Acta Cryst. (2011). D67, 584–591
Acta Crystallographica Section D
High-resolution neutron crystallographic studies of
the hydration of the coenzyme cob(II)alamin
Gerwald Jogl,aXiaoping Wang,b
Sax A. Mason,cAndrey
Mustyakimov,dZo ¨e Fisher,d
Kratkyeand Paul Langanb,d*
aDepartment of Molecular Biology, Cell
Biology and Biochemistry, Brown University,
Providence, RI 02912, USA,bSpallation
Neutron Source, Oak Ridge National
Laboratory, Tennessee, USA,cInstitute
Laue–Langevin, 6 Rue Jules Horowitz, BP 156,
38042 Grenoble CEDEX 9, France,dBioscience
Division, Los Alamos National Laboratory,
Los Alamos, NM 87545, USA, andeInstitute of
Molecular Biosciences, University of Graz,
8010 Graz, Austria
Correspondence e-mail: firstname.lastname@example.org
# 2011 International Union of Crystallography
Printed in Singapore – all rights reserved
using high-resolution monochromatic neutron crystallographic
data collected at room temperature to a resolution of 0.92 A˚
on the original D19 diffractometer with a prototype 4?? 64?
detector at the high-flux reactor neutron source run by the
Institute Laue–Langevin. The resulting structure provides
hydrogen-bonding parameters for the hydration of biomacro-
molecules to unprecedented accuracy. These experimental
parameters will be used to define more accurate force fields
for biomacromolecular structure refinement. The presence of
a hydrophobic bowl motif surrounded by flexible side chains
with terminal functional groups may be significant for the
efficient scavenging of ligands. The feasibility of extending
the resolution of this structure to ultrahigh resolution was
investigated by collecting time-of-flight neutron crystallo-
graphic data during commissioning of the TOPAZ diffracto-
meter with a prototype array of 14 modular 2?? 21?detectors
at the Spallation Neutron Source run by Oak Ridge National
Received 5 March 2011
Accepted 20 April 2011
Cobalamin (Cbl) is a large heterocyclic molecule in which a
central cobalt ion is equatorially coordinated by four donor N
atoms from a corrin ring and axially coordinated at its lower
(?) position by an N atom from 5,6-dimethylbenzimidazole
(DMB) as shown in Fig. 1. In the Co(III) state the upper (?)
axial position can be occupied by a large number of different
ligands and several liganded Cbl X-ray crystal structures
have been reported (Hannibal et al., 2010; Gruber et al., 1999;
Randaccio et al., 2006). Cbls cannot be synthesized by higher
organisms. In dietary vitamin B12(CNCbl) a cyanide group is
the ?-axial ligand, but this form is enzymatically hydrolyzed to
cob(II)alamin [Cbl(II)] and then transformed into one of two
biologically active forms, AdoCbl and MeCbl, in which the ?
axial ligand is either a 50-deoxyadenosyl (Ado) group or a
methyl (Me) group, respectively (Chu et al., 1993; Froese et al.,
2009; Kim et al., 2008).
In mammals, several proteins are involved in the uptake,
transport and storage of dietary Cbls (Banerjee et al., 2009).
X-ray crystallographic structures of complexes of Cbl with the
intrinsic factor (IF; Mathews et al., 2007) and trans-Cbl (TC;
Wuerges et al., 2006) transport proteins have been reported.
AdoCbl and MeCbl are the largest and most complex cofac-
tors in biology and are required by two mammalian enzymes:
MeCbl is the cofactor of methionine synthase (MES), whose
mechanism involves generating alkyl cations by heterolytic
cleavage of the Co—C bond, while AdoCbl is the cofactor
of methylmalonyl coenzyme A mutase (MCAM), whose
mechanism involves generating an Ado radical by homolytic
dissociation of the Co—C bond. X-ray crystallography has
shown that although DMB is coordinated to the ?-axial
position in the ‘base-on’ configuration in the free coenzyme
and in complexes with dehydratase enzymes and the transport
proteins IF and TC, it is found in the ‘base-off’ configuration
in complexes with the enzymes MES and MCAM, displaced
from the cobalt ion by a histidine amino-acid side chain of the
protein (Banerjee et al., 2009).
