James W. Pflugrath

Crystal structure of the Hhal DNA methyltransferase complexed with S-adenosyl-l-methionine

Abstract The first three-dimensional structure of a DNA methyltransferase is presented. The crystal structure of the DNA (cytosine-5)-methyltransferase, M.Hhal (recognition sequence: GCGC), complexed with S-adenosyl-l-methionine has been determined and refined at 2.5 A resolution. The core of the structure is dominated by sequence motifs conserved among all DNA (cytosine-5)-methyltransferases, and these are responsible for cofactor binding and methyltransferase function.

The 2 Å resolution structure of the sulfate-binding protein involved in active transport in Salmonella typhimurium

The crystal structure of the liganded form of the sulfate-binding protein, an initial receptor for active transport of sulfate in Salmonella typhimurium, has been solved and refined at 2.0 A resolution (1 A = 0.1 nm). The final model, which consists of 2422 non-hydrogen atoms, one sulfate substrate and 143 water molecules, yields a crystallographic R-factor of 14.0% for 16,959 reflections between 8 and 2 A. The structure deviates from ideal bond lengths and angle distances by 0.015 A and 0.037 A, respectively. The protein is ellipsoid with overall dimensions of 35 A x 35 A x 65 A and consists of two similar globular domains. The two domains are linked by three distinct peptide segments, which though widely separated in the amino acid sequence, are in close proximity in the tertiary structure. As these connecting segments are located near the periphery of the molecule, they further serve as the base or a boundary of the deep cleft formed between the two domains. Despite the unusual interdomain connectivity, both domains have similar supersecondary structure consisting of a central five-stranded beta-pleated sheet sandwiched by alpha-helices on either side. The arrangement of the two domains gives rise to the ellipsoidal shape and to the cleft between the two domains wherein the sulfate substrate is found and completely engulfed. A discovery of considerable importance is that the sulfate substrate is tightly held in place primarily by seven hydrogen bonds, five of which are donated by main-chain peptide NH groups, another by a serine hydroxyl and the last by the indole NH moiety of a tryptophan side-chain; there are no positively charged residues, nor cations, nor water molecules within van der Waals distance to the sulfate dianion. All the main-chain peptide units associated with the sulfate are in turn linked (via the peptide CO group) to arrays of hydrogen bonds. Three of these arrays are composed of alternating peptide units and hydrogen bonds within the solvent-exposed part of three alpha-helices and two are linked to a histidine and an arginine residue. The sulfate-binding protein bears strong similarity to the structures of four other periplasmic binding proteins solved in our laboratory which are specific for L-arabinose, D-galactose/D-glucose, leucine/isoleucine/valine and leucine. The similarity includes the ellipsoidal shape and the two globular domain structures, each domain consisting of a central beta-pleated sheet flanked by alpha-helices.(ABSTRACT TRUNCATED AT 400 WORDS)

CCD-based detector for protein crystallography with synchrotron X-rays☆

A detector with a 114 mm aperture, based on a charge-coupled device (CCD), has been designed for X-ray diffraction studies in protein crystallography. The detector was tested at the National Synchrotron Light Source with a beam intensity, through a 0.3 mm collimator, of greater than 10(9) X-ray photons/s. A fiberoptic taper, an image intensifier, and a lens demagnify, intensify, and focus the image onto a CCD having 512 x 512 pixels. The statistical uncertainty in the detector output was evaluated as a function of conversion gain. From this, a detective quantum efficiency (DQE) of 0.36 was derived. The dynamic range of a 4 x 4 pixel resolution element, comparable in size to a diffraction peak, was 10(4). The point-spread function shows FWHM resolution of approximately 1 pixel, where a pixel is 160-mu-m on the detector face. A data set collected from a chicken egg-white lysozyme crystal, consisting of 495 0.1-degrees frames, was processed by the MADNES data reduction program. The symmetry R-factors for the data were 3.2-3.5%. In a separate experiment a complete lysozyme data set consisting of 45 1-degrees frames was obtained in just 36 s of X-ray exposure. Diffraction images from crystals of the myosin S1 head (a = 275 angstrom) were also recorded; the Bragg spots, only 5 pixels apart, were separated but not fully resolved. Changes in the detector design that will improve the DQE and spatial resolution are outlined. The overall performance showed that this type of detector is well suited for X-ray scattering investigations with synchrotron sources.

Developments in X-ray detectors

Abstract Two types of integrating X-ray detectors, imaging plates and charge-coupled devices, represent the forefront of design in position-sensitive X-ray detectors. Their high dynamic range, spatial resolution, large active area, low noise, and ability to count at high rates make them excellent detectors for X-ray crystallography, fiber diffraction and low-angle scattering data collection.

Preliminary crystallographic data of receptors for transport and chemotaxis in Escherichia coli: d-galactose and maltose-binding proteins

Abstract We have obtained single crystals of maltose-binding protein ( M r = 40,500) and d -galactose-binding protein ( M r = 32,000), chemoreceptors for active transport and chemotaxis in Escherichia coli . This brings to a total of five the binding proteins that we have thus far crystallized; they include the l -arabinose-binding protein, the leucine, isoleucine, valine-binding protein from Escherichia coli , and a sulfate-binding protein from Salmonella typhimurium . The crystal structure of the l -arabinose-binding protein has been determined at 2.8 A resolution (Quiocho et al. , 1977 a ).

Large-aperture CCD x-ray detector for protein crystallography using a fiber-optic taper

A detector with a 114 mm aperture, based on a charge-coupled device (CCD), has been designed for x-ray diffraction studies in protein crystallography. The detector was tested on a beamline of the National Synchrotron Light Source at Brookhaven National Laboratory with a beam intensity greater than 109 x-ray photons/s. A fiber-optic taper, an image intensifier, and a lens demagnify, intensify, and focus the image onto a CCD having 512 X 512 pixels. A detective quantum efficiency (DQE) of 0.36 was obtained by evaluating the statistical uncertainty in the detector output. The dynamic range of a 4 X 4 pixel resolution element, comparable in size to a diffraction peak, was 10 4. The point-spread function shows FWHM resolution of approximately 1 pixel, where a pixel on the detector face is 160 micrometers . A complete data set, consisting of forty-five 1 degree(s) rotation frames, was obtained in just 36 s of x-ray exposure to a crystal of chicken egg-white lysozyme. In a separate experiment, a lysozyme data set consisting of 495 0.1 degree(s) frames, was processed by the MADNES data reduction program, yielding symmetry R-factors for the data of 3.2- 3.5. Diffraction images from crystals of the myosin S1 head (a equals 275 angstroms) were also recorded. The Bragg spots, only 5 pixels apart, were resolved but were not sufficiently separated to process these data. Changes in the detector design which will improve the DQE and spatial resolution are outlined. The overall performance showed that this type of detector is well suited for x-ray scattering investigations with synchrotron sources.