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PubMed1991

Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus) and herring (Clupea harengus) bone.

High-voltage (1.0 MV) electron microscopy and stereomicroscopy, electron probe microanalysis, electron diffraction and three-dimensional computer reconstruction, have been used to examine the spatial relationship between the inorganic crystals of calcium phosphate and the collagen fibrils of pickerel and herring bone. High-voltage stereo electron-micrographs were obtained of cross-sections of the cylinder-shaped intramuscular bones in uncalcified regions, in regions where only one or only several crystals had been deposited in some of the fibrils, and in successive sections containing progressively more mineral crystals until the stage of full mineralization was reached. High-resolution electron probe microanalysis confirmed that the electron-dense particles contained calcium and phosphorus. In the earliest stages of mineralization and progressing throughout the mineralization process, the crystals are located only within the collagen fibrils; crystals are not observed free in the extracellular spaces between collagen fibrils. The progressive increase in the mass of mineral deposited in the bone tissue with time occurs, essentially, completely within the collagen fibrils including the stage of full mineralization. At this stage, cross-sectional profiles of collagen fibrils are completely obliterated by mineral. A small number of crystals that are located on or close to the surface of the fibrils appear to extend a very short distance into the spaces between the fibrils. These ultrastructural observations of the very onset of calcification in which nucleation of the calcium phosphate crystals is clearly shown to begin within specific volumes of collagen fibrils, and of the subsequent temporal and spatial sequences of this phenomenon, which shows that calcification continues wholly within the collagen fibrils until maximum calcification is achieved, add important information on the basic physical chemical mechanism of the calcification and the structural elements that are involved. The spatial and temporal independence of the sites where mineralization is initiated establishes that such ultrastructural locations within individual collagen fibrils represent independent, physical chemical nucleation loci. The findings are totally inconsistent with the proposal that crystals must first be deposited in matrix vesicles, or other components such as mitochondria, and subsequently released and propagated in the interfibrillar space, until they eventually reach and impregnate the hole zone regions of the collagen fibrils. Three-dimensional computer reconstruction of serial transverse and longitudinal sections demonstrates periodic swellings along the collagen fibrils, corresponding to the hole zone region of their axial period as mineralization proceeds.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1982

A new approach for the visualization of molecular arrangement in biological micro-crystals.

Biological molecules often form micro-crystals that are in a size range that is unsuited for study either by X-ray crystallography or by electron microscopy. The method described here allows the use of most of these crystals. It consists of cutting sections through the crystal deposited on a grid after a preliminary observation in the electron microscope to determine its precise location. It allows visualization of the crystal molecular packing in any plane normal to the grid plane at the resolution usually obtained by negative staining. If the crystal is thin enough, two orthogonal views of the same crystal can be obtained. The orientation and the position of the sections along the crystal axis are precisely determined using latex particles as reference points. Crystals as small as 1 micrometer in length can be studied.

Biopolymers↗
PubMed1993

Low-dose thickness measurement of glucose-embedded protein crystals by electron energy loss spectroscopy and STEM dark-field imaging.

Electron energy loss spectroscopy and dark-field imaging in a scanning transmission electron microscope were used to determine the thickness of glucose-embedded crotoxin complex crystals. The results demonstrate the feasibility of identifying protein crystals with a thickness of half a unit cell (12.8 nm) under low-dose and low-temperature conditions. The accuracy of this method is limited by the amount of surface coating of the crystal's embedding glucose used for preserving the high-resolution structure of the protein. The histogram of the crystal thickness distribution and the spread of the anticipated crystal thickness allow us to make an estimate of the uncertainty in the glucose layer thickness. This approach can be incorporated as part of the experimental procedure in the three-dimensional data collection for structure determination of protein crystals with variable thicknesses. The measurement can be done on areas approximately 200 nm in diameter so that crystals of suitable thickness can be pre-selected before the high-resolution data is recorded. Accurate determination of the crystal thickness will optimize the data collection efficiency by avoiding the collection and subsequent analysis of unmatchable data for the three-dimensional reconstruction.

Crotoxin↗
PubMed1996

Activating piezoelectric crystal surface by silanization for microgravimetric immunobiosensor application.

