Acta Cryst. (2008). F64, 318–322
Acta Crystallographica Section F
Crystallization and preliminary X-ray diffraction
analysis of recombinant hepatitis E virus-like
Tetsuo Yamashita,d,e‡ Akifumi
Der-Ming Liou,cYen-Jen Sung,c,h
and R. Holland Chenga,b*
aMolecular and Cellular Biology, University of
California, Davis, CA 95616, USA,bKarolinska
Institute Structural Virology, F68 Karolinska
University Hospital, SE-14186 Stockholm,
Sweden,cInstitute of Public Health, National
Yang-Ming University, 112 Taipei, Taiwan,
dInstitute for Protein Research, Osaka University,
3-2 Yamadaoka, Suita, Osaka 565-0871, Japan,
eInstitute for Microbial Diseases, Osaka
University, 3-2 Yamadaoka, Suita,
Osaka 565-0871, Japan,fDepartment of
Virology II, National Institute of Infectious
Diseases, Tokyo, Japan,gCrystal Research AB,
22370 Lund, Sweden, andhInstitute of Anatomy
and Cell Biology, National Yang-Ming
University, 112 Taipei, Taiwan
‡ These authors contributed equally to the
Correspondence e-mail: firstname.lastname@example.org
Received 3 March 2008
Accepted 16 March 2008
Hepatitis E virus (HEV) accounts for the majority of enterically transmitted
hepatitis infections worldwide. Currently, there is no specific treatment for or
vaccine against HEV. The major structural protein is derived from open reading
frame (ORF) 2 of the viral genome. A potential oral vaccine is provided by the
virus-like particles formed by a protein construct of partial ORF3 protein
(residue 70–123) fused to the N-terminus of the ORF2 protein (residues 112–
608). Single crystals obtained by the hanging-drop vapour-diffusion method at
293 K diffract X-rays to 8.3 A˚resolution. The crystals belong to space group
P212121, with unit-cell parameters a = 337, b= 343, c= 346 A˚, ?= ? = ? = 90?, and
contain one particle per asymmetric unit.
The hepatitis E virus (HEV) is a naked icosahedral capsid with a
single-stranded positive-sense RNA of 7.2 kbp. The genome encodes
three open reading frames (ORFs). ORF1, mapped to the
50-terminus, encodes nonstructural proteins that are mainly involved
in virus replication and protein processing. ORF2, mapped to the
30-terminus, encodes a viral capsid protein of 660 amino acids that has
been found to elicit neutralizing antibodies (Meng et al., 2001;
Schofield et al., 2000). ORF3, mapped between ORF1 and ORF2,
encodes a protein of 123 amino-acid residues that may interfere with
control functions within the infected cell, as summarized by Panda et
al. (2007). When the N-terminally truncated ORF2 protein (residues
112–660) was expressed with a recombinant baculovirus in an insect-
cell line, self-assembling virus-like particles (VLPs) were released
into the cell supernatant (Li et al., 1997). These VLPs have been
shown to induce anti-HEV antibodies when orally administered to
experimental animals (Li et al., 2004). By N- and C-terminal
sequencing, the VLP-forming protein was found to be composed of
residues 112–608 of the ORF2 protein (VLPORF2); thus, 52 residues at
the C-terminus were cleaved during VLP formation (Li et al., 1997).
We have previously reported the structure of HEV-VLPORF2
obtained using electron cryomicroscopy (cryo-EM), which provided a
preliminary understanding of the quaternary arrangement of the viral
capsids. A three-dimensional reconstruction of VLPORF2 displays
T = 1 icosahedral symmetry and is composed of 60 copies of the
truncated ORF2 protein (Xing et al., 1999; Li et al., 2005).
Although a truncated ORF2 polypeptide is undergoing clinical
trials as a vaccine candidate (Shrestha et al., 2007), to date no specific
treatment or vaccine has been licensed for HEV (Purcell & Emerson,
2008). The viral capsid is an important form of presenting the
conformation-dependent epitopes (Maloney et al., 2005) and HEV-
VLPORF2has been proposed as a suitable candidate for an oral
vaccine (Li et al., 2004). Further investigations of the high-resolution
structural features of a VLP are required in order to establish the
folding and interactions of the viral protein in the context of the HEV
particle form, as well as to characterize the immunogenic epitopes
that are responsible for inducing the neutralizing antibodies. In the
present study, we describe the crystallization and preliminary crys-
# 2008 International Union of Crystallography
All rights reserved
tallographic characterization of a purified HEV-VLPORF3/ORF2capsid
composed of a fusion protein obtained by inserting a fragment of
ORF3 (residues 70–123) at the N-terminus of the ORF2 peptide
including residues 112–608.
