Electronic reprint (7.3 MBytes) : Directly from J. Chem. Theory Comput., 5, 3299-3311, Copyright © American Chemical Society.
BcZBP is an LmbE-like, homohexameric, zinc-dependent deacetylase from the
opportunistic pathogen Bacillus cereus with three, thus far uncharacterized,
homologues in B. anthracis. Although its specific substrate is still
unknown, the enzyme has been shown to preferentially deacetylate
N-acetylglucosamine and diacetylchitobiose via an active site based on a
zinc-binding motif of the type HXDDXnH. In the crystal structure, the active
site is located at a deep and partially blocked cleft formed at the
interface between monomers related by the molecular 3-fold axis, although
the major, in structural terms, building block of the enzyme is not the
trimer, but the intertwined dimer. Here, we report results from a 50 ns
molecular dynamics simulation of BcZBP in explicit solvent with full
electrostatics and show that (i) the view of the intertwined dimer as the
major structural and functional building block of this class of hexameric
enzymes is possibly an oversimplification of the rather complex dynamics
observed in the simulation, (ii) the most mobile (with respect to their
atomic fluctuations) parts of the structure coincide with three surface
loops surrounding the active site, and (iii) these mobile loops define the
active site's accessibility, and may be implicated in the determination of
the enzyme's specificity.
Electronic reprint (3.7 MBytes) : Local copy, or directly from J. Struct. Biol., 166, 214-225, Copyright © Elsevier B.V.
The HrcQB protein from the plant pathogen Pseudomonas syringae is a core
component of the bacterial type III secretion apparatus. The core consists
of nine proteins widely conserved among animal and plant pathogens which
also share sequence and structural similarities with proteins from the
bacterial flagellum. Previous studies of the carboxy-terminal domain of
HrcQB (HrcQB-C) and its flagellar homologue, FliN-C, have revealed
extensive sequence and structural homologies, similar subcellular
localization, and participation in analogous protein–protein interaction
networks. It is not clear however whether the similarities between the two
proteins extend to the level of quaternary association which is essential
for the formation of higher-order structures within the TTSS. Even though
the crystal structure of the FliN is a dimer, more detailed studies support
a tetrameric donut-like association. However, both models, dimer and
donut-like tetramer, are quite different from the crystallographic elongated
dimer of dimers of the HrcQB-C. To resolve this discrepancy we performed a
multidisciplinary investigation of the quaternary association of the
HrcQB-C, including mass-spectrometry, electrophoresis in non-reductive
conditions, gel filtration, glutaraldehyde cross-linking and small angle
X-ray scattering. Our experiments indicate that stable tetramers of
elongated shape are assembled in solution, in agreement with the results of
crystallographic studies. Circular dichroism data are consistent with a
dimer-dimer interface analogous to the one established in the crystal
structure. Finally, molecular dynamics simulations reveal the relative
orientation of the dimers forming the tetramers and the possible differences
from that of the crystal structure.
Electronic reprint (605 KBytes) : Local copy, or directly from Anal. Biochem., 373, 404-406, Copyright © Elsevier B.V.
Many biochemical and biophysical methods can be used to
characterize the oligomerization state of proteins. One of the most widely
used is glutaraldehyde crosslinking, mainly because of the minimum equipment
and reagents required. However, the crosslinking procedures currently in use
are impaired by the low specificity of the reagent, which can chemically
bond any two amino groups that are close in space. Thus, extensive and
time-consuming investigation of the reaction conditions is usually required.
Here we describe two approaches based on glutaraldehyde that readily give
reliable results.
Electronic reprint (574 KBytes) : Local copy, or directly from BioSystems, 92, 61-68, Copyright © Elsevier B.V.