The development of a microgravimetric immunobiosensor using a piezoelectric quartz crystal as a detector requires a stable and reproducible immobilization method for ligand binding. The method of silanization using 3-aminopropyltriethoxysilane (APTES) has been widely used for activating the carrier surface. In the present study, APTES deposition on a piezoelectric crystal surface was studied under various solvent conditions. A fluorescence method, using fluorescence isothiocyanate as a dye, was demonstrated for the quantification of amino groups on the silanized piezoelectric crystal surface. The optimum binding conditions of APTES deposition on a piezoelectric crystal surface were incorporated for the covalent immobilization of protein on the crystal surface in developing a stable and sensitive microgravimetric immunobiosensor. Determination of immunoglobulin G (IgG) concentration was performed using APTES modified piezoelectric crystals coated with protein G. The resonant frequency shift, resulting from the formation of protein G-IgG complex on the crystal surface, correlated with the concentration of IgG in the range 10 ng/ml to 0.1 mg/ml. The APTES modified, protein G coated crystal were found to be quite stable and did not show a significant loss of sensitivity even after 12 weeks of storage at 4 degrees C in a desiccator.

Biosensing Techniques↗
PubMed2006

Increasing use and associated harms of crystal methamphetamine injection in a Canadian setting.

BACKGROUND: There is a growing concern surrounding crystal methamphetamine use in Canada despite surprisingly little empirical data to support such claims. We evaluated the trends in crystal methamphetamine injection and factors associated with injection of the drug among a cohort of injection drug users (IDU) in Vancouver. METHODS: We conducted a prospective analysis of factors associated with crystal methamphetamine injection among participants enrolled in the Vancouver Injection Drug Users Study (VIDUS). Since serial measures for each individual were available, variables potentially associated with crystal methamphetamine injection were evaluated using generalized estimating equations (GEE) with logit link for binary outcomes. RESULTS: Overall, 1587 IDU were enrolled into the VIDUS cohort between May 1996 and December 2004. The proportion of IDU who reported injecting crystal methamphetamine during the last 6 months increased during the study period (Cochran-Armitage trend test, p<0.001). In multivariate GEE analyses, crystal methamphetamine injection was independently associated with younger age (adjusted odds ratio [AOR]: 4.77, 95% confidence interval [CI]=3.40-6.70), Caucasian ethnicity (AOR=2.21, 95% CI=1.57-3.12), syringe borrowing (AOR=1.62, 95% CI=1.22-2.13), and syringe lending (AOR=1.40, 95% CI=1.02-1.86). INTERPRETATION: There was a significant trend towards increasing crystal methamphetamine injection in this setting and elevated HIV risk behavior and younger age were independently associated with crystal methamphetamine injection. Given that banning precursor chemicals has had a limited effect on reducing methamphetamine supply in other jurisdictions, pragmatic and effective interventions are needed to address the growing use of this drug.

Adult↗
PubMed2006

Relationship between bone tissue strain and lattice strain of HAp crystals in bovine cortical bone under tensile loading.

Cortical bone is a composite material composed of hydroxyapatite (HAp) and collagen. As HAp is a crystalline structure, an X-ray diffraction method is available to measure the strain of HAp crystals. However, HAp crystals in bone tissue have been known to have the low degree of crystallization. Authors have proposed an X-ray diffraction method to measure the lattice strain of HAp crystals from the diffusive intensity profile due to low crystallinity. The precision of strain measurement was greatly improved by this method. In order to confirm the possibility of estimating the bone tissue strain with measurements of the strain of HAp crystals, this work investigates the relationship between bone tissue strain on a macroscopic scale and the lattice strain of HAp crystals on a microscopic scale. The X-ray diffraction experiments were performed under tensile loading. Strip bone specimens of 40x6x0.8mm in size were cut from the cortical region of a shaft of bovine femur. A stepwise tensile load was applied in the longitudinal direction of the specimen. By detecting the diffracted X-ray beam transmitted through the specimen, the lattice strain was directly measured in the loading direction. As a result, the lattice strain of HAp crystals showed lower value than the bone tissue strain measured by a strain gage. The bone tissue strain was described with the mean lattice strain of the HAp crystals and the elastic modulus.

Animals↗
PubMed2004

Development of one-dimensional nanostructures through the crystallization of amorphous colloids.

