OXG-RCBH has a loop around the +2 site that blocks one end of the active site cleft, which accounts for its exo mode of action.. At the1 site in OXG-RCBH, Asn488 interacts with the xylos
Trang 1glucanase from Geotrichum sp M128 xyloglucanase
reveals a key amino acid residue for substrate specificity Katsuro Yaoi1,*, Hidemasa Kondo2,*, Ayako Hiyoshi1, Natsuko Noro2, Hiroshi Sugimoto3,
Sakae Tsuda2,4and Kentaro Miyazaki1,5
1 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan
2 Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), Toyohira, Sapporo, Hokkaido, Japan
3 Riken SPring-8 Center, Harima Institute, Hyogo, Japan
4 Division of Biological Science, Graduate School of Science, Hokkaido University, Sapporo, Japan
5 Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tsukuba, Ibaraki, Japan
Introduction
Xyloglucan is a major hemicellulose in the primary cell
wall of plants, where it associates with cellulose
micro-fibrils via hydrogen bonds to form a
cellulose–xyloglu-can network Xyloglucellulose–xyloglu-can consists of a cellulose-like main chain of b-1,4-glucan, which is frequently branched with xylose side chains to form
a-d-Xylp-Keywords
endo-b-1,4-glucanase; glycoside hydrolase
family 74; xyloglucan; xyloglucanase;
b-1,4-glucan
Correspondence
K Yaoi, Institute for Biological Resources
and Functions, National Institute of
Advanced Industrial Science and Technology
(AIST), Tsukuba Central 6, 1-1-1 Higashi,
Tsukuba, Ibaraki 305-8566, Japan
Fax: +81 29 861 6733
Tel: +81 29 861 6065
E-mail: k-yaoi@aist.go.jp
*These authors contributed equally to this
study
Database
The coordinates of the structure for XEG
have been deposited in the Protein Data
Bank under the accession number 3A0F
(Received 14 May 2009, revised 3 July
2009, accepted 8 July 2009)
doi:10.1111/j.1742-4658.2009.07205.x
Geotrichumsp M128 possesses two xyloglucan-specific glycoside hydrolases belonging to family 74, xyloglucan-specific endo-b-1,4-glucanase (XEG) and oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH) Despite their similar amino acid sequences (48% identity), their modes of action and substrate specificities are distinct XEG catalyzes the hydrolysis
of xyloglucan polysaccharides in endo mode, while OXG-RCBH acts on xyloglucan oligosaccharides at the reducing end in exo mode Here, we determined the crystal structure of XEG at 2.5 A˚ resolution, and compared
it to a previously determined structure of OXG-RCBH For the most part, the amino acid residues that interact with substrate are conserved between the two enzymes However, there are notable differences at subsite posi-tions)1 and +2 OXG-RCBH has a loop around the +2 site that blocks one end of the active site cleft, which accounts for its exo mode of action
In contrast, XEG lacks a corresponding loop at this site, thereby allowing binding to the middle of the main chain of the substrate At the)1 site in OXG-RCBH, Asn488 interacts with the xylose side chain of the substrate, whereas the)1 site is occupied by Tyr457 in XEG To confirm the contri-bution of this residue to substrate specificity, Tyr457 was substituted by Gly in XEG The wild-type XEG cleaved the oligoxyloglucan at a specific site; the Y457G variant cleaved the same substrate, but at various sites Together, the absence of a loop in the cleft and the presence of bulky Tyr457 determine the substrate specificity of XEG
Abbreviations
GH74, glycoside hydrolase family 74; Glc, glucose; OXG-RCBH, oligoxyloglucan reducing-end-specific cellobiohydrolase; XEG, xyloglucan-specific endo-b-1,4-glucanase; Xyl, xylose.
