Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Crystallization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,783 records · Page 99Linked to original sources
PubMed2004

Cloning, expression, purification, crystallization and preliminary X-ray analysis of human liver glyceraldehyde-3-phosphate dehydrogenase.

Human liver glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified and crystallized using PEG 3350 as a precipitant. However, the crystals were extremely fragile towards osmotic shock. A 1% change in PEG 3350 content causes destruction of the crystals. After many trials for freezing the crystals, X-ray diffraction data from a native crystal were collected at 2.8 A resolution using as a cryoprotectant a mixture consisting of paraffin oil and Paratone-N in a 3:1 ratio and a cryoloop covered with Formver film. Crystals belong to space group P2(1), with unit-cell parameters a = 63.23, b = 97.84, c = 84.23 A, beta = 104.1 degrees. Molecular replacement with a starting model consisting of a homology model based on the low-resolution structure of human skeletal muscle GAPDH, which has 90% identical residues with the liver protein, led to a solution. Most of the current model was assigned properly in the electron-density map, but the map corresponding to some important regions containing the phosphate-binding loop was ambiguous. It is planned to crystallize human liver GAPDH in the presence of phosphate ions and/or some kind of inhibitor in order to fix the flexible region.

Cloning, Molecular↗
PubMed2004

Crystallization and preliminary crystallographic analysis of 2-keto-3-deoxygluconate kinase from Thermus thermophilus.

2-Keto-3-deoxygluconate kinase (KDGK) catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phosphogluconate. Two crystal forms of KDGK from Thermus thermophilus were obtained by vapour-diffusion and microbatch methods. Crystals in the form of triangular plates (TtKDGK-1) were obtained that belong to space group P3, with unit-cell parameters a = b = 145.83, c = 74.63 A, and diffract to 3.2 A. These crystals exhibited nearly perfect hemihedral twinning. Assigning six subunits of TtKDGK to the asymmetric unit of the crystal corresponds to a 46.2% solvent content. A single plate-like crystal (TtKDGK-2) belonged to space group P6(3), with unit-cell parameters a = b = 84.83, c = 168.49 A, and diffracts to 2.25 A. This crystal exhibits only partial hemihedral twinning, with a twin fraction of 24.4%. Diffraction-quality crystals of TtKDGK with bound ATP (TtKDGK-ATP), a = b = 84.72, c = 321.61 A and with bound KDG plus the ATP analogue AMP-PNP (TtKDGK-ATP-KDG), with unit-cell parameters a = b = 84.32, c = 168.7 A, were also prepared and characterized.

Adenosine Triphosphate↗
PubMed2004

DNA variability in five crystal structures of d(CGCAATTGCG).

The deoxyoligonucleotide d(CGCAATTGCG) has previously been crystallized in four different space groups. The crystals diffract to moderate resolution (2.3-2.9 A). Here, a fifth crystal form that diffracts to higher resolution (1.6 A) is presented which was obtained thanks to the use of Co2+ and cryogenic temperatures. The availability of five different crystal structures allows a thorough analysis of the conformational variability of this DNA sequence. It is concluded that the central hexamer sequence CAATTG has a practically constant conformation under all conditions, whilst the terminal base pairs at both ends vary considerably as a result of differing interactions in the crystals. The new crystal structure presented here is stabilized by guanine-Co2+-guanine interactions and the formation of C1+ -G8.C3 triplexes between neighbouring duplexes. As a result of the higher resolution of the crystal structure, a more regular structure was obtained and a clear definition of the spine of hydration was observed which was not visible in the four previous structures.

Base Pairing↗
PubMed2004

Facile crystallization of Escherichia coli ketol-acid reductoisomerase.

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of branched-chain amino acids. The reaction involves an Mg2+ -dependent alkyl migration followed by an NADPH-dependent reduction of the 2-keto group. Here, the crystallization of the Escherichia coli enzyme is reported. A form with a C-terminal hexahistidine tag could be crystallized under 18 different conditions in the absence of NADPH or Mg2+ and a further six crystallization conditions were identified with one or both ligands. With the hexahistidine tag on the N-terminus, 20 crystallization conditions were found, some of which required the presence of NADPH, NADP+, Mg2+ or a combination of ligands. Finally, the selenomethionine-substituted enzyme with the N-terminal tag crystallized under 15 conditions. Thus, the enzyme is remarkably easy to crystallize. Most of the crystals diffract poorly but several data sets were collected at better than 3.2 A resolution; attempts to phase them are currently in progress.

Alcohol Oxidoreductases↗
PubMed2004

Structure of apo acyl carrier protein and a proposal to engineer protein crystallization through metal ions.

