(kcal/mol/rad2)
restraints
COLL
radius of gyration
1000
1000
(kcal/mol/Å2)
restraint
RAMA
knowledge-based
1
1
(kcal/mol)
dihedral restraints
HBDB
knowledge-based
2/1
2/1
(kcal/mol)
hydrogen bond restraints
ResidueAffPot
knowledge-based
50/1
50/1
(kcal/mol/Å)
contact potential
BOND
bond length
1000
1000
(kcal/mol/Å2)
ANGL
bond angle
200
ramped: 200 Æ 500
(kcal/mol/rad2)
IMPR
improper dihedral angle
50
ramped: 50 Æ 5000
(kcal/mol/rad2)
DIHE
dihedral angle
50
ramped: 50 Æ 5000
(kcal/mol/rad2)b
VDW
quartic atom-atom
0.004
ramped: 0.004 Æ 4
(kcal/mol/Å4)c
repulsion
Cα-only
all atoms
a
Entries with two slash-separated values correspond to the anneal value and refine value, respectively.
Only applied to the EDTA-Cu2+ sidechain dihedral angles for which the RAMA term contains no information.
c
In addition atomic radii are scaled by a value of 1.2 during initial dynamics and minimization and scaled by a value
ramped from 0.9 to 0.78 during simulated annealing.
b
9
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