Trang 2(1 fi 6)-b-d-Glcp Other sugars such as galactose,
arabinose and fucose may also be present on the side
chains in various branching patterns, depending on the
plant species Structural studies on xyloglucans suggest
that most consist of repeating units of either XXXG
(XXXG-type) or XXGG (XXGG-type) [1], where G
refers to an unbranched Glc residue and X represents
the xylose (Xyl)-branched Glc a-d-Xylp-(1fi
6)-b-d-Glcp [2] Thus, XXXG-type xyloglucans have three
consecutive backbone residues with Xyl side chains
and a fourth unbranched Glc residue, and XXGG-type
xyloglucans have two consecutive branched backbone
residues and two unbranched backbone residues
Various microorganisms that degrade the plant cell
wall produce endo-b-1,4-glucanases that hydrolyze
xyloglucan Most of them cleave the glycosidic bond
of the unbranched Glc residues in the backbone chain
Typically, treatment of an XXXG-type xyloglucan
with these endoglucanases generates oligosaccharides
with a tetrasaccharide backbone (XXXG) For many
years, endo-b-1,4-glucanases have been considered to
be a subgroup of cellulases (EC 3.2.1.4) Recently,
however, it has been clarified that some
endo-b-1,4-glucanases display high activity toward xyloglucan but
have limited or no activity against cellulose These
xyloglucan-specific endo-b-1,4-glucanases have been
classified as a new enzyme family (EC 3.2.1.151),
desig-nated xyloglucan-specific endo-b-1,4-glucanases (XEG)
or xyloglucanases Many xyloglucanases belonging to
glycoside hydrolase families 5, 12, 44 and 74 (http://
www.cazy.org/) have now been identified Of these,
glycoside hydrolase family 74 (GH74) enzymes exhibit
high specificity towards xyloglucan [3–9]
Previously, we have reported the cloning,
purifica-tion and characterizapurifica-tion of two GH74 enzymes,
oligo-xyloglucan reducing-end-specific cellobiohydrolase
(OXG-RCBH) [6] and xyloglucan-specific
endo-b-1,4-glucanases (XEG) [7], from Geotrichum sp M128
OXG-RCBH (EC3.2.1.150) is a unique exo-type
enzyme that recognizes the reducing end of xyloglucan
oligosaccharides Its substrate recognition mechanism
has been investigated using various oligosaccharide
substrates [6], and mutational and detailed structural
studies have revealed its unique mechanism [10,11]
The exo activity of OXG-RCBH is based on a loop at
one side of the active site cleft In addition, residue
Asn488 in the active site cleft recognizes the Xyl side
chain at the )1 site, conferring the unique substrate
specificity On the other hand, XEG showed typical
endo activity [12] It hydrolyzes xyloglucan polymers
randomly In addition, XEG catalyzes hydrolysis of
the glycosidic bond of unbranched Glc residues,
sug-gesting a difference between the two enzymes in the
three-dimensional structures of the active sites In this study, we determined the crystal structure of XEG in order to determine the structural basis for its substrate specificity
Results and discussion
Overall structure of XEG Crystals were grown using 0.1 m MES buffer, pH 5.8– 6.0, and 3–6% w⁄ v PEG 8000 They were elongated rod- or needle-shaped crystals, belonging to the P3221 trigonal space group, with unit-cell parameters of
a= b = 135.2 A˚ and c= 119.9 A˚ One protein molecule of XEG existed in an asymmetric unit of the crystal X-ray diffraction data were collected at 2.5 A˚ resolution, with an Rmerge of 0.096 and 98.4% com-pleteness The structure was determined using the molecular replacement method and was refined against 20–2.5 A˚ intensity data The crystallographic R factor and free R factor were 0.236 and 0.276, respectively Table 1 summarizes the statistics of X-ray data collec-tion, and the results for the structural refinement of
Table 1 Statistics for data collection using the Beamline BL44B2
at SPring-8 and structure refinement of XEG.
Data collection
R mergea,b 0.096 (0.199)
Refinements
Number of non-hydrogen atoms
Root mean square deviations from ideality (bond length) (A ˚ )
0.015 Root mean square deviations from
ideality (angle) ()
1.64
a
Numbers in parentheses are values for the highest-resolution shell: 2.59–2.5 A ˚ for the data collection and 2.56–2.5 A˚ for the refinement b R merge ¼ P
h
P
j <IðhÞ> I j ðhÞ
= PhPjI j ðhÞ, where
<I(h)> is the mean intensity of a set of equivalent reflections.
c R factor ¼ P
h j FobsðhÞ F calc ðhÞ j= P
h j FobsðhÞ j, where F obs and Fcalc are the observed and calculated structural factors, respectively.