A topic of current interest is engineering surface mutations in order to improve the success rate of protein crystallization. This report explores the possibility of using metal-ion-mediated crystal-packing interactions to facilitate rational design. Escherichia coli apo acyl carrier protein was chosen as a test case because of its high content of negatively charged carboxylates suitable for metal binding with moderate affinity. The protein was successfully crystallized in the presence of zinc ions. The crystal structure was determined to 1.1 A resolution with MAD phasing using anomalous signals from the co-crystallized Zn(2+) ions. The case study suggested an integrated strategy for crystallization and structure solution of proteins via engineering surface Asp and Glu mutants, crystallizing them in the presence of metal ions such as Zn(2+) and solving the structures using anomalous signals.

Acyl Carrier Protein↗
PubMed2005

Combined use of AFM and X-ray diffraction to analyze crystals of an engineered, domain-deleted antibody.

A genetically engineered humanized C(H)2-domain-deleted monoclonal antibody lacking any interchain-hinge disulfide bonds has been crystallized in the presence of detergent in a form suitable for X-ray diffraction analysis. The crystals were grown from 4 M formate along with Triton X-100 and had P2(1)2(1)2 space-group symmetry, with unit-cell parameters a = 83, b = 224, c = 167 A. The crystals diffract to beyond 2.8 A resolution. A disordered crystal form of larger size and more attractive habit was also grown from 4 M formate, but in the presence of the Anapoe series of detergents. Preliminary X-ray data, in conjunction with atomic force microscopy images, are consistent with asymmetric units consisting of two intact antibodies forming a circular dimeric ring. The crystallizing unit, which must contain a twofold axis, is a toroidal assembly of four antibodies (two dimeric rings). Competition between dimers and tetramers to enter the lattice, along with a unique kind of planar defect of packing, may be responsible for the unusually high defect density and the disorder of the X-ray diffraction pattern exhibited by the second crystal form. An approach to crystallizing proteins showing phase separation, particularly intact antibodies, that uses a preliminary detergent test set is described.

Antibodies, Neoplasm↗
PubMed2005

A modified vapor-diffusion crystallization protocol that uses a common dehydrating agent.

In the vapor-diffusion protein-crystallization method, a small drop containing protein sample mixed with a crystallization solution is equilibrated against a reservoir solution in a sealed chamber. Whereas the chemical composition of the crystallization solution is critical for success, the primary role of the reservoir solution is to slowly concentrate the crystallization drop in a controlled fashion. Accordingly, it might be possible to use any reservoir solution of appropriate dehydrating strength. The important practical consequence is that many different experiments can share the same reservoir solution. This approach, called the ;shared reservoir solution' method, significantly simplifies manual and robotic experiment setup, reduces cost and allows a completely new design of optically superior and higher density crystallization plates. Although this research was motivated by these practical advantages, recent reports and the authors' results indicate that this method may actually increase crystallization success. The authors suggest that this may indicate that a protein has a preferred water activity for crystallization. Here, present practical and theoretical considerations as well as experimental tests of the shared reservoir solution method are presented.

Aldose-Ketose Isomerases↗
PubMed2005

Impact of natural variation in bacterial F17G adhesins on crystallization behaviour.

Since the introduction of structural genomics, the protein has been recognized as the most important variable in crystallization. Recent strategies to modify a protein to improve crystal quality have included rationally engineered point mutations, truncations, deletions and fusions. Five naturally occurring variants, differing in 1-18 amino acids, of the 177-residue lectin domain of the F17G fimbrial adhesin were expressed and purified in identical ways. For four out of the five variants crystals were obtained, mostly in non-isomorphous space groups, with diffraction limits ranging between 2.4 and 1.1 A resolution. A comparative analysis of the crystal-packing contacts revealed that the variable amino acids are often involved in lattice contacts and a single amino-acid substitution can suffice to radically change crystal packing. A statistical approach proved reliable to estimate the compatibilities of the variant sequences with the observed crystal forms. In conclusion, natural variation, universally present within prokaryotic species, is a valuable genetic resource that can be favourably employed to enhance the crystallization success rate with considerably less effort than other strategies.

Adhesins, Bacterial↗
PubMed2005

Surface-entropy reduction used in the crystallization of human choline acetyltransferase.

Human choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine (ACh) from choline and acetyl-CoA. A crystal structure of human ChAT has been a long-standing goal in the neuronal signalling field. Milligram quantities of pure ChAT can be purified [Kim et al. (2005), Protein Expr. Purif. 40, 107-117], but exhaustive crystallization efforts failed to produce any crystals suitable for high-resolution structural studies. To obtain high-quality crystals of human ChAT, a truncation was made in a large poorly conserved loop region and high-entropy side chains were removed from the surface of the protein. The resulting 'entropy-reduced' ChAT (MR = 68.1 kDa) crystallizes readily and reproducibly and the crystals diffract X-rays to approximately 2.2 A. The availability of these crystals will allow us to study the structure of human ChAT on its own as well as in complex with its substrates and inhibitor molecules, leading to a greater understanding of its catalytic mechanism and regulation.