In this paper we have demonstrated that the crystallization method of amorphous colloids is convenient and feasible in the large-scale production of one-dimensional (1D) nanostructures. For the crystals with highly anisotropic structures, such as orthorhombic, trigonal, and hexagonal crystals, the crystallization generally tends to occur along the (001) axis. The preparation of orthorhombic bismuth sulfide (Bi2S3) nanorods and trigonal selenium ( t-Se) nanowires by the crystallization route was used as typical examples to illustrate the process and mechanism of crystallization. The as-prepared products were characterized with transmission electron microscopy, field-emission scanning electron microscopy, X-ray diffraction, and selected area electron diffraction. Additionally, the detailed crystal growth processes involved in the crystallization of amorphous Bi2S3 colloid were investigated by studying the morphology and structure of intermediates. It demonstrates that the growth of the nanorods is through two key steps: (1) the formation of multiple activated sites on the surface of spherical Bi2S3 colloid and (2) the subsequent preferential growth along these sites.

Journal Article↗
PubMed2004

Crystal structure of calf spleen purine nucleoside phosphorylase with two full trimers in the asymmetric unit: important implications for the mechanism of catalysis.

The crystal structure of the binary complex of trimeric purine nucleoside phosphorylase (PNP) from calf spleen with the acyclic nucleoside phosphonate inhibitor 2,6-diamino-(S)-9-[2-(phosphonomethoxy)propyl]purine ((S)-PMPDAP) is determined at 2.3A resolution in space group P2(1)2(1)2(1). Crystallization in this space group, which is observed for the first time with a calf spleen PNP crystal structure, is obtained in the presence of calcium atoms. In contrast to the previously described cubic space group P2(1)3, two independent trimers are observed in the asymmetric unit, hence possible differences between monomers forming the biologically active trimer could be detected, if present. Such differences would be expected due to third-of-the-sites binding documented for transition-state events and inhibitors. However, no differences are noted, and binding stoichiometry of three inhibitor molecules per enzyme trimer is observed in the crystal structure, and in the parallel solution studies using isothermal titration calorimetry and spectrofluorimetric titrations. Presence of phosphate was shown to modify binding stoichiometry of hypoxanthine. Therefore, the enzyme was also crystallized in space group P2(1)2(1)2(1) in the presence of (S)-PMPDAP and phosphate, and the resulting structure of the binary PNP/(S)-PMPDAP complex was refined at 2.05A resolution. No qualitative differences between complexes obtained with and without the presence of phosphate were detected, except for the hydrogen bond contact of Arg84 and a phosphonate group, which is observed only in the former complex in three out of six independent monomers. Possible hydrogen bonds observed in the enzyme complexed with (S)-PMPDAP, in particular a putative hydrogen bonding contact N(1)-H cdots, three dots, centered Glu201, indicate that the inhibitor binds in a tautomeric or ionic form in which position N(1) acts as a hydrogen bond donor. This points to a crucial role of this hydrogen bond in defining specificity of trimeric PNPs and is in line with the proposed mechanism of catalysis in which this contact helps to stabilize the negative charge that accumulates on O(6) of the purine base in the transition state. In the present crystal structure the loop between Thr60 and Ala65 was found in a different conformation than that observed in crystal structures of trimeric PNPs up to now. Due to this change a new wide entrance is opened into the active site pocket, which is otherwise buried in the interior of the protein. Hence, our present crystal structure provides no obvious indication for obligatory binding of one of the substrates before binding of a second one; it is rather consistent with random binding of substrates. All these results provide new data for clarifying the mechanism of catalysis and give reasons for the non-Michaelis kinetics of trimeric PNPs.

Adenine↗
PubMed2004

Conformational flexibility of the C terminus with implications for substrate binding and catalysis revealed in a new crystal form of deacetoxycephalosporin C synthase.

Deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus catalyses the oxidative ring expansion of the penicillin nucleus into the nucleus of cephalosporins. The reaction requires dioxygen and 2-oxoglutarate as co-substrates to create a reactive iron-oxygen intermediate from a ferrous iron in the active site. The active enzyme is monomeric in solution. The structure of DAOCS was determined earlier from merohedrally twinned crystals where the last four C-terminal residues (308-311) of one molecule penetrate the active site of a neighbouring molecule, creating a cyclic trimeric structure in the crystal. Shortening the polypeptide chain from the C terminus by more than four residues diminishes activity. Here, we describe a new crystal form of DAOCS in which trimer formation is broken and the C-terminal arm is free. These crystals show no signs of twinning, and were obtained from DAOCS labelled with an N-terminal His-tag. The modified DAOCS is catalytically active. The free C-terminal arm protrudes into the solvent, and the C-terminal domain (residues 268-299) is rotated by about 16 degrees towards the active site. The last 12 residues (300-311) are disordered. Structures for various enzyme-substrate and enzyme-product complexes in the new crystal form confirm overlapping binding sites for penicillin and 2-oxoglutarate. The results support the notion that 2-oxoglutarate and dioxygen need to react first to produce an oxidizing iron species, followed by reaction with the penicillin substrate. The position of the penicillin nucleus is topologically similar in the two crystal forms, but the penicillin side-chain in the new non-twinned crystals overlaps with the position of residues 304-306 of the C-terminal arm in the twinned crystals. An analysis of the interactions between the C-terminal region and residues in the active site indicates that DAOCS could also accept polypeptide chains as ligands, and these could bind near the iron.

Amino Acid Sequence↗
PubMed2005

The limit of accuracy of protein modeling: influence of crystal packing on protein structure.

The size of the protein database (PDB) makes it now feasible to arrive at statistical conclusions regarding structural effects of crystal packing. These effects are relevant for setting upper practical limits of accuracy on protein modeling. Proteins whose crystals have more than one molecule in the asymmetric unit or whose structures were determined at least twice by X-ray crystallography were paired and their differences analyzed. We demonstrate a clear influence of crystal environment on protein structure, including backbone conformations, hinge-like motions and side-chain conformations. The positions of surface water molecules tend to be variable in different crystal environments while those of ligands are not. Structures determined by independent groups vary more than structures determined by the same authors. The use of different refinement methods is a major source for this effect. Our pair-wise analysis derives a practical limit to the accuracy of protein modeling. For different crystal forms, the limit of accuracy (C(alpha), root-mean-square deviation (RMSD)) is approximately 0.8A for the entire protein, which includes approximately 0.3A due to crystal packing. For organized secondary elements, the upper limit of C(alpha) RMSD is 0.5-0.6A while for loops or protein surface it reaches 1.0A. Twenty percent of exposed side- chains exhibit different chi(1+2) conformations with approximately half of the effect also resulting from crystal packing. A web based tool for analysis and graphic presentation of surface areas of crystal contacts is available (http://ligin.weizmann.ac.il/cryco).

Computational Biology↗
PubMed2006

Comparison of characteristics of patients with and without calcium pyrophosphate dihydrate crystal deposition disease who underwent total knee replacement surgery for osteoarthritis.

OBJECTIVE: To compare characteristics of patients with severe osteoarthritis with and without calcium pyrophosphate dihydrate (CPPD) crystal deposition disease. STUDY METHODS: Patients undergoing total knee replacement surgery participated in this study and completed questionnaires. Radiographs of the index knee (extended anteroposterior, lateral and skyline) were reviewed for the presence of chondrocalcinosis. Synovial fluids were obtained during surgery and analyzed under compensated polarized light microscopy. RESULTS: The presence of CPPD crystals was identified in 52.9% of 102 patients. The use of both radiographs and synovial fluid analysis increased the identification of crystals. There was no difference in the following characteristics of the patients with and without CPPD crystals: age of pain onset, gender, difficulty in performing daily functions (including cooking, standing up from chairs, using restroom, going upstairs, and going shopping), history of previous joint inflammation, use of walking aids, and number and types of medications ever used. CPPD patients underwent knee arthroplasty at older age compared to non-CPPD patients (70.3+/-6.37 and 67.5+/-7.15 years old, respectively) (P = 0.037). All but one CPPD patients were unaware of the presence of crystals. CONCLUSION: High prevalence of CPPD crystals was found in patients undergoing total knee replacement surgery. All but one CPPD patients were unaware of calcium deposition in the index joints. Patients with these crystals experienced similar difficulties in performing daily activities and received similar treatment to patients without CPPD crystals. CPPD patients did not undergo knee arthroplasty at earlier age than non-CPPD patients.

Aged↗
PubMed2004

Elevated silver, barium and strontium in antlers, vegetation and soils sourced from CWD cluster areas: do Ag/Ba/Sr piezoelectric crystals represent the transmissible pathogenic agent in TSEs?