Trang 3XEG Figure 1 shows the electron density map of
XEG for the region corresponding to the exo-loop of
OXG-RCBH The coordinates and structure factors
have been deposited in the Protein Data Bank (PDB)
(accession code 3A0F)
The structure of XEG was compared with those of
other GH74 xyloglucan-specific enzymes, OXG-RCBH
and Clostridium Xgh74A The overall structures of
XEG (this study), OXG-RCBH in complex with
substrate XXXG (PDB code 2EBS) and Xgh74A in
complex with substrates XLLG and XXLG (L refers to
a b-d-Galp-(1fi 2)- a-d-Xylp-(1 fi 6)-b-d-Glcp) (PDB
code 2CN3) [13] are illustrated in Fig 2 All enzymes
have two structurally similar domains, each consisting
of a seven-bladed b-propeller These are located
tandemly in the N- and C-terminal halves of the
poly-peptide, and are joined on each edge of the domain to
form a bivalve-like shape XEG can be superimposed
onto OXG-RCBH with an RMSD of 1.07 A˚ by the
corresponding 683 Ca atoms among 756 residues,
indi-cating close similarities between their domain structures
as well as the relative positions of the two domains
The active site cleft is located between the N- and
C-domains One apparent difference between XEG
and OXG-RCBH is at the active site In OXG-RCBH,
a loop comprising 11 amino acid residues protrudes
from the N-domain, blocking one end of the cleft However, XEG lacks a corresponding loop because of deletion of those residues The loop structure is responsible for the exo-activity of OXG-RCBH, and the absence of the loop is associated with endo-activity
in XEG Therefore, it is most likely that the endo-activity of XEG is primarily attributable to the active site cleft being open at both ends In the case of Xgh74A, the active cleft is open Although a precise anal-ysis of the mode of action of Xgh74A has not been performed, Xgh74A appears to be an endoglucanase because it has an open cleft
Comparison of the XEG and OXG-RCBH active sites
Figure 3 shows a close-up view of the active sites of XEG and OXG-RCBH The main chain of XEG exhibits close similarity to that of OXG-RCBH, and the side chain conformations involved in substrate interactions are very well conserved, with some excep-tions (see below for details) Previously, we have iden-tified the catalytic residues Asp35 (base) and Asp465 (acid) in OXG-RCBH [10] Equivalent residues were identified in the active site of XEG (Asp34 and Asp458) Thus, it is conceivable that XEG and OXG-RCBH recognize the backbone of the b-1,4-glu-can in a similar manner, despite their differences in substrate specificity and mode of hydrolysis
Two notable differences, located at the )1 and +2 sites, were observed OXG-RCBH functions in exo mode owing to its unique loop structure around the +2 site at one end of the cleft (Figs 2B and 3, shown
in red) The Xyl side chain at the +2 site inhibits enzymatic activity due to steric hindrance between the side chain and the exo-loop Previously, we demon-strated that deletion of the loop region leads to loss of specificity, as the resultant enzyme could catalyze cleavage at various sites of the substrate by endo activ-ity [10] XEG does not have a corresponding loop; both sides of the cleft are open This result, and those
of the previous experiment with loop-deleted OXG-RCBH [10], strongly suggest that the basis for the endo activity of XEG is the absence of the exo loop The second difference was observed at the )1 site
In OXG-RCBH, Asn488 interacts with a Xyl side chain at the )1 site Previously, we have shown that recognition of the Xyl side chain at the)1 site is a key determinant for the substrate specificity of OXG-RCBH [6] However, the corresponding position in XEG is occupied by the bulky side chain of Tyr457, which appears to hinder binding of the Xyl side chain
at this position This may explain why XEG prefers
Fig 1 Stereoview of the rA-weighted 2m|Fobs| ) D|F calc | map of
XEG at 2.5 A ˚ resolution, contoured at 1.5r The map shows the
vicinity of the region corresponding to the exo loop, which is
pres-ent in RCBH and abspres-ent in XEG The XEG molecule is also shown
as a stick model The exo loop of RCBH is drawn as a trace of Ca
atoms in orange The image was produced using the program
CCP4MG [22].
