RESUMO
A previously described laser ultrasonic technique known as spatially resolved acoustic spectroscopy (SRAS) can be used to image surface microstructure, using the local surface acoustic wave (SAW) velocity as a contrast mechanism. It is shown here that measuring the SAW velocity in multiple directions can be used to determine the crystallographic orientation of grains. The orientations are determined by fitting experimentally measured velocities to theoretical velocities. Using this technique the orientations of 12 nickel and 3 aluminum single crystal samples have been measured, and these are compared with x-ray Laue backreflection (LBR) measurements with good agreement. The root mean square difference between SRAS and LBR measurements in terms of an R-value is less than 4.1°. The influence of systematic errors in the SAW velocity determination due to instrument miscalibration, which affects the accurate determination of the planes, is discussed. SRAS has great potential for complementary measurements or even for replacing established orientation determination and imaging techniques.
Assuntos
Alumínio/química , Cristalografia , Níquel/química , Análise Espectral , Ultrassom , Calibragem , Cristalografia/instrumentação , Cristalografia/normas , Cristalografia por Raios X , Elasticidade , Lasers , Modelos Químicos , Estrutura Molecular , Movimento (Física) , Reprodutibilidade dos Testes , Análise Espectral/instrumentação , Análise Espectral/normas , Propriedades de Superfície , Ultrassom/instrumentação , Ultrassom/normasRESUMO
In the course of a crystallographic study of the Methanosarcina mazei CorA transporter, the membrane protein was obtained with at least 95% purity and was submitted to crystallization trials. Small crystals (<100 microm) were grown that diffracted to 3.42 A resolution and belonged to space group R32, with unit-cell parameters a = b = 145.74, c = 514.0 A. After molecular-replacement attempts using available CorA structures as search models failed to yield a solution, it was discovered that the crystals consisted of an Escherichia coli contaminating protein, acriflavine resistance protein B (AcrB), that was present at less than 5% in the protein preparations. AcrB contamination is a major problem when expressing membrane proteins in E. coli since it binds naturally to immobilized metal-ion affinity chromatography (IMAC) resins. Here, the structure is compared with previously deposited AcrB structures and strategies are proposed to avoid this contamination.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Cristalografia/métodos , Cristalografia/normas , Contaminação de Medicamentos , Escherichia coli/química , Proteínas de Escherichia coli/análise , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificaçãoRESUMO
A standard sample holder and vial for cryocooled macromolecular crystals has been defined for use with robotic sample changers. This SPINE standard sample holder is a modified version, with added features and specifications, of sample holders in common use. In particular, the SPINE standard meets the precision required for automatic sample exchange and includes a cap that is identified by a two-dimensional datamatrix code as well as an optional vial. At the ESRF, the sample holder standard is in use with the EMBL/ESRF/BM14 robotic sample changer (SC3) which is installed on eight beamlines. The SC3 can hold up to 50 crystals stored in five baskets. A datamatrix reader in the SC3 ensures safe management of the sample flow and facilitates fully automatic screening and characterization of samples. Tools for handling and transporting 50 samples in a dry shipping dewar have been developed. In addition to the SC3, the SPINE sample holder is currently compatible with a number of other robotic sample changers.