High levels of Silver (Ag), Barium (Ba) and Strontium (Sr) and low levels of copper (Cu) have been measured in the antlers, soils and pastures of the deer that are thriving in the chronic wasting disease (CWD) cluster zones in North America in relation to the areas where CWD and other transmissible spongiform encephalopathies (TSEs) have not been reported. The elevations of Ag, Ba and Sr were thought to originate from both natural geochemical and artificial pollutant sources--stemming from the common practise of aerial spraying with 'cloud seeding' Ag or Ba crystal nuclei for rain making in these drought prone areas of North America, the atmospheric spraying with Ba based aerosols for enhancing/refracting radar and radio signal communications as well as the spreading of waste Ba drilling mud from the local oil/gas well industry across pastureland. These metals have subsequently bioconcentrated up the foodchain and into the mammals who are dependent upon the local Cu deficient ecosystems. A dual eco-prerequisite theory is proposed on the aetiology of TSEs which is based upon an Ag, Ba, Sr or Mn replacement binding at the vacant Cu/Zn domains on the cellular prion protein (PrP)/sulphated proteoglycan molecules which impairs the capacities of the brain to protect itself against incoming shockbursts of sound and light energy. Ag/Ba/Sr chelation of free sulphur within the biosystem inhibits the viable synthesis of the sulphur dependent proteoglycans, which results in the overall collapse of the Cu mediated conduction of electric signals along the PrP-proteoglycan signalling pathways; ultimately disrupting GABA type inhibitory currents at the synapses/end plates of the auditory/circadian regulated circuitry, as well as disrupting proteoglycan co-regulation of the growth factor signalling systems which maintain the structural integrity of the nervous system. The resulting Ag, Ba, Sr or Mn based compounds seed piezoelectric crystals which incorporate PrP and ferritin into their structure. These ferrimagnetically ordered crystals multireplicate and choke up the PrP-proteoglycan conduits of electrical conduction throughout the CNS. The second stage of pathogenesis comes into play when the pressure energy from incoming shock bursts of low frequency acoustic waves from low fly jets, explosions, earthquakes, etc. (a key eco-characteristic of TSE cluster environments) are absorbed by the rogue 'piezoelectric' crystals, which duly convert the mechanical pressure energy into an electrical energy which accumulates in the crystal-PrP-ferritin aggregates (the fibrils) until a point of 'saturation polarization' is reached. Magnetic fields are generated on the crystal surface, which initiate chain reactions of deleterious free radical mediated spongiform neurodegeneration in surrounding tissues. Since Ag, Ba, Sr or Mn based piezoelectric crystals are heat resistant and carry a magnetic field inducing pathogenic capacity, it is proposed that these ferroelectric crystal pollutants represent the transmissible, pathogenic agents that initiate TSE.

Acoustic Stimulation↗
PubMed2006

Purification, crystallization and preliminary X-ray diffraction analysis of the phage T4 vertex protein gp24 and its mutant forms.

The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6A resolution compared to wild-type gp24 at 3.80A resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.

Amino Acid Substitution↗
PubMed2003

Frequency of biliary crystals in patients with suspected sphincter of Oddi dysfunction.

BACKGROUND: The passage of gallstones (macro- or microlithiasis) is theorized to play a role in inducing sphincter of Oddi dysfunction. This study examined the frequency at which biliary crystals are found in patients with suspected type II and type III sphincter of Oddi dysfunction. METHODS: A total of 85 patients (66 women, 19 men; mean age 38 years) with unexplained abdominal pain of suspected pancreatobiliary origin and no prior episode of pancreatitis underwent ERCP with sphincter of Oddi manometry and bile collection for crystal analysis. Eighty-one patients had a gallbladder in situ. No patient had evidence of stones or sludge on prior abdominal imaging. Sphincter of Oddi manometry was performed in standard retrograde fashion by using an aspirating catheter. Patients were classified by sphincter of Oddi dysfunction type by using a modified Hogan-Geenen classification system. Patients with type I sphincter of Oddi dysfunction were excluded. Bile was collected directly from the gallbladder (n=23) or common bile duct (n=62) after an infusion of 3.5 microg of cholecystokinin and was examined by light and polarizing microscopy for cholesterol crystals or calcium bilirubinate granules. RESULTS: The proportion of patients with crystals was 3.5% (3/85). Thirty-five patients (41%) had elevated biliary and/or pancreatic sphincter pressure (type II, 16; type III, 19), of whom one (3%) had cholesterol crystals. Fifty patients had normal sphincter pressure, of whom two (4%) had cholesterol crystals (p=0.6). All 3 patients with cholesterol crystals had a gallbladder in situ. Calcium bilirubinate granules were not found in any patient. CONCLUSIONS: Microlithiasis appears to be rare in patients suspected to have type II or type III sphincter of Oddi dysfunction. Evaluation of bile for crystals appears unproductive in this group of patients.