Change of DNA sequence that fuels evolution is, to a certain
extent, a deterministic process because mutagenesis does not occur in an
absolutely random manner. So far, it has not been possible to decipher the
rules that govern DNA sequence evolution due to the extreme complexity of
the entire process. In our attempt to approach this issue we focus solely on
the mechanisms of mutagenesis and deliberately disregard the role of natural
selection. Hence, in this analysis, evolution refers to the accumulation of
genetic alterations that originate from mutations and are transmitted
through generations without being subjected to natural selection. We have
developed a software tool that allows modelling of a DNA sequence as a
one-dimensional cellular automaton (CA) with four states per cell which
correspond to the four DNA bases, i.e. A, C, T and G. The four states are
represented by numbers of the quaternary number system. Moreover, we have
developed genetic algorithms (GAs) in order to determine the rules of CA
evolution that simulate the DNA evolution process. Linear evolution rules
were considered and square matrices were used to represent them. If DNA
sequences of different evolution steps are available, our approach allows
the determination of the underlying evolution rule(s). Conversely, once the
evolution rules are deciphered, our tool may reconstruct the DNA sequence in
any previous evolution step for which the exact sequence information was
unknown. The developed tool may be used to test various parameters that
could influence evolution. We describe a paradigm relying on the assumption
that mutagenesis is governed by a near-neighbour-dependent mechanism. Based
on the satisfactory performance of our system in the deliberately
simplified example, we propose that our approach could offer a starting
point for future attempts to understand the mechanisms that govern
evolution. The developed software is open-source and has a user-friendly
graphical input interface.
Electronic reprint (1.2 MBytes) : Local copy, or directly from FEBS J., 274, 3044-3054, Copyright © Blackwell Publishing, Inc.
Bacillus cereus is an opportunistic pathogenic bacterium closely related to
B. anthracis, the causative agent of anthrax in mammals. A
significant portion of the B. cereus chromosomal genes are common to
B. anthracis, including genes which in B. anthracis code for putative virulence
and surface proteins. B. cereus thus provides a convenient model organism
for studying proteins potentially associated with the pathogenicity of the
highly infectious B. anthracis. The zinc-binding protein of B. cereus,
BcZBP, is encoded from the bc1534 gene which has three homologues to
B. anthracis. The protein exhibits deacetylase activity with the N-acetyl
moiety of the N-acetylglucosamine and the diacetylchitobiose and
triacetylchitotriose. However, neither the specific substrate of the BcZBP
nor the biochemical pathway have been conclusively identified. Here, we
present the crystal structure of BcZBP at 1.8 A resolution. The
N-terminal part of the 234 amino acid protein adopts a Rossmann fold whereas
the C-terminal part consists of two beta strands and two alpha helices. In the
crystal, the protein forms a compact hexamer, in agreement with solution
data. A zinc binding site and a potential active site have been identified
in each monomer. These sites have extensive similarities to those found in
two known zinc-dependent hydrolases with deacetylase activity, MshB and
LpxC, despite a low degree of amino acid sequence identity. The functional
implications and a possible catalytic mechanism are discussed.
(2ixd.pdb).
Electronic reprint (1.4 MBytes) : Local copy, or directly from Acta Cryst. D63, 705-713, Copyright © International Union of Crystallography.
Comparison of crystallographically determined and molecular dynamics
simulation-derived parameters for a small (26 kDa) homotetrameric
four-alpha-helical bundle protein revealed an unexpected pattern of similarities
and differences between experiment and simulation. On one hand, the protein
structure per se is exceptionally well preserved during the simulations,
with a root-mean-square deviation between the Ca atoms of the crystal
structure and the simulation-derived average structures of only 0.58 Angstrom,
which is not very different from the expected coordinate error of the
experimentally determined structure. On the other hand, comparison of the
temperature factors showed a large discrepancy, with the experimental B-factors
being approximately three times higher than the simulation-derived B-factors.
Closer examination of this discrepancy appears to validate the
molecular dynamics prediction and to implicate as its source static disorder
at the crystalline state, as indicated by the strong diffuse scattering and
pronounced anisotropy of the diffraction pattern of the protein crystals.
A posteriori re-refinement of the structure using a new TLS parameterization
scheme based on the results obtained from the simulations led to a further
reduction of the R factor and the free R value by 0.4% and 0.8%,
respectively, indicating that molecular-dynamics simulations have matured to
the point that they can be used to aid the selection of TLS groups for
macromolecular refinement.
Electronic reprint (284 KBytes) : Local copy, or directly from Wiley InterScience, Copyright © Wiley InterScience.
