2007 SWMS Session
Abstracts
Authors: Richard H. G. Baxter[1], Chung-I Chang[1], Yogarany Chelliah[1],
Stéphanie
Blandin[2], Elena A. Levashina[2], and Johann Deisenhofer[1]
[1] Howard Hughes Medical Institute and Department of Biochemistry, University
of
Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX
75390-9050.
[2] Institut de Biologie Moléculaire et Cellulaire, UPR9022 du
Centre National de la
Recherche Scientifique, Équipe "Avenir" Institut National de la Santé
et de la
Recherche Médicale, 67084 Strasbourg, France.
Abstract: Thioester-containing proteins (TEPs) are a major component of
the innate
immune response of insects to invasion by bacteria and protozoa. TEPs form a
distinct
clade of a superfamily that includes the pan-protease inhibitors
alpha2-macroglobulins
and vertebrate complement factors. The essential feature of these proteins is a
sequestered thioester bond that, after cleavage in a protease-sensitive region
of the
protein, is activated and covalently binds to its target. Recently, TEP1 from
the
malarial vector Anopheles gambiae was shown to mediate recognition and killing
of
ookinetes from the malarial parasite Plasmodium berghei, a model for the human
malarial parasite Plasmodium falciparum. Here, we present the crystal structure
of the
TEP1 isoform TEP1r. Although the overall protein fold of TEP1r resembles that
of
complement factor C3, the TEP1r domains are repositioned to stabilize the
inactive
conformation of the molecule (containing an intact thioester) in the absence of
the
anaphylotoxin domain, a central component of complement factors. The structure
of
TEP1r provides a molecular basis for the differences between TEP1 alleles TEP1r
and
TEP1s, which correlate with resistance of A. gambiae to infection by P.
berghei.
Chad A. Brautigam1, Ranjit Deka2, Diana R. Tomchick1, Mischa Machius1,
Farol L. Tomson2, Sarah B. Lumpkins1, and Michael V.
Norgard2.
Departments of 1Biochemistry
and 2Microbiology, The University of Texas Southwestern Medical
Center at Dallas, Dallas, TX, 75390.
Treponema
pallidum, an obligate human pathogen and the causative agent of syphilis,
is not cultivatable in vitro, making it intractable to conventional
genetic approaches to discovering the functions of gene products. This
difficulty has prompted our labs to purse a structure-to-function approach to
study the lipoproteins of T. pallidum. One target is the ~20 kDa
protein called Tp34 (TP0971). The crystal structure of a nonacylated,
recombinant form of Tp34 (rTp34), determined at 1.9Å resolution, revealed two
metal-occupied binding sites within a dimer; the identity of the ion most
likely was zinc. Residues from both of the monomers contributed to the
interfacial metal-binding sites. Previous reports had implied that Tp34 could
bind human lactoferrin (hLF). Analytical ultracentrifugation showed that, in solution,
rTp34 formed a metal-stabilized dimer and that rTp34 bound hLF with a
stoichiometry of 2:1. Isothermal titration calorimetry further revealed that
rTp34 bound hLF at high (submicromolar) affinity. Finally, membrane topology
studies revealed that native Tp34 is not located on the outer surface (outer
membrane) of T. pallidum but, rather, is periplasmic. How the propensity
of Tp34 to bind zinc and the iron-sequestering hLF may relate overall to the
biology of T. pallidum infection in humans is discussed.
Crystal
Structures of Multidrug Resistance Regulator EbrR
Jinhui Dong, Lisheng
Ni, and Richard G. Brennan
Department of
Biochemistry and Molecular Biology, MD Anderson Cancer Center, Houston, TX
In
bacteria, Multidrug resistance transporters are tightly controlled by
transcription regulators. EbrR is a putative DNA binding protein of the
AcrR/TetR family. It is suggested that EbrR function as the repressor of its
upstream ebrA gene, a putative multidrug resistance transporter of small
multidrug resistance (SMR) family. Preliminary data indicate that EbrR can bind
multiple structurally dissimilar chemicals. We use EbrR as a model protein to
study the structural mechanisms of multidrug recognition.
Crystal structures of EbrR in complex with three different chemicals have been solved with
MAD data or molecular replacement. Overall fold of EbrR structure is similar to
those of other TetR family members. Like another multidrug resistance regulator
of TetR family QacR, EbrR also has a large flexible substrate-binding pocket,
which utilizes aromatic and negative-charged residues to form specific
interactions with substrates. Substrate binding causes large conformational
changes. Morevoer, the structures might also explain the interactions between “drugs”
observed in drug binding.