Adolescent↗
PubMed1986

Crystal-matrix interrelations in brushite and uric acid calculi.

Brushite and uric acid calculi were studied by means of scanning electron microscopy with the partial dissolution method and transmission electron microscopy. Brushite calculi consist of radially oriented columnar crystals which have sheet-like substructure. The organic matrix is identified chiefly at the outside of the crystals but partly included between the substructure. The concentric matrix bands are often dislocated between the neighbouring crystals. Uric acid calculi also consist of radially oriented columnar crystals, and a fine meshwork of the organic matrix is incorporated within the crystals. The concentric matrix layers of different density are angled according to the crystal lattice. These findings indicate that the organic matrix arose from a mucinous surface coat, at least in the radially striated calculi. The crystals continued to grow in this gel-state milieu, either thrusting the matrix aside or incorporating it within the crystals.

Calcium Oxalate↗
PubMed1998

X-ray studies on cross-linked lysozyme crystals in acetonitrile-water mixture.

Tetragonal crystals of hen egg white lysozyme were cross-linked and subjected to X-ray diffraction study in acetonitrile-water media with different acetonitrile concentrations. Crystals in neat acetonitrile did not scatter X-ray well. Structures of crystals in neat water, in 90% and 95% acetonitrile, and crystal back-soaked from acetonitrile to water, were determined to about 2 A resolution. For crystals in both 90% acetonitrile, and crystal back-soaked from acetonitrile to water, were determined to about 2 A resolution. For crystals in both 90% and 95% acetonitrile, only one protein-bond acetonitrile molecule is found in the active site cleft, and its location and binding-protein mode is similar to the C subunit of polysaccharide. The alteration in conformation and hydrogen-bond pattern involving water as solvent causes the reduction of the protein's flexibility in organic media. The back-soaked crystal regained its ordinary three-dimensional structure in water.

Acetonitriles↗
PubMed2000

AFM characterization of domain structure of ferroelectric TGS crystals on a nanoscale

Atomic force microscopy has been used for the study of the surface topography and domain structures of triglycin sulfate (TGS) crystals. The images of various types of domains at the polar (0 1 0) surfaces of as-cleaved TGS crystals have been obtained (in contact mode and resonant mode). The crystals grown old, after annealing, gamma-irradiated by various doses, were studied. It has been found that the surface relief of crystals after annealing differs from that of crystals grown old and irradiated: in the first case the peak-to-peak value is nearly constant whereas in the second case it varies in wide limits. The parameters of domain structure were determined. These parameters have been shown to be different for crystals grown old, crystals after annealing and irradiated crystals. It has been shown that, after cantilever scanning after a long period in different AFM modes the TGS surface experiences a partial polarization reversal.

Journal Article↗
PubMed2000

Atomic force microscopy of the three-dimensional crystal of membrane protein, OmpC porin.

Three-dimensional microcrystals of OmpC osmoporin were air-dried slowly and imaged in air with an atomic force microscope (AFM). The overall structural features in AFM images are in good agreement with the X-ray diffraction data of these OmpC osmoporin crystals: monoclinic P2(1) with the unit cell constants a=117.6 Å, b=110 Å, c=298.4 Å, beta=97 degrees. Such a good correspondence between X-ray diffraction and AFM data suggests that the slow and mild air-drying of these crystals did not induce any significant alterations in the crystal lattices as expected upon crystal dehydration. At the (100) crystal face, individual trimeric protein-detergent complexes were resolved. These results show the potential for studying the molecular structure of microcrystals of integral membrane proteins. This study also suggests that the crystal grew in a fashion of rapid two-dimensional expansion along the bc plane followed by a slow deposition along the a axis, perhaps as a rate-limiting nucleation process. Thus, AFM imaging of air-dried crystals would also be of considerable use in the early stages of a project to grow large three-dimensional crystals of membrane proteins suitable for high-resolution X-ray diffraction studies.

Journal Article↗