Understanding how an enzyme facilitates controlled and
reversible cleavage of the Co—C bond and therefore transi-
tion between Co(III) and Co(I,II) is an outstanding problem
in biochemistry. Although early ideas from classical coordi-
nation chemistry suggested the importance of the Co—N bond
to the DMB ?-ligand in modulating cleavage, more recent
X-ray crystallographic studies and electronic structure calcu-
lations of enzyme–Cbl complexes (Jensen & Ryde, 2009) and
similarities between the structures of MeCbl and its homolysis
product Cbl(II) (Kra ¨utler al., 1989) suggest that enzymes play
a dominant role in pulling the adenosyl radical away from the
corrin ring. However, the exact architecture of the corrin ring,
with its seven amide side chains (three acetamides and four
propionamides) projecting above and below the plane, and
coordination with DMB are likely to contribute to stabilizing
the low-spin Co(I), Co(II) and Co(III) states involved in
catalysis and recombination (Jensen & Ryde, 2009).
In addition to its role as a coenzyme, Cbl has also been
implicated in the inhibition of nitric oxide-induced physi-
ologies and pathologies (Hassanin et al., 2010). NO is a short-
lived radical that is produced by tumor and host cells and is
thought to function as an important modulator of tumor
progression and angiogenesis. It has been suggested that in
vivo Cbl(II) directly scavenges NO to form NOCbl, which
has renewed interest in the exact structure of Cbl(II) and its
?-ligand binding. Recent X-ray crystal structures of NOCbls
have featured remarkably long Co—N(DMB) ?-axial bond
lengths, suggesting that this bond may well be correlated with
direct ?-ligand scavenging (Hassanin et al., 2010). Cbls have
also been implicated in the dechlorination by anaerobic
organisms of the toxic compounds perchloroethylene and
trichloroethylene (Randaccio et al., 2006).
Cbls possess a number of functional groups found in bio-
macromolecules and can be crystallized in forms that contain
several ordered solvent molecules and which diffract X-rays
to high resolution. They have therefore been suggested as
particularly useful models for studying the interaction of water
with proteins and nucleic acids (Bouquiere et al., 1994;
Randaccio et al., 2006; Savage et al., 1987; Savage, 1986).
Neutrons offer important advantages for determining solvent
structures hydrating biomacromolecules because the relatively
strong scattering power of hydrogen (H) and deuterium (D)
can allow the location of all atoms in a water molecule even
at medium resolution (>1.5 A˚; Blakeley et al., 2008); D atoms
(neutron scattering length 6.67 ? 10?15m) appear as strong
peaks in neutron scattering density maps, thereby revealing
the location of isotopically exchanged H atoms, while H atoms
(?3.74 ? 10?15m) appear as negative troughs.
The water structure in crystals of vitamin B12has been
studied in detail previously using high-resolution monochro-
matic neutron data collected at 279 K to a resolution of 0.95 A˚
on the former instrument D8 with a point detector at the
high-flux reactor neutron source run by the Institute Laue–
Langevin (ILL; Savage et al., 1987). This study revealed
intricate networks of partially disordered solvent running
throughout the crystal (Savage, 1986). A subsequent high-
resolution study on the original D19 at 15 K extended the
resolution to 0.90 A˚(Bouquiere et al.,
1994). We have previously studied the
water structure in Cbl(II) at room
temperature using quasi-Laue neutron
data collected using the original LADI
diffractometer also at ILL (Langan et
al., 1999). LADI has recently been
significantly upgraded (to LADI-III) in
(Blakeley et al., 2010). Limitations set
by the wavelength band used on the
original LADI (which had a peak at
2.8 A˚and a FWHH of 19%) restricted
the resolution of the data to d > 1.42 A˚.