2. Experimental procedures and results
2.1. Expression and purification of recombinant HEV-VLPs
Recombinant HEV-VLPORF3/ORF2was produced using a similar
approach to those described previously (Li et al., 1997; Xing et al.,
1999) except that an ORF3/ORF2 fusion protein containing a frag-
ment of the ORF3 protein (residues 70–123) attached without an
intervening sequence to the N-terminus of a truncated ORF2 protein
(residues 112–608) was used in the construct for expression. The
transfer vector was co-transfected with insect Sf9 cells (Riken Cell
Bank, Tsukuba, Japan) to produce the recombinant baculovirus. The
recombinant baculovirus obtained was plaque-purified three times.
For large-scale expression, an insect-cell line from Trichoplusia ni,
BTL-Tn 5B1-4 (Tn5; Invitrogen, San Diego, California, USA), was
used and was infected with recombinant baculovirus at a multiplicity
of infection of 10. The cells were incubated in EX-CELL-405 medium
(JRH Biosciences, Lenexa, Kansas, USA) for 7 d at 300 K. The VLPs
were harvested from the supernatant. The recombinant baculovirus
and cell debris were removed by centrifugation at 10 000g for 90 min
at 277 K. The VLPs in the supernatant were then spun down at
100 000g for 2 h at 277 K. The resulting VLP pellets were then
resuspended in EX-CELL-405 medium and further purified in a CsCl
equilibrium density gradient. On inspection by negative-staining EM,
the morphology of VLPORF3/ORF2appeared to be similar to that of
VLPORF2, except for an extra density within the particles.
Prior to crystallization or cryo-electron microscopy (EM) experi-
ments, the purified VLPs were pelleted through a 5%(w/v) sucrose
cushion in 50 mM potassium–MES buffer pH 6.2 at 110 000g in a
Beckman SW 55-Ti rotor at 277 K for 1 h. The pellet was resuspended
in 50 mM potassium–MES buffer pH 6.2 and maintained at 277 K for
10 min. The concentration of recombinant HEV-VLP was adjusted to
10 mg ml?1according to the standard concentration curve deter-
mined from the light absorbance at 260 and 280 nm. The quality of
the purified particles was routinely verified by EM using 2%(w/v)
uranyl acetate negative-stain contrast (Agar Scientific Ltd, Stansted,
England) and SDS–PAGE performed on 8–25% acrylamide gels
under denaturing conditions (Gong et al., 1990; Cheng et al., 1992).
2.2. Cryo-EM and three-dimensional reconstruction of purified
Cryo-EM sample preparation followed previously established
procedures (Xing et al., 1999). Briefly, a 3.5 ml drop of VLPORF3/ORF2
suspension was applied onto a glow-discharged ‘holey’ carbon-coated
grid, blotted with filter paper and vitrified by rapidly plunging the
grids into liquid ethane cooled by liquid nitrogen. The grids, with the
frozen VLPORF3/ORF2physically fixed to fill in the holes of the carbon
film after rapid freezing, were transferred to an FEI CM-120 micro-
scope using a Gatan 626DH cryoholder and all subsequent steps were
carried out with the sample maintained at 95 K. The electron
microscope was operated at 120 kVand low-dose (<7 e?A˚?2) images
were recorded on Kodak SO163 films at a magnification of 45 000?.
Selected micrographs with a defocus level of 1000 nm were digitized
using a Zeiss microdensitometer (Z/I imaging) at a step size of 14 mm,
which corresponds to 3.1 A˚per pixel at the level of the specimen. The
first zeroof thecontrast transfer function wasat aspatial frequencyof
?0.056 A˚?1. Isolated VLP images were extracted from the digitized
micrographs, normalized andcombined into one single image-stacked
file for subsequent processing. Determination of the structure was
carried out using a model-based polar Fourier transform (PFT)
method (Cheng et al., 1994; Baker & Cheng, 1996). As the PFT
algorithm requires a three-dimensional model to start with, a cryo-
EM density map of VLPORF2was used as an initial model (Xing et al.,
1999). The model was low-pass filtered to 40 A˚resolution in order to
reduce the influence of noise bias included in image processing. The
starting model was back-projected at 1?angular increments to create
an image database that covered all possible views of the model at the
orientations within one half of the icosahedral asymmetric unit.