Jonathan P.
Schuermann, Angela J. Rodriguez, Xiaohang Cao, Alexander B. Taylor, and P. John
Hart
Department of
Biochemistry and the X-ray Crystallography Core Laboratory, The University of
Texas Health Science Center at San Antonio, San Antonio, TX USA
Approximately 115 distinct mutations in
human copper-zinc superoxide dismutase (SOD1) cause an inherited form of
amyotrophic lateral sclerosis (ALS, motor neuron disease). A helper protein,
the copper chaperone for SOD (CCS), modifies nascent SOD1 by inserting the catalytic
copper and oxidizing the disulfide bond in each subunit. These
posttranslational alterations impart enormous stability to the enzyme and are
therefore critical for the understanding of SOD1-linked fALS, which is now
accepted to be a protein misfolding and aggregation disease.
One emphasis of this talk will be the significance of the
crystal structure of an immature, pathogenic human SOD1 variant that is
recognized, but cannot be acted upon by CCS. The asymmetric unit contains one
canonical Zn-bound SOD1 homodimer and one completely novel, metal-free,
disulfide reduced homodimer. The unanticipated “open” conformation appears
accessible only to newly synthesized SOD1 polypeptides. Gel shift and
solution biophysical data strongly support the notion that the open
conformation of SOD1 is bound tightly by CCS. Modeling studies using existing
CCS structures suggest a mechanism of copper delivery different from that
currently accepted in the field. The enhanced understanding of SOD1-CCS
interactions that arises from the structure suggests, for the first time, a
unifying characteristic shared by each of the ~115 pathogenic SOD1 mutations
identified to date.
The
other emphasis of this talk will be on complications caused by crystals with
perfect hemihedral twinning combined with pseudosymmetry. The two distinct
pathogenic SOD1 dimers in the asymmetric unit in space group P312 pack in a
six-fold “honeycomb” arrangement (top right) in alternating bilayers such that
each subunit is related to the others by both crystallographic and
pseudosymmetric axes of rotation. The convoluted path leading to the eventual
final structure will be described in the hope that others will not have to
endure the trials and tribulations we experienced in this process. In the end,
the observed differences in pathogenic SOD1 structure, and the insight into CCS
action and fALS etiology derived from them, would have been missed completely
had the twinning issues not been detected and handled properly.
Lenong Li, Luzia V. Modolo†,
Luis L. Escamilla-Trevino,
Lahoucine Achnine, Richard A. Dixon, and Xiaoqiang Wang
Plant Biology Division, Samuel Roberts Noble Foundation,
2510 Sam Noble Parkway, Ardmore, OK 73401, USA
(Iso)flavonoids are a diverse
group of plant secondary metabolites with important implications for plant,
animal and human health. They exist in various glycosidic forms. Glycosylation,
which may determine their bioactivities and functions, is controlled by
specific plant uridine diphosphate glycosyltransferases (UGTs). A new
multifunctional (iso)flavonoid glycosyltransferase, UGT85H2, was identified from
the model legume Medicago truncatula with activity towards a number of
phenylpropanoid-derived natural products including the flavonol kaempferol, the
isoflavone biochanin A, and the chalcone isoliquiritigenin. The crystal
structure of UGT85H2 has been determined with
molecular replacement using program Phaser and the structure of UGT71G1 (PDB
ID: 2ACV) as a search model, and refined at 2.1 Å resolution to an R
factor of 19.7% and Rfree of 24.2%.UGT85H2
displays a similar GT-B fold observed previously for UGT71G1, and consists of two
N- and C-terminal domains with similar Rossmann-type folds. The structure of UGT85H2 reveals distinct structural features
which are different from those of other UGTs and related to the enzyme’s
functions and substrate specificities. Structural and comparative analyses
revealed the putative binding sites for the donor and acceptor substrates which
are located in a large cleft formed between the two domains of the enzyme, and
indicated that Trp360 may undergo a conformational change after sugar donor binding
to the enzyme. Further substrate docking combined with enzyme activity assay
and kinetic analysis provided structural insights into this substrate
specificity and preference.