The final nominal resolution (at which
the completeness fell below 70%) of the
data set collected in 36 h was around
1.8 A˚. Even at this medium resolution
we were able to obtain coordinates for
all of the atoms involved in seven
ordered water and two acetone solvent
Acta Cryst. (2011). D67, 584–591 Jogl et al.
(a) Structural formula of Cbl(II). (b) Atom numbering and side-chain labeling used for description.
determined that contributed to an improved general under-
standing and modeling of the hydration of biomacromolecules.
In addition to the data set collected on LADI, another more
complete neutron data set was collected from Cbl(II) at room
temperature on the original D19 with its small prototype 4??
64?area detector. We were able to collect data to a higher
resolution with a data-collection time of 28 d. The resolution
of the data was still limited to d > 0.92 A˚because of the use of
a wavelength of 1.54 A˚and the geometry of the original D19.
Recently, a new single-crystal diffractometer called TOPAZ
has been built at the Spallation Neutron Source (SNS) run by
Oak Ridge National Laboratory. At the SNS neutrons are
produced by bombarding a mercury target with pulses of
high-energy protons. Neutrons produced by proton pulses are
‘time-stamped’ and travel as a function of their energy so that
neutrons of different energies are detected at different arrival
times. By recording the time-of-flight (TOF) information, the
corresponding energy and wavelength of each neutron can
be calculated. TOF techniques therefore allow wavelength-
resolved Laue patterns to be collected using all of the avail-
able useful neutrons. In particular, the tunable wavelength
range on TOPAZ in combination with an array of position-
sensitive detectors will offer the possibility of collecting data
to ultrahigh resolution from biological molecules with unit-cell
parameters of up to about 70 A˚in length.
In this study, we report the structure of Cbl(II) determined
from the monochromatic neutron data set collected on the
original D19 at the H11 thermal beam at the ILL to 0.92 A˚
resolution. We also report preliminary studies of the feasibility
of extending the resolution of this structure even further by
collecting TOF neutron data during a short commissioning
experiment on TOPAZ. Particular attention is given to a
description of the data-collection and integration process on
TOPAZ since this is a new instrument and it has not been
reported in full before. The results from D19 provide detailed
hydrogen-bonding parameters for the hydration of different
functional groups in a biological molecule at room tempera-
ture to unprecedented resolution and accuracy. The features
of the Cbl(II) structure may promote the capture and
scavenging of small molecules such as NO. The results from
TOPAZ demonstrate that neutron data can be collected from
biological molecules rapidly to high resolution but that there is
also the potential to collect to ultrahigh resolution with longer
2. Experimental methods
The preparation of Cbl(II) and its crystallization with hexa-
deuteroacetone in D2O have been described previously
(Langan et al., 1999). A crystal of dimensions 4.5 ? 1.4 ?
1.3 mm was mounted in a sealed quartz capillary with its
mother liquor. Data were collected on the original D19 using
a vertically mounted cylindrical electronic position-sensitive
detector which subtended 4?? 64?at the sample position at
room temperature with a wavelength of 1.54 A˚in normal-
beam geometry with a goniometer step size in ! of 0.1?
and over a period of about 28 d [space group P212121;
unit-cell parameters a = 15.9795 (9), b = 21.8889 (13),
c = 26.8095 (18) A˚; V = 9377.26 A˚3; 11 986 measured reflec-
tions, 7970 unique reflections and 5619 unique reflections with
Fo> 4?; R?= 0.1009; Rmerge= 0.042 for 6625 unique reflections
with positive intensities]. Reflections were integrated with the
three-dimensional procedure of Wilkinson et al. (1988), which
uses an empirical library of peak shapes from the stronger
reflections to improve the integration for the weak peaks. D19
has recently been upgraded with a new horizontally mounted
30?? 120?cylindrical detector with corresponding gains of up
to 50-fold in data-collection efficiency from biological samples
(Nishiyama et al., 2008; Kovalevsky et al., 2010), so that high-
resolution data sets on macromolecules can be measured in
days rather than weeks.
