The protein CheZ, which has the last unknown structure in the
Escherichia coli chemotaxis pathway, stimulates the dephosphorylation of
the response regulator CheY by an unknown mechanism. Here we report the
co-crystal structure of CheZ with CheY, Mg2+ and the phosphoryl
analog, BeF3-. The predominant structural
feature of the CheZ dimer is a long four-helix bundle composed of two helices
from each monomer. The side chain of Gln 147 of CheZ inserts into the CheY
active site and is essential to the dephosphorylation activity of CheZ. Gln 147
may orient a water molecule for nucleophilic attack, similar to the role of the
conserved Gln residue in the RAS family of GTPases. Similarities between the
CheY−CheZ and Spo0F−Spo0B structures suggest a general mode of
interaction for modulation of response regulator phosphorylation chemistry.
Over the past 25 years, the molecular events governing chemotaxis in
Escherichia coli have emerged in detail1,
2. As a
consequence, bacterial chemotaxis is one of the most thoroughly characterized
biological information processing networks, making it an excellent model for
computer simulations in the field of systems biology3,
4.
Bacterial chemotaxis has also served as a prototype for two-component
regulatory systems, a signaling transduction strategy widely used in
prokaryotes, plants and fungi5 for response to a broad range of
extracellular stimuli. In chemotaxis, chemical attractants or repellants bound
to transmembrane receptors regulate the autophosphorylation of the sensor
kinase CheA. The phosphoryl group is transferred from a histidyl residue on
CheA to an aspartyl residue (Asp 57) on the response regulator CheY.
Interaction of phospho-CheY (CheY-P) with the flagellar motor dictates cellular
swimming behavior. Efficient removal of the phosphoryl group from CheY-P is
essential for the continuous response to environmental changes. Although CheY
can catalyze its own dephosphorylation, the protein CheZ in enteric bacteria
such as E. coli stimulates this rate and is critical for the rapid
response of bacteria to stimuli6, which is essential for
chemotaxis.
An invaluable component of our current level of understanding of
chemotaxis is the atomic structures of six of the seven proteins in the
pathway. Genetic and biochemical studies have generated considerable, but
fragmented, data about the seventh protein, CheZ, which could be put in context
by the determination of its structure. Protease sensitivity suggests that CheZ
has at least two structural domains7,
8. Disabling single-site
substitutions cluster in six regions of the CheZ primary structure8,
9. Except for the C-terminus of CheZ, which is involved in binding
to the 455 surface of
CheY-P10,
11, the functional roles and spatial relationships of
the CheZ regions identified by mutagenesis are unknown. Genetic studies suggest
that 112 and the
55 loop13 of CheY bind to as
yet unidentified portions of CheZ. The mechanism by which CheZ catalyzes the
dephosphorylation of CheY is also not known. CheY autodephosphorylation is
believed to proceed through a direct reversal of the phosphorylation
reaction14, which involves an in-line substitution mechanism
mediated by an active site Mg2+ (ref. 15). Whether CheZ acts as an allosteric effector of CheY
autodephosphorylation activity or as a conventional phosphatase is not known.
Possible regulation of CheZ activity by CheY-P16 or a truncated
form of CheA17 have been reported but their molecular mechanisms
have not been elucidated.
The lack of detailed structural or mechanistic information concerning
CheZ has made our understanding of chemotaxis and, hence, its use as a model
system, incomplete. Our goal was to determine the three dimensional structure
of CheZ with the hope that the structure would provide insight into its
catalytic mechanism. The recent discovery that
BeF3- acts as a stable functional18 and structural analog19 of the phosphoryl group in
CheY-P has allowed us to determine the co-crystal structure of CheZ and
CheY−BeF3- −Mg2+.
The structure completes the set of atomic structures for the seven chemotaxis
proteins and reveals the fundamental mechanism of CheZ-stimulated
dephosphorylation of CheY.
Overall structure The co-crystal structure of CheZ and
CheY−BeF3-−Mg2+ was
obtained at 2.9 Å resolution (Fig.
1a,b). The ribbon representation (Fig.