Alex Taylor, Gang Hu, Lee McAlister-Henn, and P. John
Hart
Department of Biochemistry and the X-ray
Crystallography Core Laboratory
University of Texas Health Science Center, San Antonio, TX, USA
Mitochondrial NAD+-specific isocitrate dehydrogenases
(IDHs) are key regulators of flux through biosynthetic and oxidative pathways
in response to cellular energy levels. The affinity of the yeast IDH enzyme
for isocitrate is enhanced upon the binding of AMP and diminished by ATP and
NADH. This metabolite-mediated control contributes to the inverse relationship
between rates of energy production by oxidative pathways and glycolysis. Under
conditions of energy sufficiency, i.e. when relative cellular ratios of
[ATP]/[AMP] and of [NADH]/[NAD] are high, flux through the tricarboxylic acid
(TCA) cycle is attenuated at the level of IDH, rates of glycolysis increase,
and the tricarboxylic acids, citrate and isocitrate, are diverted into
biosynthetic pathways. Unlike the homodimeric bacterial enzymes that contain
two equivalent IDH subunits, yeast IDH is composed of four IDH1 and four IDH2
subunits that are 47% identical. Each IDH1 and IDH2 subunit associates to form
a heterodimer, and four heterodimers assemble into the biologically relevant
~300 kDa heterooctamer. IDH2 has retained the catalytic isocitrate/Mg2+
and NAD binding sites, while IDH1 has diverged to bind isocitrate and AMP,
thereby acting as a regulatory subunit. Although well-characterized
kinetically, the molecular basis for allosteric regulation of eukaryotic IDH
enzymes has been elusive because, until now, no structures of eukaryotic IDH
enzymes were known. To further our understanding of regulatory mechanisms, we
have determined the X-ray crystal structures of the functional yeast IDH heterooctamer
in the presence and absence of regulatory ligands. The results suggest that the
eukaryotic enzymes are exquisitely tuned to ensure that allosteric activation occurs
only when concentrations of isocitrate are elevated. This fine-control stands
in stark contrast to the on-off regulation of non-allosteric bacterial
isocitrate dehydrogenases via phosphorylation.
John M. Humphreys,1
Seung-Jae Lee,1 Haixia He,1 Prashanti Madhavapeddi,2
Yue Chen,1 Yingming Zhao,1 and Elizabeth J. Goldsmith||1
1 Department of Biochemistry, The University of Texas
Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas,
Texas 75390-8816
2 AstraZeneca India Private Ltd., Bellary Road, Hebbal, Bangalore - 560024 India
How
MAP kinase modules (comprised of a MAP3K, a MAP2K and a MAPK) induce
switch-like sigmoid responses has remained unclear. The switch depends upon
four kinase reactions, two each on the activation loops of MAP2Ks and MAPKs, to
activate first a dual specificity MAP2K, then a Ser/Thr MAPK. Here we show
that the dual specificity of the central MAP2Ks depends upon phosphorylation
status, like dedicated Tyrosine and Ser/Thr kinases. The MAP2K MEK6 adopts
different conformations as it becomes phosphorylated, the first is active as a
Ser/Thr kinase, but inactive toward its substrate MAPK p38α. The second
is competent as a Tyrosine kinase, the first step in MAPK activation. Thus,
the dual-specificity of the MAP2K is used to establish a precise order to the
kinase reactions catalyzed by the MAP kinase module, taking a
phosphorylation-directed detour from Ser/Thr to Tyrosine kinase activity that
creates extra thresholds to MAPK activation.
Crystal Structures of GSI-a
Glutamine Synthetase from Bacillus subtilis bound to ATP with glutamate and the
ATP analogue, AMP-PCP
David Murray,
Ryan Watkins, Maria Schumacher, and Richard Brennan
Department of
Biochemistry and Molecular Biology, MD Anderson Cancer Center, Houston, TX
Glutamine synthetase (GS) catalyzes the two-step,
ATP-dependent reaction converting glutamate and ammonia into glutamine, ADP and
inorganic phosphate. Glutamine synthetases are divided into two major
categories in bacteria: GSI-a
and GSI-b.
GSI-b
is found in high G+C gram positive bacteria, proteobacteria, and others and is
controlled by protein adenylylation. GSI-a
is found in low G+C gram positive bacteria, Euryarchaeota, and some thermophilic
bacteria and is regulated by glutamine and AMP dependent feedback inhibition.
This feedback inhibition is dependent upon the glutamine bound GSI-a
forming a complex with the TnrA transcription factor, which in turn cannot
repress GlnR, the repressor of the gene encoding GSI-a
The structure of GSI-a
in B. subtilis has been solved in its APO form , bound to the ATP analogue
AMP-PCP, and with both ATP and glutamate at 2.55, 2.74, and 3.10 Å resolution,
respectively.