Data were then collected during the commissioning of
TOPAZ at room temperature in TOF Laue mode (Hoffmann
et al., 2009) using neutrons with wavelengths in the range
0.65–3.4 A˚. For commissioning purposes, the instrument was
equipped with a prototype array of 14 modular area detectors
at distances of 39.5–42.5 cm from the sample. Each detector
module had a square active area of 150.2 ? 150.2 mm
(corresponding to an angular coverage of about 21?? 21?)
that was divided into 256 ? 256 pixels (0.578 mm per pixel).
The detector modules were positioned on the surface of a
spherical detector array with a total vertical angular coverage
of ?32?from the horizontal plane and 18.0–144.0?in the
horizontal (2?) plane, as shown in Fig. 2. In its final config-
uration TOPAZ will have 48 detectors and therefore more
than three times the detector coverage used in this short
commissioning experiment. Since this experiment, significant
gains have also been made in the signal-to-noise ratio of data
collected on TOPAZ by reducing the instrument scattering
Raw data were recorded in neutron event mode with a time
resolution of 1 ms, which is equivalent to a neutron wavelength
resolution of 2.15 ? 10?4A˚at 1.0 A˚for a sample at 18.0 m
Jogl et al.
Acta Cryst. (2011). D67, 584–591
The geometrical arrangement of the initial 14 detector modules in the
detector array during the commissioning of TOPAZ.
from the neutron source. The detected neutron events were
recorded as a function of TOF, detector number, detector
pixel address (ID) and scattering angle. Positions of detector
pixel IDs were mapped initially from their engineering values
and then calibrated using a standard sapphire crystal with
known unit-cell parameters. The calibrated detector infor-
mation was stored in Nexus format (Konnecke, 2006), in which
the Z axis is along the neutron beam direction, the Y axis is up
and the X axis follows the right-handed rule. During data
collection, the neutron event data were saved and broadcast
to a listening server in a local EventViewer program in ISAW
(Mikkelson et al., 2009) for live viewing of the measured
diffraction data in three-dimensional reciprocal q-space, as
shown in Fig. 3. The EventViewer program is capable of
indexing peaks, generating an orientation matrix with user
input and carrying out data integration in real time.
An initial orientation matrix was determined by indexing
the diffraction data after the first few minutes of data collec-
tion. The matrix was then imported into the CrystalPlan
program (Zikovsky et al., 2011) for data-collection strategy
optimization. Event data were collected at 11 crystal gonio-
meter settings over the course of 2 d (42 h of neutron beam
time) and translated into Nexus frames for data integration.
During translation, TOF data were binned into neutron image
frames logarithmically with a constant ?t/t resolution of 0.004
(0.4%). Integrated diffraction intensities were obtained using
reduction and absorption correc-
tion were carried out with the
(Schultz et al., 1984) launched
from within the ISAW program
suite (Mikkelson et al., 2005).
The Becker–Coppens procedure
implemented in GSAS (Larson &
Von Dreele, 2004) was used for
the neutron wavelength-depen-
dent extinction correction. The
resulting reflection data were
converted to SHELX format for
structural analysis [space group
a = 16.005 (2), b = 21.931 (3),
c = 26.911 (4) A˚, V = 9446 (2) A˚3;
10 706 observed reflections with
I > 2?(I); Rmerge= 0.1008 for 7323
The structure was refined first
against the D19
SHELX (Sheldrick, 2008; C68H/
D114CoN13O23P; R= 0.1232 for all
7970 data and R = 0.0923 for 5904
reflections with Fo > 4?) and
included seven water (D2O) and
two acetone [(CD3)2CO] solvent
molecules. All atoms except those
of the acetone molecules were refined with anisotropic atomic
displacement parameters. The occupancies of the D atoms
bound to the amide groups (0.77), the acidic C10 corrin atom
(0.69) and the hydroxyl groups (0.71) were allowed to refine.