Assuntos
Cristalografia/instrumentação , Automação , Cristalografia/normas , Eletrônica , Magnetismo , Padrões de Referência , Robótica , Software , SíncrotronsRESUMO
The performance of the GROMOS96 parameter set 45A3 developed for aliphatic alkanes is tested on a bilayer of dipalmitoylphosphatidylcholine (DPPC) in water in the liquid-crystalline L(alpha) phase. Variants of the force-field parameter set as well as different sets of simulation conditions or simulation parameter sets are evaluated. In the case of the force-field parameters, the van der Waals constants for the non-bonded interaction of the ester carbonyl carbon and the partial charges and charge group definition of the phosphatidylcholine head group are examined. On the methodological side, different cut-off distances for the non-bonded interactions, use of a reaction-field force due to long-range electrostatic interactions, the frequency of removal of the centre of mass motion and the strength of the coupling of the pressure of the system to the pressure bath are tested. The area per lipid, as a measure of structure, the order parameters of the chain carbons, as a measure of membrane fluidity, and the translational diffusion of the lipids in the plane of the bilayer are calculated and compared with experimental values. An optimal set of simulation parameters for which the GROMOS96 parameter set 45A3 yields a head group area, chain order parameters and a lateral diffusion coefficient in accordance with the experimental data is listed.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Cristalografia/normas , Eletroquímica/métodos , Eletroquímica/normas , Transferência Linear de Energia , Bicamadas Lipídicas/química , Modelos Moleculares , 1,2-Dipalmitoilfosfatidilcolina/normas , Simulação por Computador , Cristalografia/métodos , Campos Eletromagnéticos , Bicamadas Lipídicas/normas , Lipídeos/química , Conformação Molecular/efeitos da radiação , Movimento (Física) , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Eletricidade Estática , Estresse MecânicoRESUMO
The emergence of structure-determination initiatives that employ high-throughput protein crystallography emphasizes the need to establish quality-control methods for screening the resulting models prior to deposition with the public data banks. An in-house database of 26 new protein structures, associated diffraction data and high-quality experimentally determined electron-density maps have been used to develop (i) a set of minimal global quality criteria that a structure must meet before the refinement may be considered completed and (ii) a reliable set of indicators for detecting local errors in protein structures. These criteria have been applied to detecting local errors to a set of structures recently deposited in the Protein Data Bank and it is estimated that about 3% of amino acids are incorrectly modeled.
Assuntos
Bases de Dados de Proteínas , Proteínas/química , Software , Gestão da Qualidade Total/métodos , Cristalografia/métodos , Cristalografia/normas , Elétrons , Modelos Moleculares , Conformação Proteica , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
The resolution of images or density maps produced by electron microscopy and electron crystallography can be objectively defined in terms of the spatial frequency of the highest resolution diffraction spot, or Fourier coefficient, included in the data processing. In practice, this objective definition of resolution is expected to be too optimistic if the amplitudes of the highest resolution structure factors are too weak, if the population of high resolution reflections is too sparse, or if the signal-to-noise ratio of the high resolution data is too low. Calculated examples are presented here which illustrate how the apparent resolution in images of a membrane protein, bacteriorhodopsin, can be reduced from a nominal value of 3.5 A by weak amplitudes, sparse data or high noise levels. These calculations provide concrete examples which can serve as a guide when estimating whether the objective definition of image resolution is likely to correspond to a practical, structurally useful estimate of image resolution.
Assuntos
Cristalografia/métodos , Bacteriorodopsinas/ultraestrutura , Simulação por Computador , Cristalografia/normas , ElétronsRESUMO
We describe a technique for embedding drops of fresh synovial fluid in mounting resin to prepare test slides that retain crystal morphology for at least 2 weeks. Hospital and private rheumatologists were solicited nationwide to test themselves and their staff with 5 unknown specimens. Eighty-nine percent identified monosodium urate (MSU) crystals from a tophus but only 71% identified smaller and less frequent MSU. Seventy-five percent correctly identified calcium pyrophosphate dehydrate and 42% detected steroid crystals. Apatite crystal clumps included as control slides with no birefringent material were suspected by only 7%. This or another method which simulates actual practice by also testing the equipment and handling of the microscope is proposed as an important component of quality control testing programs for synovianalysis.
Assuntos
Cristalografia/normas , Preservação Biológica/métodos , Líquido Sinovial , Birrefringência , Fosfatos de Cálcio , Humanos , Controle de Qualidade , Ácido ÚricoRESUMO
Now that some protein X-ray structures have been proved to contain major errors, the question of the precision of 3-dimensional structures is taken seriously by crystallographers and NMR spectroscopists. Errors which cannot be avoided during model building in electron density maps, should correct themselves during crystallographic refinement, and the precision of the refined model should reach 0.15 to 0.25 A depending on the resolution of the data. Independent estimates based on homologous protein structures confirm that better than 0.5 A precision is commonly achieved, at least for C alpha and main chain atoms. The precision of NMR structures is less easily evaluated, but it should be better than 2 A when a sufficient number of NOE distance constraints are available. One may deplore the fact that not all published structures meet these standards, but possible errors should not be an excuse for not depositing atomic co-ordinates in data banks.