The structure
GSI-a
was solved in its functional dodecameric state, with each subunit containing 444
amino acids (5328 residues in total). Between 440 and 444 residues were built
into each subunit. This structure was solved by molecular replacement, using
the M. tuberculosis GSI-b
as a search model. The structure of B. subtilis GSI-a
has two domains, a large C-terminal domain containing the active site and a
smaller N-terminal domain. The binding pocket has a bifunnel structure with ATP
entering the “top” and glutamate and NH4+ entering at the
“bottom” and “side”. Two residues seem to be most important in binding ATP and
AMP-PCP: Y201 and R331. The tyrosine and arginine form a
p-stacking
interaction with the adenine ring, with R331 forming additional electrostatic
interactions with the phosphate groups. Glutamate is held in place by a
histidine and arginine residue and by the gamma-phosphate of ATP. Early
analysis of the difference maps indicates the presence of the gamma-glutamyl
phosphate intermediate.
Aditya Hindupur, Deqian Liu, Yonghong Zhao, Henry D.
Bellamy, Mark A. White, and Robert O. Fox
Department of Human Biological Chemistry & Genetics University of Texas Medical Branch, Galveston, TX
Bacteria are capable of responding to a variety of stress
conditions. These conditions include heat, osmotic shock, acid/alkali. The
heat shock response results in production of a variety of proteins ranging from
the molecular chaperons GroEL, ClpA, ClpB, to specific proteins whose
functions are now being elucidated. Here we report the structure and potential
biochemical function of two such proteins: YedU and YciF. The yedU mRNA is
induced 31-fold on heat shock and is enhanced in an hns-deletion strain that is
known to derepress the stress response. It is a 31 kDa protein that forms
dimers in solution. The structure shows alpha-beta characteristics and is a
member of the Class I glutamine amidotransferase superfamily that includes
proteases, catalases and transcriptional regulators. YciF is an 18.5 kDa
protein found as a dimer in solution. The protein is conserved across
eubacterial species. The structure of the apo-protein was solved at 2.0 Å
resolution. The protein forms 4 helix bundle with an additional 5th helix.
Structurally YciF was found to be close to the archaeal diiron protein
rubrerythrin. Another archaeal protein sulerythrin shows similar features to
YciF in lacking a rubedoxin domain found on rubrerythrin.
Kate J. Newberry1,
Mayuree Fuangthong2, Skorn Mongkolsuk2 and Richard G.
Brennan1
1Department
of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer
Center, Houston, TX; 2Laboratory of Biotechnology, Chulabhorn
Research Institute, Lak Si, Bangkok 10210, Thailand
OhrR,
a member of the MarR family of transcriptional regulators is widely conserved
amongst Gram negative and Gram positive bacteria, and activates expression of
the ohr peroxidase gene in response to organic hydroperoxides (OHP), but
not hydrogen peroxide. The soil bacterium Xanthamonas campestris (Xc)
contains an OhrR that is a specific sensor of lipid hydroperoxides, such as
linoleic acid hydroperoxide, that are highly toxic compounds generated by the
plant host defense response. These lipid hydroperoxides are sensed via
oxidation of a conserved cysteine residue near the N-terminus of the protein.
The recently described crystal structures of Bacillus subtilis OhrR in
both the apo- and DNA-bound forms describes the structure a single cysteine
containing OhrR in the reduced form and although these structures give valuable
insight into the DNA-binding mechanism of the MarR family of transcriptional
regulators, the atomic details of the mechanism of oxidation-induced
derepression by either family of OhrR remains unclear. We have recently
determined the crystal structures of both reduced and oxidized OhrR, each to
1.9Å resolution. In the reduced form the disulfide forming cysteine residues
C22 and C127 are 15.5Å apart. Upon oxidation by OHP, OhrR undergoes a dramatic
structural rearrangement of helices 5 and 6 that brings
residues C22 and C127 into proximity, thereby allowing disulfide bond formation
and a reconfiguration of the dimer interface, which disrupts DNA binding.
Moreover, these structures in combination with in vivo and in vitro
studies on a series of Xc OhrR point mutants provide an OHP-induction roadmap
that is likely utilized by the vast majority of the MarR family.
Maria A. Schumacher1, Tiffany C. Glover1, Anthony J.
Brzoska2, Slade O.
Jensen2, Thomas Dunham1, Ronald A. Skurray2
& Neville Firth2
1Department of Biochemistry and Molecular Biology, University of
Texas, M.D. Anderson Cancer Center, Unit 1000, Houston TX 77030, U.S.A.