Individual unique views of model projections in the database were
interpolated onto a polar grid to form a polar projection (PRJ) image
and the PRJ image was then Fourier transformed to produce a PFT
image; these PRJand PFTimages were stored in two separate files for
use as references for alignment with individual images of PFTs and
PRJs from the selected VLP projections (Cheng et al., 1994; Baker &
Cheng, 1996). In addition, the alignment was performed with
enhanced accuracy by initially including a band-pass filter (spatial
frequency between 1/90 A˚?1and 1/30 A˚?1) of the PFT images to
optimize the search for origins and orientations. A list of origins and
orientations corresponding to each particle was obtained and a noise-
filtered three-dimensional reconstruction was computed using the
Fourier–Bessel algorithm implemented with a cylinder expansion
method and imposed 522 symmetry (Crowther, 1971; Cheng et al.,
1992; Fuller et al., 1996). The presence of the threefold symmetry in
the three-dimensional model validated the accuracy of the recon-
struction. The search model was subsequently updated with the newly
computed three-dimensional density map of VLPORF3/ORF2through
individual cycles of refinement to make the orientations and origins
of the image data to be included in the averaging of the subsequent
density maps more accurate. The cryo-EM structural density of
VLPORF3/ORF2for use in initial phasing of the X-ray diffraction data
was achieved by the progressive addition of data at higher spatial
frequency. The iterations were continued by re-projecting the model
at a finer angular increment (0.5?) and by progressively extending the
low-pass filter from 30 to 20 A˚. The cycles of refinement stopped
when no major improvement was observed in the three-dimensional
reconstruction. Fourier shell correlations of the reconstruction
yielded an estimated resolution of 24 A˚ for recombinant HEV-
VLPORF3/ORF2basedonFourieraveraging of134VLPimages (Fig.1).
VLPORF3/ORF2has a diameter of ?270 A˚and the capsid shell was
composed of 30 dimer-like protrusions arranged in a T = 1 icosahe-
dral surface lattice. Analysis of the density-distribution map revealed
VLPORF3/ORF2to consist of 60 subunits of the fusion protein. The
VLP structure demonstrated two distinct domains, namely the shell
domain, which forms a continuous layer of viral capsid, and the
protrusion domain, which forms protruding spikes (Cheng et al., 1992,
1995). The cryo-EM density map of VLPORF3/ORF2was subsequently
used for initial phasing of the data collected by X-ray diffraction.
2.3. Crystallization strategy and data collection
The initial crystallization trials were performed by the hanging-
drop method (McPherson, 2004a,b) with a commercially available kit,
Crystal Screen Lite, from Hampton Research (Laguna Niguel, Cali-
fornia, USA) at 293 K. The crystallization drops contained 2 ml
VLPORF3/ORF2 solution at various concentrations mixed with 2 ml
screening solution and were set up for vapor diffusion against 1 ml
screening solution in 24-well plates (Falcon). Crystals were obtained
using two different conditions: (i) 4%(w/v) polyethylene glycol
(PEG) 4000 in 100 mM sodium acetate pH 4.6 and (ii) 4%(w/v) PEG
Acta Cryst. (2008). F64, 318–322Wang et al.
? Hepatitis E virus-like particle
8000 in 100 mM Tris–HCl pH 8.5. In condition (i) a number of small
crystals appeared within a few minutes, while in condition (ii) the
crystals appeared after one week. To further assess the integrity ofthe
VLPs packed in the crystals, we selected crystals from both the pH 4.6
and pH 8.5 conditions, dissolved them in the respective reservoir
solution and performed negative-staining EM with 2%(w/v) uranyl
acetate (Gong et al., 1990). The VLPs had remained intact within the
crystals in both conditions (Fig. 2). The quality of these crystals was
assessed using an in-house X-ray generator. The crystals obtained at
pH 4.6 diffracted to a lower resolution (40 A˚) compared with those
obtained at pH 8.5 (20 A˚). Based on this result, the crystallization
conditions were further optimized by changing the PEG 8000 and
VLP concentrations. Good-quality crystals were obtained with
3.5%(w/v) PEG 8000 in 100 mM Tris–HCl pH 8.5; these crystals were
rod-shaped and reached a maximum length of 1 mm after 14 d
The HEV-VLPORF3/ORF2 crystals were immersed for 2 min in
reservoir solution containing 20, 30 or 40%(v/v) ethylene glycol as a
cryoprotectant. A single crystal was picked up with a cryoloop and
directly flash-frozen in liquid nitrogen. The HEV-VLPORF3/ORF2
crystals were found to be very fragile and cracked in most cases.