1a) shows that, consistent with solution studies20,
CheZ is a dimer (CheZ2) composed of two monomers (Z1 and Z2), which
are related by a two-fold rotational axis. The predominant structural feature
of CheZ2 is a long (105 Å) four-helix bundle
('CheZcore'). Residues 35−168 from each CheZ monomer form two
amphipathic helices with a single hairpin turn (residues 100−104) and
assemble in a 'head-to-head' orientation to form the bundle. Many inter- and
intrachain interactions within the bundle, including 33 pairs of residues
involved in hydrophobic interactions and 26 hydrogen bonds, suggest a highly
stable dimer interface. An additional helix (residues 5−34) extends from
the four-helix bundle at an angle of 100° and contains a majority of
the interdimer crystal contacts. A 13-residue helix ('CheZc'),
believed to correspond to the extreme C-terminus of CheZ (residues
201−213; see below), is in proximity to the
455 region of CheY but is
unattached to the rest of the visible CheZ. In all, coordinates for 177 out of
the total 214 residues of CheZ were determined. The remaining residues
(1−4 and 169−200) are disordered in the crystal.
Figure 1. Overall structure of
(CheY−BeF3-−Mg2+)2CheZ2.
a, Ribbon diagram showing the topology of the
CheY−CheZ structure. The CheZ2 chains are cyan and orange and
the CheY molecules are gray. BeF3- (green) and
Mg2+ (red) are in space-filling representation. The assignment
of CheZc with the nonproximal CheZ5−168 chain was
arbitrary. b, Stereo representation (BobScript38) of the
electron density (2Fo− Fc map contoured at 1
level) near the hairpin turn of the CheZ monomer. c, Positions
of CheZ loss-of-function (red) and gain-of-function (blue) mutants8 are mapped onto a GRASP39 surface representation of
CheZ2. Only one CheY molecule (magenta) is shown for clarity.
d, Co-crystal structure of the phosphotransfer domain of the histidine
phosphotransferase Spo0B and response regulator Spo0F29 (PDB
entry 1F51). The two chains of the Spo0B
dimer are cyan and orange. The Mg2+ ions are in red. (a)
and (d) were created using RIBBONS40.
Intermolecular interactions between CheY and CheZ Each CheY binds to CheZ through two distinct interaction surfaces (Fig. 1a). The interface between CheY and
CheZcore has a buried surface of 1,215 Å2, which
is comparable to the surface buried between a typical antibody and antigen21. This interface involves residues from 1, the
55 and 44
loops, and the active site surface of CheY and residues between 136−151
of Z1 and 67−71 of Z2 of CheZ (Fig. 2a;
Table 1), about halfway down the four-helix bundle.
Consistent with this binding interface, replacement of Asn 23 in
1 of CheY with Asp diminishes CheZ binding by
30−50-fold12,
22. Likewise, the
55 loop, containing Lys 109 of CheY, which
interacts with CheZ (Fig. 2a; Table 1), has been implicated by mutagenesis studies to be
involved in the CheZ interaction13.
Figure 2. Interactions between CheY and CheZcore and potential
catalytic mechanism.
a, Residues involved in
CheY−CheZcore interactions. Side chains from CheY (gray), Z1
(cyan) and Z2 (orange) that are involved in interactions are shown in
ball-and-stick representation. BeF3- is green
and Mg2+ is light purple. Labels for CheY residues are black and
labels for CheZ residues are cyan (Z1) or orange (Z2). See Table 1 for details of interactions. b, Close-up view
of the CheY active site in the co-crystal. A modeled water molecule (2.0
Å from the Be atom) is in the correct geometry for in-line attack (narrow
black stippled line) and within hydrogen bonding distance of the amide nitrogen
of Gln 147 of CheZ (2.8 Å; thick black stippled line). Labels for CheY
residues are black and CheZ residues are cyan. Coordination of the active site
Mg2+(magenta) is displayed as magenta stippled lines. (a)
and (b) were created using RIBBONS40. c,
Biochemical data showing that CheZ Q147A is inactive and can still bind to
CheY-P. Upper panel: the effect of CheZ Q147A (circles) and wild type CheZ
(inset, squares) on the rate of Pi release from mixtures containing
CheY and phosphoimidazole. Note the difference in scales of the abcissa. Rates
were determined using the Enzchek Pi Assay (Molecular Probes)22. Lower panel: binding of wild type CheZ (squares), CheZ Q147A
(circles) or CheZ1−181 (triangles) to CheY, assessed by their
ability to compete with fluoresceinated wild type CheZ for CheY N59R-P.