Trang 4substrates with an unbranched Glc residue at the )1
site and catalyzes cleavage of the glycosidic bond of
unbranched Glc residues
Activity of the XEG mutant Y457G
To confirm the role of Tyr457, we constructed a XEG
variant, Y457G, which was expressed in Escherichia
coli, purified, and characterized The kinetic constants
of Y457G against tamarind seed xyloglucan were determined The apparent Km values of the wild-type and Y457G enzymes were 0.47 and 0.43 mgÆmL)1, respectively, and their specific activities were 15.7 and 3.97 unitÆmg)1protein, respectively Next, the substrate specificity was investigated using a tetradecasaccharide, XXXGXXXG Wild-type XEG generated only XXXG (Fig 4), because of its strict specificity for glycosidic bonds of the unbranched Glc residue By contrast, the
Fig 2 Schematic drawing of the entire
structures (traces of Ca atoms) of XEG (A),
the OXG-RCBH ⁄ substrate (XXXG) complex
(B) and the Xgh74A ⁄ substrate (XLLG and
XXLG) complex (C) The substrates are
shown as stick models The exo loop of
OXG-RCBH (Gly375–His385) is shown
in red.
Trang 5Y457G mutant released various oligosaccharides from
cleavage of XXXGXXXG The structures of these
products were X, XG, GX, XX, XXG, XXX and
XXXG, as confirmed by MALDI-TOF MS combined
with enzymatic treatments of the reaction products
using isoprimeverose-producing oligoxyloglucan
hydro-lase from Oerskovia [14] and b-glucosidase from
almond, Prunus dulcis, as described previously [8]
(data not shown) On the basis of the product patterns,
we propose the possible cleavage sites shown in Fig 4
Therefore, the Y457G variant can catalyze cleavage of
the glycosidic bond of branched Glc residues, i.e the
Glc residue at the )1 site can be branched The
Y457G mutant enzyme was less selective, indicating
that Y457 plays an important role in determining sub-strate specificity
Conclusion
Geotrichum M128 produces two GH74 enzymes, XEG and OXG-RCBH They share 48% amino acid identity and three-dimensional structures The majority of the residues interacting with the substrate are well-con-served However, the residues that determine substrate specificity and mode of action were distinct XEG and OXG-RCBH display endo and exo modes of action, respectively A loop structure that determines the exo action of OXG-RCBH is not present in XEG
Fig 3 Comparison of the active site from )2 to +2 for XEG and the OXG-RCBH ⁄ XXXG complex The stereo views of XEG and OXG-RCBH are shown in blue and purple, respectively The substrate XXXG and the amino acids that interact with the substrate are shown as stick models The substrate is colored blue, light green, green and orange at the )2, )1, +1 and +2 sites, respectively The exo loop of OXG-RCBH is colored in red.
Fig 4 HPLC analysis of the digestion prod-ucts of the xyloglucan oligosaccharide XXXGXXXG by wild-type XEG and the Y457G mutant The xyloglucan oligosaccha-ride (XXXGXXXG) was incubated with wild-type XEG or the Y457G mutant, and the products were analyzed by HPLC The pro-posed cleavage sites are indicated by arrows (A) Oligosaccharide marker (B) Products from cleavage by wild-type XEG (C) Products from cleavage by the Y457G mutant.