A computer program has been developed to aid the analysis of
molecular dynamics trajectories. The program is tuned for macromolecular
large-scale problems and supports features such as removal of global
translations-rotations of the solute, calculation of average distance maps
and their corresponding standard deviations, calculation of the
variance-covariance and cross-correlation matrices, and principal component
analysis of trajectories with the added ability to create artificial
trajectories based on selected eigenvectors. Limited graphics (trajectory
viewing) capabilities are also available.
Electronic reprint (2.0 MBytes) : Local copy, or directly from the American Chemical Society, Copyright © American Chemical Society.
The repressor of primer (Rop) protein has become a steady source
of surprises concerning the relationship between the sequences and the
structures of several of its mutants and variants. Here we add another piece
to the puzzle of Rop by showing that an engineered deletion mutant of the
protein (corresponding to a deletion of residues 30-34 of the wild-type
protein and designed to restore the heptad periodicity at the turn region)
results in a complete reorganization of the bundle which is converted from a
homodimer to a homotetramer. In contrast (and as previously shown), a
two-residue insertion, which also restores the heptad periodicity, is
essentially identical with wild-type Rop. The new deletion mutant structure
is a canonical, left-handed, all-antiparallel bundle with a completely
different hydrophobic core and distinct surface properties. The structure
agrees and qualitatively explains the results from functional,
thermodynamic, and kinetic studies which indicated that this deletion mutant
is a biologically inactive hyperstable homotetramer. Additional insight into
the stability and dynamics of the mutant structure has been obtained from
extensive molecular dynamics simulations in explicit water and with full
treatment of electrostatics.
(1qx8.pdb).
Electronic reprint (65 KBytes) : Local copy, or directly from J. Appl. Crystallogr. 38, 574-575, Copyright © International Union of Crystallography.
The program Queen of Spades encodes an algorithm for a stochastic multidimensional approach to
molecular replacement. The program has been shown to be capable of successfully
locating solutions even in cases as complex as a 23-dimensional, 4-body
problem. Recently, we extended our approach
to tackle the full molecular replacement problem by allowing the possibility
of using many different search models simultaneously, and showed that we
could successfully locate solutions in the case of a 17-dimensional problem
involving one DNA and two (different) protein search models.
This multimodel, multidimensional approach does have
its cost: with a few thousand unique reflections in a high symmetry space
group and with more than two search models, a typical Qs run would
take well over two to three weeks of CPU time on the fastest personal
workstations. The way forward for such computationally intensive
calculations is of course parallelisation. Here, I report the availability of
a parallel version of Qs which is based on the Message Passing
Interface (MPI) paradigm.
Electronic reprint (312 KBytes) : Local copy, or directly from Wiley InterScience, Copyright © Wiley InterScience.
The Repressor of primer (Rop) protein is the paradigm of a canonical
(left-handed, all-anti-parallel) homodimeric 4-alpha-helical bundle. It
is known (through the analysis of an orthorhombic crystal form) that a
single alanine-to-proline amino-acid substitution at the turn region of Rop
(the A31P mutant) markedly changes the topology of the protein which is
converted to a right-handed, mixed parallel and antiparallel bundle. Here we
report the structure of this mutant in a second (monoclinic) crystal form
and show that although its topology remains unchanged, the differences
between the two A31P crystal structures are unexpectedly large, with a root
mean square deviation between equivalent Ca atoms of approximately
3A. Remarkably, a 3 ns molecular dynamics simulation of A31P which was
initiated from the orthorhombic form crystal structure, sampled an ensemble
of configurations rather similar to the structure seen in the monoclinic
form. This finding suggests that the observed crystal structures may
correspond to images of two conformers taken from a structurally
heterogeneous population of molecules at equilibrium. Comparison of the
A31P simulation with a 3 ns simulation of wild-type Rop indicated that the
mutant is an inherently highly flexible molecule, both with respect to the
relative placement of its helices and the malleability of its (loosely
packed) hydrophobic core. Based on these findings, we propose that the A31P
Rop mutant is an equilibrium molten globule and we attempt to interpret its
thermodynamic properties based on this assumption. Furthermore, we present
results from a 3 ns molecular dynamics simulation of a hypothetical
structure which was constructed by artificially mutating the alanine at
position 31 of the wild-type Rop structure to a proline. This hypothetical
A31P structure appears to be significantly more stable than the
experimentally determined one, leading us to propose that the observed A31P
structure may correspond to a kinetically trapped molten globule.