The second interface between CheY and CheZ involves interaction of the
455 face of CheY with
CheZc (Fig. 1a). The electron density
for CheZc is poor (see Methods), but the
following evidence supports the identity of CheZc as the C-terminus
of CheZ. The tryptic fragment CheZ196−214 binds to CheY in a
phosphorylation-dependent manner11, and CheY residues perturbed
by CheZ196−214binding in NMR experiments10
correlate well with the CheY-binding surface for CheZc. Furthermore,
CheZ196−214 shares significant sequence identity with
FliM1−16, a peptide that corresponds to the N-terminus of the
flagellar switch protein FliM; the two peptides bind to CheY-P with nearly
identical affinities10 and FliM1−16 binds at a
similar location on activated CheY as CheZc23. The
assignment of the 169−200 region of CheZ as a disordered linker is
consistent with secondary structure predictions using several programs (data
not shown), proteolysis data7,
8,
11 and amino acid alignments of
CheZ sequences from different species8. With two large
independent areas of interactions, the hinged CheZ molecule 'clamps down' on
the globular CheY molecule, consistent with the tight binding constant
(50−250 nM) between CheY-P and CheZ22.
Locations of loss- and gain-of-function mutants Mapping the positions of loss-of-function CheZ mutants, which fall
predominantly in six clusters on the primary amino acid sequence of CheZ8, onto the three-dimensional structure of CheZ allows recognition of
functionally important regions (Fig. 1c). The
largest two clusters of mutants correlate with the two regions of
CheZcore that interact with CheY and a smaller mutant cluster
correlates with the CheZc domain. Therefore, CheY interactions with
both CheZcore and CheZc are critical for CheZ function.
Two additional mutant clusters are located on either side of the hairpin region
such that residues from one cluster interact with residues from the other
within a monomer. This region may, therefore, be important in maintaining the
conformational integrity of the helical hairpin. Finally, a small cluster of
mutants maps to the linker region of CheZ (data not shown), implying that
although disordered in the crystal, the linker has functional significance.
CheZ dephosphorylation mechanism Whether CheZ acts as an allosteric activator of the intrinsic
autodephosphorylation activity of CheY or as a true phosphatase in its own
right has long been debated. A striking feature of
CheZcore−CheY interactions (Fig.
2a,b; Table 1) is the presence of
CheZ residues at the active site of CheY, suggesting that residue(s) from CheZ
may participate directly in the catalysis of CheY dephosphorylation. Most
prominent is the side chain of Gln 147 of CheZ, which inserts directly into the
CheY active site, filling one coordination site of the Mg2+ and
making additional van der Waals interactions with Phe 14 of CheY and
BeF3-. One helical turn away, Asp 143 of CheZ
forms a salt bridge with CheY active site residue Lys 109. Gln 147 from CheZ is
conserved among all eight known CheZ sequences, and its central position in the
active site strongly suggests that it could have a catalytic role. Cells
containing a CheZ Q147A mutant display exclusively clockwise flagellar
rotation, consistent with severely diminished CheZ function. Although the Q147A
mutant of CheZ still binds CheY-P with moderate affinity (Fig.
2c) and is capable of forming co-crystals with CheY (data not
shown), it displays no detectable (<0.02% wild type) phosphatase activity
(Fig. 2c). These data support an essential
catalytic role for Gln 147 of CheZ. A water molecule could be modeled into the
active site (Fig. 2b), satisfying the geometry
required for in-line attack of the beryllium atom (analog of the phosphoryl
phosphorous) and concomitant hydrogen bond formation with the amide nitrogen of
Gln 147, suggesting a possible role for CheZ Gln 147 in orienting the water for
nucleophilic attack.
The current model of CheY autodephosphorylation involves in-line attack
of water on the phosphoryl phosphorous, generating a pentavalent trigonal
bipyramidal transition state that collapses into products14,
24.
The Mg2+, positioned in the active site via interactions
with Asp 13, Asp 57, Asn 59 and the phosphoryl group19, is
required for catalysis14, perhaps for transition state
stabilization25. There may also be other active site functional
groups that contribute to catalysis which have not yet been identified. The
overall architecture of the CheY active site (including the positions of Asp
12, Asp 13, Asp 57, Asn 59, Lys 109, BeF3- and
divalent cation) are similar in
CheY−BeF3-−Mg2+
bound to CheZ and free
CheY−BeF3-−Mn2+
(ref. 19), suggesting that the catalytic
determinants for CheY autodephosphorylation are still in place in the CheZ
mechanism. Therefore, CheZ, with the insertion of Gln 147 into the active site,
seems to use the existing mechanism of CheY autodephosphorylation and render it
more efficient by positioning the attacking water molecule in the appropriate
geometry for in-line attack. The proposed role does not fall neatly into either
the allosteric effector or phosphatase category but, rather, contains features
of both.