Trang 6A further difference in the active site residues
involves recognition of a Xyl side chain at the
)1 site OXG-RCBH recognizes a Xyl side chain at
the )1 site (Asn488), whereas XEG does not In
XEG, residue Tyr457, which corresponds to Gly464
of OXG-RCBH, appears to protrude into the)1 site,
providing a basis for the substrate specificity of XEG
In fact, a single amino acid substitution of Tyr457 by
Gly caused a significant change in substrate
recogni-tion by XEG These results suggest that the substrate
specificities of XEG and OXG-RCBH depend on a
limited number of residues in the substrate binding
cleft
Experimental procedures
Purification of XEG
The gene encoding the mature region of XEG [7] was
subcl-oned into pET14-b (Novagen, Madison, WI, USA) using
NdeI and BamHI sites, resulting in fusion of a His6tag to
the N-terminus The resultant product was transformed
into E coli BL21-CodonPlus (DE3) RP (Stratagene, La
Jolla, CA, USA) Expression was induced by addition of
isopropyl-b-d-thio-galactopyranoside (final concentration
0.1 mm) for 16 h at 20C The soluble, intracellular
recom-binant protein was extracted using BugBuster protein
extraction reagent (Novagen), and was purified using a
HiTrap chelating column (GE Healthcare, Little
Chal-font, UK) Solid ammonium sulfate was added to the active
fractions from the column to a final concentration of
0.75 m, and the fractions were loaded onto a Resource
PHE hydrophobic interaction chromatography column
(Amersham Biosciences) equilibrated with 25 mm
imidaz-ole⁄ HCl buffer (pH 7.4) containing 0.75 m (NH4)2SO4
Bound proteins were eluted using a linear gradient of
0.75–0 m (NH4)2SO4 in 25 mm imidazole⁄ HCl buffer
(pH 7.4) Finally, XEG was resolved on a HiLoad 16⁄ 60
Superdex 200 pg column (Amersham Biosciences) in 25 mm
imidazole⁄ HCl buffer (pH 7.4) Before crystallization,
purified XEG was concentrated to 8 mgÆmL)1 using an
Ultrafree-15 centrifugal filter device (Millipore, Bedford,
MA, USA)
Crystal structure determination of XEG
Crystallization of XEG was performed at 20C using the
hanging-drop vapor-diffusion method [15] The
crystalliza-tion condicrystalliza-tions were initially screened using the screens
Index and PEG⁄ Ion (both Hampton Research, Aliso Viejo,
CA), and Wizard I, Cryo I and Cryo II (all DeCODE
Genetics, Reykjavik, Iceland), and were refined by varying
the pH of the buffer and the concentration of the
precipi-tant Prior to data collection, a crystal was transferred into
Paratone-N (Hampton Research) and mounted on a CryoLoop (Hampton Research) of 20 lm diameter The mounted crystal was immersed in liquid nitrogen Diffrac-tion data were collected at 100 K on Beamline BL44B2 at SPring-8 (Harima Institute, Hyogo, Japan) using an ADSC Quantum 210 CCD detector (Area Detector Systems Corporation, Poway, CA, USA) with 1.0000 A˚ radiation, and were processed using HKL2000 [16] and the ccp4 pro-gram suite [17] The structure of XEG was solved by means of the molecular replacement method, using the AMORE program [18] in the ccp4package The coor-dinates of OXG-RCBH (PDB code 1sqj) were used as
a search model The cns program [19] was used for refinement against the 20–2.5 A˚ intensity data A ran-domly selected portion of the diffraction data (5.0%) was used to calculate the free R factor [20] The pro-gram coot [21] was used to display and correct the structure Figures were created using pymol (DeLano Scientific LLC, Palo Alto, CA; http://www pymol.org) The coordinates were deposited in the Pro-tein Data Bank (3A0F)
Construction of the Y457G mutant
The Y457G mutant was constructed using the QuikChange procedure (Stratagene) with primers 5¢-TCTTCAGCGGC ATGGGCGACCTCGGCGGCAT-3¢ and 5¢-ATGCCGC CGAGGTCGCCCATGCCGCTGAAGA-3¢ The recombi-nant protein was purified using a HiTrap chelating column and Resource PHE column (both Amersham Biosciences)
Analysis of kinetic parameters
The kinetic parameters were determined at various concen-trations of the substrate, tamarind seed xyloglucan (Dainip-pon Sumitomo Pharma, Osaka, Japan), in 50 mm sodium acetate buffer (pH 5.5) at 45C for 15 min The bicinchoni-nate assay was used to quantify reducing sugars The Michaelis constant (Km) and specific activity were calcu-lated from a plot of initial reaction rates versus substrate concentration using Prism (GraphPad Software, San Diego,
CA, USA) One unit was defined as the amount of enzyme that released 1 lmol of glucose equivalent as reducing sug-ars from xyloglucan per minute
Analysis of substrate specificity
Substrate specificity was analyzed using xyloglucan tetra-decasaccharide, XXXGXXXG, prepared as described previ-ously [8] The oligosaccharide (0.2 mg) was incubated with each enzyme in 20 lL of 50 mm sodium acetate buffer (pH 5.5) at 45C for 16 h The resulting products were analyzed by HPLC using an Amide-80 normal-phase
Trang 7column (4.6 mm I.D.· 250 mm; TOSOH, Tokyo, Japan)
using 60% acetonitrile (isocratic) at a flow rate of
0.8 mLÆmin)1
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