(1gmg.pdb).
Electronic reprint (539 KBytes) : Local copy, or directly from Acta Cryst. D60, 1334-1337, Copyright © International Union of Crystallography.
Detailed knowledge of the influence of various parameters on
macromolecular solubility is essential for crystallization. The concept
of so-called ionic strength reducers provides insight into the changes
in solubility induced by organic solvents and hydrophilic polymers in
aqueous electrolytic solutions. A simple and efficient procedure is
presented which exploits the properties of ionic strength reducers in
the purification and crystallization of proteins. Using two designed
variants of the Rop protein as model systems, superior crystals have
been obtained compared with conventional techniques. This
procedure is particularly useful in cases where excessive nucleation
leads to the growth of a large number of tiny crystals that are useless
for crystallographic analysis.
Electronic reprint (1.1 MBytes) : Local copy, or directly from J. Appl. Crystallogr. 37, 159-161, Copyright © International Union of Crystallography.
Classical molecular replacement methods and the newer six-dimensional searches
treat molecular replacement as a succession of sub-problems of reduced dimensionality.
Due to their divide-and-conquer approach, these methods necessarily ignore (at
least during their early stages) the very knowledge that a target crystal structure may
comprise, for example, more than one copy of a search model, or, several models of
different types. We have previously described an algorithm for a stochastic multidimensional
molecular replacement search and showed that it can successfully locate
solutions even in cases as complex as a 23-dimensional, 4-body search. The original
description of the method only dealt with a special case of molecular replacement,
namely with the problem of placing n copies of only one search model in the
asymmetric unit of a target crystal structure. Here we present a natural generalisation of
this algorithm to deal with the full molecular replacement problem, that is, with the
problem of determining the orientations and positions of a total of n
copies of m different models which are assumed to be present in the asymmetric unit
of a target crystal structure. The generality of this approach is illustrated through its
successful application to a 17-dimensional, 3-model problem involving one DNA and
two protein molecules.
Electronic reprint (629 KBytes) : Local copy, or directly from Proc. Natl. Acad. Sci. USA, 101, 70-75, Copyright © National Academy of Sciences USA.
Type III secretion systems enable plant and animal bacterial pathogens
to deliver virulence proteins into the cytosol of eukaryotic
host cells, causing a broad spectrum of diseases including
bacteremia, septicemia, typhoid fever, and bubonic plague in mammals,
and localized lesions, systemic wilting, and blights in plants. In
addition, type III secretion systems are also required for biogenesis
of the bacterial flagellum. The HrcQB protein, a component of the
secretion apparatus of Pseudomonas syringae with homologues in
all type III systems, has a variable N-terminal and a conserved
C-terminal domain (HrcQB-C). Here, we report the crystal structure
of HrcQB-C and show that this domain retains the ability of the
full-length protein to interact with other type III components. A 3D
analysis of sequence conservation patterns reveals two clusters of
residues potentially involved in proteinprotein interactions.
Based on the analogies between HrcQB and its flagellum homo-
logues, we propose that HrcQB-C participates in the formation of
a C-ring-like assembly.
(1o9y.pdb).
Electronic reprint (3.8 MBytes) : Local copy, or directly from Acta Cryst. D59, 709-718, Copyright © International Union of Crystallography.
The crystal structure of a 4-alpha-helical bundle protein has been
determined through the application of a 23-dimensional molecular replacement
search performed with a stochastic method. The search model for the
calculation was a 26 residue-long poly-alanine helix amounting to less than
13% of the total number of atoms in the asymmetric unit of the target
crystal structure. The crystal structure determination procedure is
presented in detail, with emphasis on the molecular replacement
calculations
(1gmg.pdb).
Electronic reprint (1.6 MBytes) : Local copy, or directly from Acta Cryst. D58, 421-430, Copyright © International Union of Crystallography.
In the gram positive bacterium Bacillus subtilis the concentration of the amino acid L-arginine is controlled
by the transcriptional regulator AhrC. The hexameric AhrC protein binds in an L-arginine-dependent manner to
pseudo-palindromic operators within the prometer regions of arginine biosynthesic and catabolic gene clusters.