Similarities to Ras GTPases The proposed mechanism for CheZ-mediated dephosphorylation shares
several similarities with the GTPase mechanism in the Ras/G
families. First, these GTPases also contain an active site Gln residue, which
has been proposed to orient a nucleophilic water molecule for attack of
GTP26, although in Ras it is the amide carbonyl oxygen rather
than the amide nitrogen that directly interacts with the water. The codon for
the conserved Gln residue in p21ras is a common site of
mutation, which results in cellular transformation27. Second, the
general mechanism of CheZ activity — stimulation of an existent
hydrolysis mechanism by the insertion of a catalytic side chain — is
directly analagous to the mechanism of the GAPs (GTPase activating proteins),
which insert an Arg residue into the Ras active site, assisting the existing
GTPase mechanism through transition state stabilization28.
Possible generality of CheY−CheZcore
interactions Although CheZ does not show amino acid sequence similarity to any other
proteins, the interactions between CheY and CheZcore are strikingly
similar to the interactions between the Bacillus subtilis histidine
phosphotransferase Spo0B and response regulator Spo0F29 (Fig. 1d). Like CheZ, Spo0B contains a four-helix bundle
composed of two helical hairpins assembled in a head-to-head orientation.
Although the helical bundle is much shorter in Spo0B than CheZ, the relative
positions of the bundle and the response regulator are similar in the two
complexes, as is the surface of the response regulator, which interacts with
the bundle. As with CheZ and CheY, two residues from Spo0B insert into the
active site of Spo0F. His 30 from Spo0B, the site of phosphorylation, inserts
into the Spo0F active site (resembling Gln147 of CheZ) and Asn 34 from Spo0B
forms a hydrogen bond with Lys 104 of Spo0F (resembling the CheZ
Asp143−CheY Lys 109 interaction). A helical bundle structure is probably
conserved among the phosphohistidine-containing domains in phosphotransferases
and kinases of two-component systems24, although the overall
topology can vary30. Thus, the nature of the interactions
observed in the CheY−CheZ and Spo0F−Spo0B structures may represent
a general mode of interaction for modulation of phosphotransfer reactions of
response regulators, including HisAsp or Aspwater phosphotransfer.
Secondary structure prediction suggests that each of the nine Rap proteins,
which have phosphatase activity towards response regulators involved in B.
subtilis sporulation31, are all-helical proteins (data not
shown). The Rap proteins may form helical bundles that interact with response
regulators in a similar manner.
Roles of C-terminal and N-terminal helices of CheZ Whereas the core domain of CheZ provides a catalytic residue for the
dephosphorylation reaction, CheZc is likely to provide binding and
selectivity for activated CheY. CheZ196−214 binds to CheY-P
and CheY with Kd values of 26 and 440 M, respectively10. The structural basis for this selectivity is probably the same as
that for FliM1−16, whereby CheY activation induces rotation of
Thr 87 and Tyr 106, removing Tyr 106 from the surface to an internal
hydrophobic pocket and exposing the binding site23. In the
CheY−CheZ structure, Tyr 106 from CheY is in the internal conformation,
and the external conformation is sterically occluded by CheZc. In
light of the structural information, it is surprising that
CheZ1−181 (Fig. 2c) and
CheZ1−201 (ref. 11) cannot bind
to CheY-P, and CheZ1−181 has no detectable phosphatase
activity (R.E.S., unpub. results). Thus, CheZc is critical for the
binding and activity of CheZ, possibly through one of the following mechanisms.
Binding of CheZc may induce a conformational change on CheY-P,
enhancing binding to CheZcore. Alternatively, binding of
CheZc might bring CheZcore and CheY-P to a high local
concentration, which is critical for CheZ binding and activity. Attempts to
generate phosphatase activity in vitro by simultaneous addition of
CheZ1−181 and CheZ196−214 were unsuccessful
(data not shown), arguing against the first model.
The role of the N-terminal helix in CheZ function remains to be
determined. It is striking, however, that the 20 known gain-of-function
missense CheZ mutants contain amino acid substitutions that are concentrated on
one face of this helix, as well as on regions in the 50s and 160s of
CheZcore9 (Fig. 1c). This
suggests that, in the absence of CheY-P, the N-terminal helix may fold against
CheZcore, blocking access to the Gln 147 region. Gain-of-function
substitutions may destabilize this interaction, thereby enhancing activity.