Therefore, the VLPs were subsequently crystallized under the same
conditions with the addition of 20–40%(v/v) ethylene glycol to the
reservoir. The crystals obtained had a similar appearance to those
obtained without the addition of ethylene glycol. One of the resulting
crystals was successfully frozen and diffracted X-rays to beyond 7.8 A˚
resolution at 100 Kon a DIP6040 imaging-plate/CCD hybrid detector
(MacScience, Bruker-AXS) with a crystal-to-detector distance of
700 mm, an oscillation angle of 1.0?and an exposure time of 10 s
using synchrotron radiation at SPring-8 (Hyogo, Japan) beamline
BL44XU (Fig. 4). As the crystal decayed during data collection, the
data set was only processed to 8.3 A˚resolution. The diffraction
images were indexed, reduced, scaled and merged using the HKL-
2000 package (Otwinowski & Minor, 1997). The intensities were
converted into the structure-factor amplitudes using TRUNCATE
from the CCP4 package (Collaborative Computational Project,
Number 4, 1994). The space group was determined to be P212121by
scaling in the resolution range 70–8.3 A˚assuming Laue group 222
(Rmerge= 13.6% from SCALEPACK) and was assigned on the basis
of systematic absences of odd reflections along the h00, 0k0 and 00l
axes. The unit-cell parameters were a = 337, b = 343, c = 346 A˚,
? = ? = ? = 90?. The statistics of data collection are summarized in
Table 1. The value of I/?(I) was found to be 8.3 and 2.2 for the
resolution ranges 70–8.3 and 8.6–8.3 A˚, respectively. The Rmergefor
the outermost resolution shell was slightly worse than for most low-
symmetry protein structure determinations. In this case of viral
(60-fold) and the solvent flattening provided the phasing power
required to successfully employ the diffraction data to 8.3 A˚resolu-
tion. While four particles were found in one unit cell (with a mole-
cular weight of 3.2 ? 106Da), there is only one complete VLP per
Wang et al.
? Hepatitis E virus-like particle
Acta Cryst. (2008). F64, 318–322
Three-dimensional structure of recombinant HEV-VLPORF3/ORF2at a resolution of
24 A˚determined by cryo-EM and image reconstruction. An isosurface representa-
tion of the outer surface of recombinant HEV-VLPORF3/ORF2is shown viewed along
the icosahedral twofold axis. The surface density was contoured at a level
corresponding to 100% mass of the expected particle volume. The particle is color-
coded to differentiate two distinct domains: the shell domain (white) and the
protrusion domain (yellow). The surface capsid conforms to T = 1 icosahedral
symmetry in which the 60 subunits are arranged into 30 protruding spikes with the
homodimers as the basic building blocks at each icosahedral twofold axis. The scale
bar represents 100 A˚.
Purity and integrity of recombinant HEV-VLPORF3/ORF2crystals grown in 4%(w/v)
PEG 8000, 100 mM Tris–HCl pH 8.5 analyzed by SDS–PAGE (a) and negative-
stained electron microscopy (b). In (a), the left lane contains molecular-weight
markers (kDa) and the right lane contains the protein band of recombinant HEV-
VLP from a dissolved crystal. In (b), a recombinant HEV-VLPORF3/ORF2crystal was
crushed with a nylon loop and stained with 2% uranyl acetate; the VLPs remained
with intact capsid morphology after dissolving from the crystal. The bar represents
Recombinant HEV-VLPORF3/ORF2crystal. The crystal was grown in Tris–HCl pH
8.5 buffer, 3.5%(w/v) PEG 8000 with the addition of 30% ethylene glycol as a
cryoprotectant. In the scale, 28 intervals represent 0.1 mm.
asymmetric unit, resulting in 60-fold noncrystallographic symmetry
redundancy. The calculated Matthews coefficient VMis 3.1 A˚3Da?1
2.4. Phase determination
A self-rotation function was computed using the program
POLARRFN from the CCP4 package in order to determine the
orientationof the icosahedral
elements. By using reflections in the resolution range 15–10 A˚, a fast
rotation function was calculated with an integration radius of 130 A˚
and a B factor of ?70 A˚2. The section corresponding to the fivefold
axis is depicted in Fig. 5(a). The fivefold rotation function was
consistent with the presence of four particles per unit cell. Six peaks
were clearly identified corresponding to one of the four particles in
the unit cell. While additional peaks were observed corresponding to
the symmetry-related particles, some unexpected peaks might arise
from the 72?rotational relationship between the icosahedral parti-
cles, as they were reproduced from the calculated data using a cryo-
EM map. Subsequently, the molecular-replacement method starting
from a cryo-EM density map (Fig. 1) was used to phase the reflec-
tions. The original cryo-EM map was rotated to superimpose the
icosahedral symmetry axes of the cryo-EM density onto the VLP
orientation determined by the rotation function using the matrix
Packing considerations suggested that the particle is situated close to
the positions in space group P212121with x = 0, y= 0, z= 0 or x = 0.25,
Acta Cryst. (2008). F64, 318–322 Wang et al.