AhrC binding results in the repression of transcription of biosynthetic genes and in the activation of transcription
of catabolic genes. We have determined the crystal structure of AhrC at 2.7A resulution. Each sununit of the protein
has two domains. The C-terminal domains are arranged with 32 point group symmetry and mediate the major inter-subunit
interactions. The N-terminal domains are located around this core, where they lie in weakly associated pairs but do not
obey strict symmetry. A structural comparison of AhrC with the arginine repressor from the thermophile
Bacillus stearothermophilus reveals close similarity in regions implicated in L-arginine binding and DNA recognition
but also some striking sequence differences, especially within the C-terminal oligomerisation domain, which may contribute
to the different thermostabilities of the proteins. Comparison of the crystal structure of AhrC with a 30A resolution
model obtained by combining X-ray structure factor amplitudes with phases derived from electron microscopic analyses of
AhrC crystals confirms the essential accuracy of the earlier model and suggests that such an approach may be more
widely useful for obtaining low resolution phase information
(1f9n.pdb).
Electronic reprint (1.9 MBytes) : Local copy, or directly from Acta Cryst. D57, 1462-1473, Copyright © International Union of Crystallography.
The ECM 20 (Krakow, 2001) presentation is also available for download (Powerpoint presentation, 1.6 MB).
A method is described which attempts to simultaneously and independently
determine the positional and orientational parameters of all molecules
present in the asymmetric unit of a target crystal structure. This is
achieved through a reverse Monte Carlo optimisation of a suitable statistic
(like the R-factor or the linear correlation coefficient between the
observed and calculated amplitudes of the structure factors) in the
6n-dimensional space defined by the rotational and translational
parameters of the n search models. Results from the application of this
stochastic method ---obtained with a space group general computer program
which has been developed for this purpose--- indicate that with present-day
computing capabilities the method may successfully be applied to molecular
replacement problems for which the target crystal structure contains up to
three molecules per asymmetric unit. It is also shown that the method may be
useful in cases where the assumption of topological segregation of the self
and cross vectors in the Patterson function is violated (as may happen, for
example, in closely packed crystal structures).
Electronic reprint (385 KBytes) : Local copy, or directly from Protein Engng. 14, 321-328, Copyright © Oxford University Press.
The distribution of the chi1, chi2 dihedral angles in a data set consisting of
twelve unrelated 4-alpha-helical bundle proteins has been determined and
compared with that observed in globular proteins. The analysis suggests that
for this tertiary motif : (i) the side-chain conformations are limited to only
a subset of the conformations observed in globular proteins and are more
constrained that side chains in helical regions of globular proteins.
(ii) The side chains of Aspartic acid and Asparagine adopt occasionally a new,
topology-specific rotamer. (iii) The rotamer preferences of Tyrosine and
Isoleucine depend on whether they are located in the hydrophobic core of the
bundle or in a more exposed position. (iv) Naturally occuring mutations in
the hydrophobic core of 4-alpha-helical bundles follow a pattern that is
consistent with the notion that the mutated and wild-type residues have at
least one of their most highly populated rotamers in common. These findings
indicate a relationship between protein topology and preferred side-chains
conformations.
Electronic reprint (69 KBytes) : Local copy, or directly from Anal. Biochem. 287, 185-186, Copyright © Academic Press.
A simple procedure for drying polyacrylamide gels is described. This
only involves soaking the gel twice in low grade
ethanol : The gel is placed in a petri dish containing 5-10 gel volumes of low grade ethanol and stirred
for 10-15 minutes. After that time, the ethanol solution is refreshed and soaking continues for 5
minutes. During this second soak the gel becomes opaque, dehydrates and shrinks uniformly (without cracking)
by a factor of about 35-40%.
In the final step, the gel is removed from the ethanol solution,
placed on a hard (non-adhesive) surface, ethanol is allowed
to evaporate from its top surface, and then it is covered with a glass plate to avoid curling during
the final stages of ethanol evaporation. After few hours the gel is ready for storage.
Electronic reprint (237 KBytes) : Local copy, or directly from Acta Cryst. D56, 1015-1016, Copyright © International Union of Crystallography.
The Rop protein is the paradigm of a highly regular 4-a-helix
bundle, and as such it has been subject to numerous structural
and mutagenesis studies. Crystals of a designed Rop variant which
establishes a continuous heptad pattern through the bend region
have been obtained by a combination of vapour diffusion and seeding
techniques. The crystals diffract to ultrahigh (0.8A)
resolution using synchrotron radiation and cryogenic conditions.