This model would account for the apparent weak affinity of
CheZ1−201 for CheY-P11. With the N-terminal
helix precluding access to CheZ, a high local concentration of
CheZcore, such as brought about by the CheZc tether, may
be required to overcome the occlusion of the Gln 147 region. Manipulation of
the conformation of the N-terminal helix — for example, by other
chemotaxis proteins17 — could provide a means to regulate
CheZ activity in the cell.
Methods Protein purification, mutagenesis and activity
assays. CheY and CheZ were purified using published protocols22,
32. CheZ E134K, which confers a wild type Che+
phenotype and displays 80% of wild type CheZ activity in vitro8, was used for crystallization because wild type CheZ did not
crystallize. Selenomethionine (SeMet)-CheY was purified from cultures
containing pRBB40 (ref. 33) transformed into the
methionine auxotroph E. coli strain B834(DE3) (Novagen) and grown in the
presence of SeMet (Sigma). The mutant cheZ Q147A was made by PCR
mutagenesis. CheZ phosphatase activity was determined by spectroscopic
measurement of the rate of Pi release in the presence of CheY and
phosphoimidazole22. The magnitude of catalytic activity of CheZ
Q147A compared to wild type CheZ was estimated by dividing the concentration of
wild type CheZ required to obtain a minimal significant increase in
Pi release rate by the maximum concentration of CheZ Q147A used.
Binding of Che Z Q147A to CheY was assessed by measuring its ability to compete
with fluoresceinated wild type CheZ using fluorescence anisotropy22. Fluoresceinated CheZ (0.20M) was mixed with varying amounts of
nonfluoresceinated CheZ (wild type, CheZ Q147A or CheZ1−181)
in the presence of 17 mM acetyl phosphate. CheY N59R (0.20 M) was added,
and the change in anisotropy was determined. CheY N59R binds nearly
quantitatively to CheZ under these conditions22.
Crystallization and data collection. Both the unsubstituted and SeMet-containing crystals were grown at 4
°C using the hanging drop vapor diffusion method. Drops contained 1 l
of the protein complex (264 M CheY, 3.6 mM BeCl2, 10 mM NaF, 10
mM MgCl2 and 264 M CheZ E134K, assembled in the stated order)
and 1 l of well solution (0.1 M bicine, pH 8.5, 0.2 M ammonium acetate and
30% (v/v) isopropanol). Crystals grew for 2−3 weeks before reaching
maximal dimensions (0.5 0.1 0.1 mm). Only the combination of
SeMet-substituted CheY and unsubstituted CheZ produced useful SeMet-containing
crystals. Crystals were transferred into 2 l of stabilization solution (0.1
M bicine, 30% (v/v) isopropanol, 1.8 mM BeCl2, 5 mM NaF and 5 mM
MgCl2), followed by the sequential addition of 2, 4 and 8 l of
cryoprotectant solution (stabilization solution plus 50% (w/v) sucrose) at 5
min intervals. The crystals were finally transferred to pure cryoprotectant
solution and flash frozen in liquid nitrogen. Tb3+-derivitized
crystals were prepared by growing the crystals using 10 mM TbCl3 in
place of the MgCl2. Native and MAD data were collected at beam lines
X4A and X25, respectively, of the National Synchrotron Light Source (NSLS) at
the Brookhaven National Laboratory. All data were processed using DENZO and
SCALEPACK34. Data statistics are shown in Table
2.
Structure determination and refinement. Initial phases were determined using SOLVE35 with the
combination of the SeMet MAD data and the TbCl3 MAD data. The SOLVE
results clearly show that P43212 is the correct space
group instead of P41212. One Tb3+ site and
five Se sites were found by SOLVE (Table 2). The
Tb3+ ion, surprisingly, binds to CheZ instead of at the expected
Mg2+-binding site in CheY. The five Se sites were further
refined in CNS36, and the SeMet MAD data alone were used to
generate the initial map (addition of the Tb3+ data did not
improve the phases), which was further improved by solvent flattening with CNS.