2School of Biological Sciences, University of Sydney, Sydney, New
South Wales 2006, Australia
The stable inheritance of genetic material depends on accurate DNA
partition. Plasmids serve as tractable model systems to study DNA
segregation because they require only a DNA centromere, a centromere-binding
protein and a force-generating ATPase. The centromeres of partition (par)
systems typically consist of a tandem arrangement of direct repeats. The
best-characterized par system contains a centromere-binding protein called ParR
and an ATPase called ParM. In the first step of segregation, multiple
ParR proteins interact with the centromere repeats to form a large
nucleoprotein complex of unknown
structure called the segrosome, which binds and recruits ParM filaments.
pSK41 ParR binds a centromere consisting of multiple 20-bp tandem repeats to
mediate both transcription autoregulation and segregation. Here we report the
structure of the pSK41 segrosome revealed in the crystal structure of a
ParR-DNA complex. In the crystals, the 20-mer tandem repeats stack pseudo-continuously
to generate the full-length centromere with the ribbon-helix-helix fold of ParR
binding successive DNA repeats as
dimer-of-dimers. Remarkably, the dimer-of-dimers assemble in a continuous
protein super-helical array wrapping the DNA about its positive convex surface
to form a large segrosome with an open, solenoid-shaped structure. Cryo-EM
studies of the complex reveals a circular helical structure with a diameter
identical to that seen in the crystal structure, supporting the segrosomal
crystal structure model. Importantly, this helical superstructure
suggests a mechanism for ParM capture and subsequent plasmid
segregation.
Mischa Machius,
Department of Biochemistry,
The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, 75390.
Histone methylation regulates diverse chromatin-templated
processes, including transcription. The recent discovery of the first
histone lysine-specific demethylase (LSD1) has changed the long-held
view that histone methylation is a permanent epigenetic mark.
Many transcriptiona, l corepressor complexes contain LSD1 and CoREST
that collaborate to demethylate mono- and dimethylated H3-K4 of nucleosomes.
LSD1 is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that
demethylates histone H3 Lys4 (H3-K4). We have determined the crystal
structure of the LSD1-CoREST complex. LSD1- CoREST forms an elongated structure
with a long stalk connecting the catalytic domain of LSD1 and the CoREST
SANT2 domain. LSD1 recognizes a large segment of the H3 tail through a
deep, negatively charged pocket at the active site and possibly a shallow
groove on its surface. CoREST SANT2 interacts with DNA. Disruption of the
SANT2-DNA interaction diminishes CoREST-dependent demethylation of
nucleosomes by LSD1. The shape and dimension of LSD1-CoREST suggest its
bivalent binding to nucleosomes, allowing efficient H3-K4 demethylation.
This spatially separated, multivalent nucleosome-binding mode may apply
to other chromatin-modifying enzymes that generally contain
multiple nucleosome binding modules. In order to elucidate the mechanism
by which LSD1 achieves its substrate specificity, we have further
determined the crystal structure of LSD1 with a propargylamine-derivatized
H3 peptide covalently tethered to FAD. H3 adopts three consecutive
gamma-turns, enabling an ideal side-chain spacing that places its N
terminus into an anionic pocket and positions methyl-Lys4 near FAD for
catalysis. The LSD1 active site cannot productively accommodate more than
three residues on the N-terminal side of the methyllysine, explaining
its H3-K4 specificity. Dysregulation of histone acetylation and
methylation leads to the silencing of tumor suppressor genes and
contributes to cancer progression. Inhibitors of enzymes that catalyze
the addition and removal of these epigenetic marks thus have therapeutic
potential for treating cancer. Because LSD1 belongs to the family of
FAD-dependent amine oxidases, certain inhibitors of monoamine oxidases
(MAOs), including the clinically used antidepressant trans-2-
phenylcyclopropylamine (PCPA; tranylcypromine; Parnate), are also capable
of inhibiting LSD1. We have characterized the kinetic parameters of the
inhibition of LSD1 by PCPA and determined the crystal structure of
LSD1-CoREST in the presence of PCPA. PCPA forms a covalent adduct with FAD
in LSD1 that is distinct from the FAD PCPA adduct of MAO B. Our study
provides the basis for designing more potent inhibitors of LSD1 with
therapeutic potential, either derived from the LSD1-bound H3 peptide or
derived from PCPA, exploiting the unique structural characteristics of the
LSD1 substrate-binding site.