The occupancies of the water and acetone solvent molecules
were also allowed to refine. Significant (>3?) density was
present in the final 2Fo? Fcmap in the large solvent channel
which could not be easily interpreted as further ordered water
or acetone molecules. There were also clear indications that
the acetone molecule sitting near the ?-axial position over the
cobalt cation is disordered. We did not attempt to model these
features as our primary interest was in determining detailed
hydrogen-bonding parameters for ordered water molecules.
The final structure is shown in Fig. 4 and a CIF file is available
as supplementary material1. Hydrogen-bond geometries
determined from these parameters are given in Table 1. The
structure was also refined against the TOPAZ data. We
investigated first using a resolution cutoff of d > 0.9 A˚and
with all atoms having isotropic atomic displacement para-
meters (R = 0.1108 for 7323 data). The resulting structure was
essentially identical to that obtained with the D19 data. When
the resolution range was extended to d < 0.9 A˚the R factor
Acta Cryst. (2011). D67, 584–591 Jogl et al.
Background, neutron events (20 out of 259 million) for one sample position as displayed by the three-
dimensional reciprocal-space EventViewer software. Upper left insert, nine of the strong peaks found in
Nexus image frames using the peak-search program in ISAW. Lower left insert, display parameters.
1Supplementary material has been deposited in the IUCr electronic archive
(Reference: TM5035). Services for accessing this material are described at the
back of the journal.
increased dramatically without significantly improving the
accuracy of the structure. This effect suggests that although
there is some diffraction at ultrahigh resolution beyond 0.9 A˚,
the nominal diffracting power of the crystal is about 0.9–1.0 A˚.
The experimental standard deviation (e.s.d.) range for bond
lengths in Cbl(II) involving all non-H atoms in the high-
resolution (0.92 A˚) D19 neutron structure reported here is
0.007–0.020 A˚. This is similar to the range of values found in
the previous high-resolution (0.95 A˚) neutron study of vitamin
B12(0.007–0.021 A˚; Savage et al., 1987) and indicates a rela-
tively high level of precision for a biomacromolecule. The
range of values obtained for the neutron structure of Cbl(II)
determined with the TOPAZ data was slightly higher (0.008–
0.030 A˚), indicating a slightly less accurate structure. How-
ever, this is only to be expected given the drastically shorter
data-collection time of 2 d on TOPAZ. The purpose of
collecting the TOPAZ data was not to improve the accuracy of
the data and therefore the structure already determined at
d > 0.9 A˚, but rather to explore the possibility of collecting
further diffraction data at ultrahigh resolutions of d < 0.9 A˚. It
is clear that both the current D19 with its newly upgraded
detector and the future as-designed TOPAZ with its larger
detector array will be capable of collecting highly accurate
data from small to medium-sized biological macromolecules at
The overall geometry of Cbl(II) has been described
previously and its corrin moiety is very similar to that of
vitamin B12(Kra ¨utler al., 1989; Fig. 4). However, significant
differences exist between the conformations adopted by some
of the side chains. In particular, the conformation of the
nucleotide F loop is different and the phosphate groups
therefore hydrogen bond to different water moieties. W1–W5
are found in the hydrophilic region at the interface of stacked
planes of Cbl(II) molecules and are involved in hydrogen
bonding to the phosphate groups (Langan et al., 1999; Fig. 4a).
Another difference is the existence of a large open solvent
channel in Cbl(II). W6 and W7 and the two acetone molecules
are found in this channel, but it is mostly occupied by dis-
ordered solvent (Langan et al., 1999) which we did not attempt
to model. In vitamin B12the DMB ligand occupies this channel
and is surrounded by a complex pattern of partially occupied
water hydrogen-bonding networks that extend throughout the
crystal (Savage, 1986). The water hydrogen-bonding para-
meters derived from this study are therefore based on a
different water structure to that found in vitamin B12
(Bouquiere et al., 1994; Randaccio et al., 2006; Savage, 1986;
Savage et al., 1987).