Electronic reprint (384 KBytes) : Local copy, or directly from Acta Cryst. D56, 1070-1072, Copyright © International Union of Crystallography.
The
distribution of the bulk solvent correction parameters (Bsol, ksol)
-as determined with an
exponential scaling algorithm based on Babinet's principle-
for 219 crystal
structures deposited with the Protein Data Bank is presented. The observed distribution
strongly suggests that the two parameters are not independent,
and a reasonable agreement
with the experimental data could be obtained through the application
of a simple exponential function.
Possible interpretations of this finding are discussed.
Electronic reprint (584 KBytes) : Local copy, or directly from J. Appl. Cryst. 33, 982-985, Copyright © International Union of Crystallography.
A maximum entropy formalism aiming at the production of a
"maximally non-committal" map is a standard method in fields of
science like radioastronomy,
but a rare exception in both X-ray crystallography and electron microscopy
(or crystallography).
This is rather unfortunate, given the wealth of information that a MAXENT map can reveal,
especially when the
map itself
is the end product (for example, low resolution electron or potential density maps,
Patterson functions, deformation maps).
The program GraphEnt
attempts to automate the procedure of calculating
maximum entropy maps, with emphasis on the calculation of
difference Patterson functions
for macromolecular crystallographic problems, while
providing a useful graphical output of the current stage of the calculation.
Electronic reprint (319 KBytes) : Local copy, or directly from Acta Cryst. D56, 101-103, Copyright © International Union of Crystallography.
Chloramphenicol acetyltransferase (CAT) is responsible for bacterial
resistance to chloramphenicol. It catalyzes inactivation of the
antibiotic by acetyl group transfer from acetyl CoA to one or
both hydroxyl groups of chloramphenicol. Type I CAT possesses
some unique properties which are not observed in other CAT variants.
Type I CAT overexpressed in E.coli was purified and crystals
with a resolution limit of 2.22 Å have been obtained
using a novel procedure which is based on the concept
of the "ionic strength reducers". The crystals have the symmetry
of spacegroup P1 and the unit cell dimensions are a=96.5, b=113.9,
c=114.2, alpha=119.9,
beta=94.1 and gamma=98.6 degrees. These
dimensions are consistent with four to six trimers per unit cell
corresponding to a solvent fraction ranging from 65 to 47%.
Electronic reprint (415 KBytes) : Local copy, or directly from Acta Cryst. D56, 169-174, Copyright © International Union of Crystallography.
The CCP4 2001 Study Weekend presentation is also available for download (Powerpoint presentation, 1 MB).
The ECM 20 (Krakow, 2001) presentation is also available for download (Powerpoint presentation, 1.6 MB).
The
classical approach to the problem of placing n copies of a search
model in the asymmetric unit of a target crystal structure, is to divide
this 6n-dimensional optimisation problem into a succession of 3-dimensional
searches (rotation function followed by translation function searches for
each of the models). Here it is shown that a structure determination method
based on a reverse Monte Carlo minimisation of the conventional crystallographic
R-factor in the 6n-dimensional space defined by the rotational
and translational parameters of the n molecules, is both feasible
and practical, at least for small n. Because all parameters of all
molecules are determined simultaneously, this algorithm should improve
the signal-to-noise ratio in difficult cases involving high crystallographic/non-crystallographic
symmetry in tightly packed crystal forms. Preliminary results from the
application of this method -obtained with a space
group general computer program which has been developed for this purpose-
are presented.
Electronic reprint (1.7 MBytes) : Local copy, or directly from Acta Cryst. D55, 1301-1308, Copyright © International Union of Crystallography.