Choline O-Acetyltransferase↗
PubMed2005

Entropy and surface engineering in protein crystallization.

Protein crystallization remains a key limiting step in the characterization of the atomic structures of proteins and their complexes by X-ray diffraction methods. Current data indicate that standard screening procedures applied to soluble well folded prokaryotic proteins yield X-ray diffraction crystals with an approximately 20% success rate and for eukaryotic proteins this figure may be significantly lower. Protein crystallization is predominantly dependent on entropic effects and the driving force appears to be the release of ordered water from the sites of crystal contacts. This is countered by the entropic cost of ordering of protein molecules and by the loss of conformational freedom of side chains involved in the crystal contacts. Mutational surface engineering designed to create patches with low conformational entropy and thereby conducive to formation of crystal contacts promises to be an effective tool allowing direct enhancement of the success rate of macromolecular crystallization.

Crystallization↗
PubMed2006

A survey of protein-protein complex crystallizations.

A survey of crystallization conditions was carried out for 650 published protein-protein complexes in the Protein Data Bank (PDB) of the Research Collaboratory for Structural Bioinformatics (RCSB). This resulted in the establishment of a Protein Complex Crystallization Database (PCCD) and a set of configuration-space boundaries for protein-complex crystallizations. Overall, polyethylene glycol (PEG) based conditions accounted for 70-80% of all crystallizations, with PEG 3000-4000, 5000-6000 and 8000 being the most frequently used. The median values of PEG concentrations were between 10 and 20% and were inversely correlated with their molecular weights. Ammonium sulfate remained the most favorable salt precipitant, with a median concentration of 1.6 M. The crystallization pH for the vast majority of protein complexes was between 5.0 and 8.0. Overall, the boundaries for the crystallization configuration space of protein complexes appear to be more restricted than those of soluble proteins. This may reflect the limited stability and solubility of protein-protein complexes. Based on statistical analysis of the database, a sparse-matrix and a systematic buffer and pH screen were formulated to best represent the crystallization of protein complexes.

Buffers↗
PubMed1998

A novel approach for the crystallization of soluble proteins using non-ionic surfactants.

Crystallization trials using three polyoxyethylene surfactants as precipitating agents are described. Of the eight soluble proteins screened, five were successfully crystallized at the first attempt. These included lysozyme, catalase, ferritin, ribonuclease A and ubiquitin. Further work suggested that these surfactants could also be suitable for cryo-crystallographic analysis of crystals. At the concentrations used in the crystallization trials [10-40%(v/v)], they are capable of promoting the formation of non-crystalline glasses at cryogenic temperatures (77K). This would facilitate crystal mounting and allow the minimization of crystal irradiation damage. Results from this study also suggest that proteins remain stable at high concentrations of these surfactants [40%(w/v)] and over long time periods (>1 month). A number of membrane proteins were also screened for crystallization. These included photosystems I and II and light harvesting complexes I and II from spinach and bacteriorhodopsin from Halobacterium halobium++. The trial s were unsuccessful both in the absence and presence of heptane-1,2,3-triol and over a wide range of surfactant concentrations.

Catalase↗
PubMed1998

Crystallization and preliminary X-ray diffraction studies of homoserine dehydrogenase from Saccharomyces cerevisiae.

Recombinant homoserine dehydrogenase from Saccharomyces cerevisiae has been crystallized in three different forms. Crystals of the apo-enzyme belong to the tetragonal space group P4 and have unit-cell-dimensions a = b = 130 and c = 240 A. The resolution limit for these crystals is 3.9 A. Crystals of homoserine dehydrogenase grown in the presence of the co-factor NAD+ have the tetragonal space group P41212 or its enantiomorph P43212. The unit-cell dimensions for these crystals are a = b = 80.4 and c = 250.2 A, and the observed resolution limit is 2.2 A. Protein crystals grown in the presence of the product L-homoserine and the inert NAD+ analogue 3-aminopyridine adenine dinucleotide belong to the monoclinic space group P21 with unit-cell parameters a = 58.8, b = 104.2, c = 120.7 A, beta = 91.9 degrees. This last crystal form has a diffraction limit of 2.7 A resolution.

Crystallization↗
PubMed1998

A description of imperfections in protein crystals.

An analysis is given of the contribution of various crystal imperfections to the rocking widths of reflections and the divergence of the diffracted beams. The crystal imperfections are the angular spread of the mosaic blocks in the crystal, the size of the mosaic blocks and the variation in cell dimensions between blocks. The analysis has implications for improving crystal perfection, defining data-collection requirements and for data-processing procedures. Measurements on crystals of tetragonal lysozyme at room temperature and 100 K were made in order to illustrate how parameters describing the crystal imperfections can be obtained. At 100 K, the dominant imperfection appeared to be a variation in unit-cell dimensions in the crystal.