The accuracy of the hydrogen-bonding parameters invol-
ving water is significantly improved in the high-resolution
neutron structure presented here compared with the pre-
viously reported neutron structure of Cbl(II) to medium
resolution (1.8 A˚; Langan et al., 1999), e.g. the mean values of
the e.s.d. of donor–acceptor distances and angles for hydrogen
bonds between water molecules are 0.03 A˚and 2.5?, respec-
tively, in this study, compared with 0.07 A˚and 5.2?in the
previous study. Interestingly, this improved accuracy reveals a
more linear geometry, with the mean hydrogen-bond angle
being 173 (7)?in this study compared with 152?in the previous
study. The former value is still significantly smaller than 180?
and is very similar to the values of 170?and 175?found in
a statistical study of the distributions of hydrogen-bond angles
for N—H???O and O—H???O, respectively, in protein–ligand
complexes (Sarkel & Desiraju, 2004). Furthermore, the
improved accuracy allows the identification of some differ-
ences in the mean solvent hydrogen-bonding parameters for
different functional groups which were not evident in the
previous study. In particular, the mean donor–acceptor
hydrogen-bond lengths decrease in the order amine–water >
water–water > water–ketone > water–phosphate [3.00 (18) >
2.88 (8) > 2.81 (16) > 2.66 (3) A˚; Fig. 5]. This trend agrees with
an increase in hydrogen-bond strength in the series N—H???
O—H > O—H???O—H > O—H???O
(Grabowski, 2006; Rozas, 2007).
The relatively small hydrogen-bond lengths for water
molecules hydrating the phosphate group reflect a larger
electrostatic contribution from the charge on the phosphate.
These hydrogen bonds fall into a group of hydrogen bonds
called negatively charge-assisted hydrogen bonds (Perrin &
C > O—H????O—P
Jogl et al.
Acta Cryst. (2011). D67, 584–591
Hydrogen-bond parameters for donor (D), acceptor (A) and deuterium
Donor Acceptor D—H (A˚)H???A (A˚)D???A (A˚)D—H???A (?)
Water–water hydrogen bonds
Amine–water hydrogen bonds
Water–phosphate hydrogen bonds
Water–ketone hydrogen bonds
Other hydrogen bonds
Nielson, 1997). There is a significant difference between the
P1—O3P and P1—O2P bond lengths [1.53 (1) and 1.47 (1) A˚,
respectively] which indicates that most of the charge is asso-
ciated with O3P, as correctly represented in Fig. 1. O3P and
O2P participate in three and two hydrogen bonds, respec-
tively. In addition to having the longest mean lengths,
hydrogen bonds donated by amine groups to water molecules
have the largest range of angles [134.7 (16)–177.2 (18)?].
Although the largest range of hydrogen-bond angles involve
the hydrogen bonds involving water molecules, there is one
hydrogen bond (N5B—O3P) which is clearly an outlier as it
has both the smallest angle (117.7?) and the longest H???A
distance (2.6 A˚).