Conventional least-squares or simulated annealing refinement of even correctly
positioned poly-alanine models of a target structure, results to a systematic
distortion of the molecular geometry and to a concomitant increase of the
mean phase difference from the correct phase set. Here it is shown that
iterative rigid-body simulated annealing refinement of poly-alanine models
employing successively fewer residues per rigid body (down to one alanine
residue per body) at a very high initial temperature (of the order of To=10000
K) and with the geometric energy terms switched on, not only preserves
the geometry of the model, but can also converge to an essentially correct
poly-alanine trace of the target structure even when the starting model
deviates systematically and significantly from the sought structure. As
an example of the application of the method we present details of the structure
determination of the alanine-31 to proline mutant of the Rop protein, where
an initial, roughly positioned poly-alanine model (giving an average phase
difference of 78.2 degrees from the final phase set) was successfully refined
against a 1.8A resolution native data set, leading to an essentially correct
model of the main chain with an average displacement of its atomic positions
from the final model of 0.275A. The phases calculated from this refined
poly-alanine model had an average difference of 43.8 degrees from the final
phase set (corresponding to a mean figure of merit of 0.72) and gave a
readily interpretable electron density map.
Electronic reprint (660 KBytes) : Local copy, or directly from J. Appl. Cryst. 32, 821-823, FREE ITEM, Copyright © International Union of Crystallography.
A computer program has been developed which
given a set of structure factor amplitudes for any centrosymmetric plane
group, displays the amplitude-weighted reciprocal lattice plane and allows
the user to interactively assign and modify the phases of the structure
factors, while observing the effect of these changes on the corresponding
electron density function. The program has the added feature of being able
to calculate and interactively display the electron density maps corresponding
to all phase combinations of a user-defined subset of structure factors.
The application of the program in both crystallographic teaching and research
are discussed.
Electronic reprint (2.2 MBytes) : Local copy, or directly from ScienceDirect, Copyright © Current Biology Publications.
The
general experience from structural studies of single amino-acid substituted
mutant proteins, is that the effect of mutation is rather localised and
minor. Here we provide a counter-example to this statement by showing that
a single alanine to proline substitution in the turn region of a 4-alpha-helical
protein leads to a complete reorganisation of the whole molecule which
is converted from the canonical left-handed all-antiparallel form, to a
right-handed, mixed parallel and antiparallel bundle, which to the best
of our knowledge and belief represents a novel topological motif for this
class of proteins. Our results suggest a possible new mechanism for the
creation and evolution of protein folds, show the importance of the loop
regions in determining the allowable folding pathways and illustrate the
malleability of protein structure
(1b6q.pdb).
Electronic reprint (2.6 MBytes) : Local copy, or directly from Acta Cryst. D54, 215-225, Copyright © International Union of Crystallography.
Attempts
to determine the X-ray crystal structure of the intact homohexameric arginine
repressor/activator from B. subtilis have so far been unsuccessful.
The major problem appears to be the lack of an isomorphous heavy atom derivative
with a manageable number of substitution sites. Here it is shown how electron
microscopy of thin three-dimensional crystals,
the same
as those used for the X-ray crystallographic
studies, made it possible (i) to obtain experimental support for some conclusions
drawn on the basis of X-ray data alone, (ii) to determine the low resolution
distribution of electron density in several different crystallographic
projections, and, (iii) to obtain a tentative low resolution model of the
whole hexamer.
Electronic reprint (7.7 MBytes)
In the presence of L-Arginine, AhrC --the Arginine-dependent
Repressor/Activator from Bacillus subtilis-- represses the
transcription of the genes encoding the anabolic and activates those
encoding the catabolic enzymes of arginine metabolism. AhrC is a homohexamer
of total molecular mass 105 kDa. It shows no homology to any of the
characterised DNA-binding motifs or DNA-binding proteins with the exception
of ArgR, the Arginine Repressor from Escherichia coli. ArgR does not
act as a transcription activator but it has been shown to be a necessary
accessory protein for the resolution --through site-specific
recombination-- of multimers of the ColE1 plasmid. Although the two
proteins share only 29% identity and are from such taxonomically distinct
prokaryotes, AhrC can complement E. coli ArgR- strains both in the
regulation of Arginine metabolism and the resolution of the ColE1 plasmid.
This thesis describes our attempts to determine the crystal structure of
AhrC. Three different crystal forms have been produced and characterised.
Useful derivatives have been prepared for two of these forms but the
determination of their heavy atom structures proved impossible. An attempt
to determine the low resolution structure of AhrC using Electron Microscopy
has been unsuccessful. Molecular Replacement using as a search model the
crystal structure of the hexameric core fragment of ArgR also failed to give
a convincing solution.