This experimental phase map revealed a clear four-helical bundle structure for
the CheZ dimer but had poor density for CheY. Activated CheY (PDB entry
1FQW)19 was fit in the
experimental phase map based on the five Se sites found in SOLVE (r.m.s.
deviation between the found Se sites and the actual S atoms of Met was
1.1 Å). A poly-Ala model for CheZ was built into the map and went through
one round of positional refinement. Side chains of CheZ were constructed based
on the CheZ Trp 94 and Trp 97 reference points, which are clear in the
2Fo− Fc and Fo− Fc maps
generated with the refined poly-Ala model. Further refinement against a native
data set (20−2.9 Å) was carried out in CNS interspersed with rounds
of manual re-building in O37 based on the 2Fo−
Fc and Fo− Fc maps. The final
Rfree is 29.8% (Table 2). Fig. 1b shows the quality of the CheZ electron density
after refinement. The side chains of the following CheZ residues are not
visible: 5, 8−10, 26, 33, 39, 46, 68, 77, 81, 108, 150, 151, 158, 168,
201−213. The CheY density after refinement was still poor. Side chains at
the CheY−CheZ interface were well defined, but many of the side chains in
the other regions of CheY were not clear. The side chains of the following
residues were visible for CheY: 4−6, 8, 12, 13, 14, 16−25, 27, 30,
35, 37, 44, 46, 51, 53, 57, 59−61, 78, 81, 106, 108−112, 116 and
119−121. Side chain conformations from activated CheY (PDB entry
1FQW) were used when there was no clear
density for CheY in the current structure. Main chain densities for the five
helices of CheY were clear, but densities for the five -strands were not
well separated. The poor density of CheY probably reflects CheY flexibility in
the crystal because it is not involved in any crystal contacts and interacts
only with CheZ. As a consequence, we do not discuss the structural differences
between CheZ-bound CheY and free CheY. Similarly, the side chain densities for
the CheZc peptide were visible but poor. There is strong evidence
that this peptide corresponds to the extreme C-terminus of CheZ (see main
text). This peptide was tentatively modeled as CheZ 201−213, which
lowered the Rfree by almost 1%. However, we cannot preclude the
possibility that this sequence could be misassigned by one or two residues at
either end of the peptide.
Coordinates. The structural coordinates have been deposited in the Protein Data
Bank (accession code 1KM1).
Acknowledgments We thank J. Snyder and M. Kimple (UNC Chapel Hill) for help
collecting the native data set; D. Wemmer and S.-Y. Lee (UC Berkeley) for
sharing the BeF3- parameter and topology files
and the coordinates for the
CheY−BeF3--FliM structure before they
were publicly accessible; G. Zhang (National Jewish Medical and Research
Center) and H. Ke, D. Worthylake and M. Redinbo (UNC Chapel Hill) for helpful
discussion; L. Betts (UNC X-ray facility) for technical support; X. Chen, M.
Churchill and the X-ray Core Facility at the University of Colorado Health
Science Center (UCHSC) for their support for the completion of R.Z.'s project
at UCHSC; and the Brookhaven beamline staff for help with data collection.
Competing interests statement:
The authors declare that they have no competing financial interests.
4
5
105 Å) four-helix bundle
('CheZcore'). Residues 35−168 from each CheZ monomer form two
amphipathic helices with a single hairpin turn (residues 100−104) and
assemble in a 'head-to-head' orientation to form the bundle. Many inter- and
intrachain interactions within the bundle, including 33 pairs of residues
involved in hydrophobic interactions and 26 hydrogen bonds, suggest a highly
stable dimer interface. An additional helix (residues 5−34) extends from
the four-helix bundle at an angle of 
level) near the hairpin turn of the CheZ monomer. c, Positions
of CheZ loss-of-function (red) and gain-of-function (blue) mutants
families. First, these GTPases also contain an active site Gln residue, which
has been proposed to orient a nucleophilic water molecule for attack of
GTP
Asp or Asp
water phosphotransfer.
Secondary structure prediction suggests that each of the nine Rap proteins,
which have phosphatase activity towards response regulators involved in B.
subtilis sporulation
M, respectively
0.1 
atoms of Met was
1.1 Å). A poly-Ala model for CheZ was built into the map and went through
one round of positional refinement. Side chains of CheZ were constructed based
on the CheZ Trp 94 and Trp 97 reference points, which are clear in the
2Fo− Fc and Fo− Fc maps
generated with the refined poly-Ala model. Further refinement against a native
data set (20−2.9 Å) was carried out in CNS interspersed with rounds
of manual re-building in O