Crystallization↗
PubMed1998

X-ray topographic studies of protein crystal perfection and growth.

The effects of solution variations during growth on the perfection of tetragonal lysozyme crystals have been characterized using X-ray topography and high angular and wavevector resolution reciprocal-space scans. X-ray images of crystals grown under nearly uniform conditions show little contrast or evidence of defects, and mosaic widths of these crystals are comparable with those reported for microgravity-grown crystals. Images of crystals for which solution conditions (temperature, pH or salt concentration) are changed after an initial period of uniform growth can show extensive contrast, indicating the presence of disorder. The X-ray mosaic widths of these crystals can be significantly broadened, but their radial widths are at most very slightly broadened, indicating that image contrast is primarily due to mosaicity. Comparison of X-ray images with mosaic scans indicates that regions grown after the change in solution conditions have broader mosaicities and are more disordered; that regions grown immediately after the change tend to have broader mosaicities than subsequent growth regions; and that the pre-change growth region is largely unaffected by solution changes. The observed disorder may arise from solution change-related transient growth instabilities, from transient liquid-liquid phase separation that can occur during the change, and from post-change relaxation of the lattice constant of the pre-change growth regions. These results suggest that solution variations during growth, including those typical of macroseeding, vapor-diffusion growth and other widely used techniques, may be an important source of disorder in some protein crystals.

Animals↗
PubMed1998

Continuous and discontinuous changes in the unit cell of HIV-1 reverse transcriptase crystals on dehydration.

A crystal form of HIV-1 reverse transcriptase (RT) complexed with inhibitors showed diffraction to a high-resolution limit of 3.7 A. Instability in the unit-cell dimensions of these crystals was observed during soaking experiments, but the range of this variability and consequent change in lattice order was revealed by a chance observation of dehydration. Deliberately induced dehydration results in crystals having a variety of unit cells, the best-ordered of which show diffraction to a minimum Bragg spacing of 2.2 A. In order to understand the molecular basis for this phenomenon, the initial observation of dehydration, the data sets from dehydrated crystals, the crystal packing and the domain conformation of RT are analysed in detail here. This analysis reveals that the crystals undergo remarkable changes following a variety of possible dehydration pathways: some changes occur gradually whilst others are abrupt and require significant domain rearrangements. Comparison of domain arrangements in different crystal forms gives insight into the flexibility of RT which, in turn, may reflect the internal motions allowing this therapeutically important enzyme to fulfill its biological function.

Crystallization↗
PubMed1999

Plasminogen activator inhibitor 1 (PAI-1) in its active conformation: crystallization and preliminary X-ray diffraction data.

Because of its intrinsic lability, wild-type plasminogen activator inhibitor 1 (PAI-1) cannot be crystallized in its active conformation. Therefore, a stable variant of PAI-1 was used to retain the active conformation during crystallization. Four different crystallization conditions were evaluated in detail and two major types of crystals were detected. Whereas solutions consisting of either (i) cacodylate and sodium acetate, (ii) lithium sulfate and polyethylene glycol 4K, or (iii) imidazole, sodium chloride and sodium potassium phosphate buffer revealed thin platelet crystals, a solution (iv) containing ammonium acetate, citrate and polyethylene glycol 4K appeared to enhance the formation of clustered brush-like crystals. Crystals grown under condition (iii) were found to be suitable for X-ray data collection and consequent structural investigation. Data collection was 79.8% complete with a maximum resolution of 2.92 A. Importantly, PAI-1 retained its functional properties under all conditions.

Crystallization↗
PubMed2000

Effects of microgravity on the crystal quality of a collagen-like polypeptide.

(Pro-Pro-Gly)(10) is one of the most widely studied collagen polypeptide models. Microgravity crystal growth of (Pro-Pro-Gly)(10) was carried out in the Advanced Protein Crystallization Facility aboard the Space Shuttle Discovery during the STS-95 mission. Crystals were successfully grown in all experiments, using both dialysis and free-interface diffusion methods. The quality of the microgravity-grown crystals and of ground-grown counterparts was assessed by X-ray synchrotron diffraction. Microgravity-grown crystals exhibited a significant improvement in terms of dimensions and resolution limit. As previously reported, crystals were orthorhombic, space group P2(1)2(1)2(1). However, the diffraction pattern showed weak reflections, never previously measured, that were consistent with new unit-cell parameters a = 26.9, b = 26.4, c = 182.5 A. This allowed the derivation of a new model for the arrangement of the triple-helical molecules in the crystals.

Amino Acid Sequence↗