James E. Knapp, Mark A. White, James C.
Lee
Department of Biochemistry and Molecular
Biology, The University of Texas Medical Branch, Galveston, TX 77555
CRP is a
homodimeric protein with each subunit having cAMP-binding and DNA-binding
domains connected by a short hinge region. Each subunit has a high affinity
cAMP and low affinity cAMP binding sites. CRP is an excellent model system to
study allosteric protein regulation because there is a direct relationship
between protein dynamics and cooperative cAMP and DNA binding. This
relationship was demonstrated by hydrogen-deuterium exchange and adiabatic
compressibility experiments on a series of mutations. Both experiments measure
global dynamics. These results showed that binding energetics (ÄÄG)
between the binding of the first and second cAMP to each sample is correlated
to the global protein dynamics1.
Furthermore, the energetics of DNA binding (ÄG) follows the
same trend1. The crystallographic analysis of WT in
the presence of differing concentrations of cAMP shows that DNA binding in
solution is correlated with flexibility of the domain-domain hinge motion as
judged by the difference in the hinge angle between NCS related subunits.
Similarly, the structural analysis of the D53H and S62F CRP mutations shows
that mutation induced changes in the energetics of DNA binding and cooperative
energetics of cAMP binding correlate with the domaindomain flexibility of each
protein. Thus, the crystallographic work presented here is consistent with the
hypothesis that the domain-domain dynamics mediates allostery within the CRP
system.
1. Gekko, K., Obu, N., Li, J., Lee, J.C.
(2004) “A linear correlation between the
energetics of
allosteric communication and protein flexibility in the Escherichia coli
cyclic AMP
receptor protein revealed by mutation-induced changes in compressibility and
amide hydrogen-deuterium exchange.” Biochemistry 43: 3844-52.
David B. Neaub, Nathaniel C. Gilberta, Sue G. Bartletta,
Adam Dasseya, and Marcia E. Newcomera
aDepartment of Biological Sciences and the bCenter for
Advanced Microstructures and Devices, Louisiana State University, Baton Rouge,
LA
Lipoxygenases (LOX) catalyze the regio- and stereo- specific dioxygenation of
polyunsaturated membrane-embedded fatty acids . A Ca2+-dependent
membrane-binding function was localized to the amino terminal C2-like domain of
8R-lipoxygenase (8R-LOX) from the soft coral Plexaura homomalla (Oldham
et al., 2005). The 3.2 Å resolution crystal structure of 8R-LOX and
spectroscopic data suggested that Ca2+ stabilizes two membrane
insertion loops. Analysis of the protein packing contacts in the crystal
lattice indicated that the conformation of one of the two loops complicated
efforts to improve the resolution of the X-ray data. A deletion mutant of
8R-LOX, [delta]40-45:GSLOX, in which the corresponding membrane insertion loop
is absent was engineered. Removal of the membrane insertion loop dramatically
increased the protein yield from bacterial cultures and the quality of the
crystals obtained, resulting in an improvement in resolution better than 1 Å
for the diffraction data.
Angela R. Criswell,
Kris F. Tesh, A. L. Dowell, Joseph D. Ferrara and J.W. Pflugrath
Rigaku, 9009 New Trails Dr., The Woodlands, TX 77381-5209
Tel: +1 (281) 363-1033 / Fax: +1 (281) 364-3628 / www.Rigaku.com
One of the primary concerns in modern crystallography laboratories is to
develop protocols for improving poorly diffracting crystals. Crystals are optimized by changing a number of parameters during crystallization (salt,
buffer concentration, pH, additives, etc.). Additionally, post-crystallization
techniques include replacing water with additives (PEG, sugars, salts),
dehydrating in air or under oil, and cryo-annealing. Much of what is done
today is not reproducible and lacks the ability to improve a very poor quality,
mounted crystal. In the case of "bad" diffraction, the usual
thought is to cut your losses and mount a new crystal; in the worst case, you
go back and attempts to grow better crystals. But, this may not need to
happen. If there is evidence of better diffraction from previous
crystals, or if there are few options due to little available protein, you may
wish to "heal" a crystal. The Free Mounting System (FMS) is
used to carefully regulate and adjust the relative humidity about a mounted
crystal, which changes the water content within the crystal. Previously
flash-cooled crystals of very poor quality will be shown to improve as a result
of FMS rescue and rehabilitation, even to the point of usable data.
Joseph
D. Ferrara
Rigaku Americas Corporation, The Woodlands, USA