? Hepatitis E virus-like particle
A diffraction pattern recorded from a recombinant HEV-VLPORF3/ORF2crystal. (a) A typical 1.0?oscillation photograph exposed for 10 s. The concentric circles indicate the
13.0 and 6.5 A˚resolution shells. (b) An enlarged image shows a diffraction spot observed at 7.8 A˚(indicated by an arrow).
Phasing X-ray data with acryo-EM density map.(a) Fivefold self-rotation function peaks calculated in POLARRFN with data inthe resolution range 15–10 A˚and aradius of
integration of 130 A˚. The positions (’, ) corresponding to one of the four particles in the unit cell are indicated. (b) Translational correlation coefficient search for the origin
of recombinant HEV-VLPORF3/ORF2. The data used in the calculations were in the resolution range 70–30 A˚(only the result at x = 0 is shown). The search grid started at the
coarse interval of 10 A˚and was refined at a finer interval of 2 A˚. The maximum correlation coefficient was observed at the point (?2, 0, ?6 A˚).
y = 0.25, z = 0.25. The correlation coefficient was computed between Download full-text
the structure factors observed from the amplitudes of the X-ray
diffraction data (Fo) and the amplitudes of the calculated structure
factor (Fc) derived from Fourier transformation of the cryo-EM
model properly rotated and positioned in the crystal unit cell around
these two positions using the programs MAVE from the RAVE
package of the Uppsala Software Factory (Kleywegt et al., 2001) and
SFALL and RSTATS from the CCP4 package. The translation search
was initially carried out with a coarse interval of 10 A˚steps using data
in the resolution range 70–30 A˚. After searching with a finer interval
of 2 A˚, a maximum correlation coefficient value of 0.41 was reached
at the origin (?2, 0, ?6 A˚) (Fig. 5b). Phase refinement and extension
were carried out in the resolution range 30–8.3 A˚using real-space
averaging and solvent flattening with the RAVE and CCP4 packages
as performed in our previous work (Nakagawa et al., 2003). The final
correlation coefficient and R factor between the Fos and the Fcs
obtained from inversion of the averaged and solvent-flattened map at
8.3 A˚resolution were 0.92 and 0.21, respectively.
We report here the detailed conditions for the crystallization of
recombinant HEV-VLPORF3/ORF2and the implementation of a 24 A˚
cryo-EM density map in the initial phasing of the X-ray diffraction
data. The diffraction data presented carry sufficient information for
determining the density map of a 270 A˚diameter VLPORF3/ORF2to a
resolution of 8.3 A˚. The availability of data with improved resolution
provides the structural information needed for the better under-
standing of virus-particle assembly and will be very valuable for HEV
We thank Drs Andy Fisher and Lena Hammar for their critical
editing of the manuscript. This project was supported by grants from
Cancer Research Center, Swedish Research Council and EC/FP6
Levmac (RHC). C-YW, NM and LX were supported by grants from
Cancer Research, STINT Foundation and Discovery Programs,
respectively. Part of the data-analysis scheme developed in this study
was supported by the National Institutes of Health through the NIH
Roadmap for Medical Research (PN2EY018230). C-YW and NM
were initially supported as exchange students by the NYMU Inter-
national Competitiveness Grant and a Grant-in-Aid for 21st Century
Centers of Excellence Program, respectively. This article is part of the
requirement for the PhD degree fulfilment of C-YW and TY.
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Acta Cryst. (2008). F64, 318–322
Crystal information and data-processing statistics.
Values in parentheses are for the outermost shell.
Unit-cell parameters (A˚,?)
a = 337, b = 343, c = 346,
? = ? = ? = 90
Resolution range (A˚)
Total No. of crystals
Total No. of reflections
measurements including symmetry equivalents.
ijIiðhklÞ ? hIðhklÞij=P
iIiðhklÞ, where Ii(hkl) is the intensity of
an individual measurement of the reflection hkl and hI(hkl)i is the mean intensity for all