The locations of H and D atoms covalently bound to Cbl(II)
are well defined by negative troughs and positive peaks in
2Fo? Fc, ?Amaps and Fo? FcOMIT density maps, respec-
tively. These atoms were refined without constraints on their
positions or atomic displacement parameters (ADP). Table 2
shows the mean ADPs for H/D atoms and the heavy atoms
that they are covalently bonded to. The values of the ratio of
the mean ADPs suggests that when refining a biomacro-
molecular structure at lower resolutions with H atoms
included using a ‘riding model’ (Sheldrick, 2008) the ADPs of
H atoms should be tied to about 1.5 or 1.1 times the ADP of a
bound C or N atom, respectively. It is surprising to find that
the ratios of the ADPs for H to C in CH, CH2and CH3groups
are similar given the different possible motions of these
groups, although we acknowledge that the sampling array is
Interestingly, the methyl H atoms that project up above the
corrin plane, together with the aliphatic H atoms from the
three acetamide side chains, form a hydrophobic perimeter
wall around the upper (?) axial ligand position, as illustrated
in Fig. 4(b). The upper side of Cbl(II) therefore has the shape
of a bowl with a hydrophobic bottom and inside, and an
opening that faces the large solvent channel. At the opening of
this barrel lies the partially disordered acetone molecule,
tethered above the cobalt cation to the functional amide group
of the A acetamide side chain by a bridging W6 water mole-
cule(Fig.4a). Only the A and C acetamide side chains (located
to the right of the cobalt ion in Fig. 4a) point upwards to the
upper side of the corrin ring.
These A and C side chains are
found in a variety of different
conformations in X-ray crystal
structures of different Cbls and
their amine functional groups are
found both pointing towards or
away from the inside of the
hydrophobic bowl. It is therefore
likely that they are highly flexible.
It seems likely that the functional
groups at the end of these flexible
side chains play a role in con-
centrating physiological ligands
around the hydrophobic bowl and
perhaps even in guiding them
towards the cobalt cation at the
bottom of the bowl (Fig. 4b).
Crystallographic refinement of
minimizes a target function that
typically a force field. Obtaining
bonding parameters for water
hydrating biological structures is
important because it provides
experimental data that can be
used to parameterize the force
Acta Cryst. (2011). D67, 584–591Jogl et al.
(a) Skeletal representation of the neutron structure of Cbl(II) with the ordered solvent molecules labeled
W1–W7. (b) Space-filling representation illustrating the presence of a hydrophobic bowl above the corrin
plane. (c) Stereogram of positive neutron scattering density in a 2Fo? Fcmap displayed at a level of 5?.
Mean (Ueq) atomic displacement parameters for H/D atoms in different
GroupNo. C or NH Ratio
field. The structure reported and the resulting water
hydrogen-bonding parameters are of unprecedented resolu-
tion for a neutron study of a biomacromolecule. Nonbonded
interactions in the force field are typically treated as a simple
repulsive term (referred to as the ‘Repel’ force field; Adams et
al., 1997). This repulsive force depends on the atomic radii of
the interacting atoms and it is able to model the larger
hydrogen-bonding distances observed in this study for N???O
interactions compared with O???O interactions. However, it
does not model the significant differences observed in the
O???O distances when different functional groups are
involved and thus does not take into account the different
strengths of hydrogen bonds. The hydrogen-bonding para-
meters reported here can therefore be used to improve the
Repel force field by taking into account the involvement of
different functional groups. We expect that this will increase
the accuracy of biomacromolecular structure refinement.
Furthermore, the hydrogen-bonding parameters reported
here provide abasis for testing the accuracy of newly emerging
force fields and approaches in biomacromolecular refinement
that include electrostatic interactions (Fenn et al., 2011).
D19 with its newly upgraded detector and the future as-
designed TOPAZ with its larger detector array will be capable
of collecting highly accurate data from small to medium-sized
biological macromolecules at unprecedented rates. Further-
more, in our exploration of the possibility of extending the
resolution of the Cbl(II) structure beyond 0.9 A˚, we observed
some diffraction at ultrahigh resolution beyond 0.9 A˚,
although the nominal diffracting power of the crystal
prevented its use in structure refinement. This suggests that
TOPAZ may be an ideal instrument for developing new
research based on ultrahigh-resolution neutron studies of
We acknowledge the ILL and SNS for provision of facilities.
TOPAZ is funded by the Office of Basic Energy Sciences of
the Department of Energy. PL, MM, AK and ZF were partly
funded by the Office of Biological and Environmental
Research of the Department of Energy. PL and MM were
partly supported by an NIH–NIGMS grant (R01GM071939-
01) to develop computational tools for neutron protein crys-
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