X-ray diffraction images from two-dimensional position-sensitive detectors can be characterized as thick or thin, depending on whether the rotation-angle increment per image is greater than or less than the crystal mosaicity, respectively. The expectations and consequences of the processing of thick and thin images in terms of spatial overlap, saturated pixels, X-ray background andI/σ(I) are discussed. Thed*TREKsoftware suite for processing diffraction images is briefly introduced, and results fromd*TREKare compared with those from another popular package.
An important question in understanding substrate binding by proteins is how charged groups are stabilized in the absence of their solvation shell. We have addressed this question here by solving the structure of the sulphate-binding protein of Salmonella typhimurium with bound substrate at 2.0 A resolution. The results are remarkable in that the charged oxygen atoms of the sulphate molecule, which is buried and completely inaccessible to the solvent, are not stabilized by the formation of salt-bridges but by hydrogen bonds donated by specific residues of the protein. These hydrogen bonds are in turn coupled via peptide units to several resonating hydrogen bonding systems. These findings may be of general significance for the role of electrostatic interactions in protein structure and function.
Routines for crystal orientation and the prediction of expected reflections which are part of the data collection software package MADNES for area-detector diffractometer systems in macromolecular crystallography are described. This package is designed to be area-detector-system independent. In addition to refining crystal cell lengths and angles, crystal orientation, crystal-to-detector distance, position of the primary beam on the detector, and rotation of the detector around the primary beam, the orientation routine also refines the effective mosaic spread of the crystal, the beam inclination angle/~, and the detector tilt angle r. A prealignment procedure is described for rapid rough orientation of the crystal. The routines are written in Fortran 77 in a modular way, so that they may be used independently of MADNES and each other.
The lysozyme of bacteriophage T7 is a bifunctional protein that cuts amide bonds in the bacterial cell wall and binds to and inhibits transcription by T7 RNA polymerase. The structure of a mutant T7 lysozyme has been determined by x-ray crystallography and rermed at 2.2-resolution. The protein folds into an a/.ssheet structure that has a prominent cleft. A zinc atom Is located in the cleft, bound directly to three amino acids and, through a water molecule, to a fourth. Zinc is required for amidase activity but not for inhibition of T7 RNA polymerase. Aligment ofthe zinc ligands of ¶7 lysozyme with those of carboxypeptidase A and thermolysin suggests structural similarity among the catalytic sites for the amdase and these zinc proteases. Mutational analysis identified presumed catalytic residues for amidase activity within the deft and a surface that appears to be the site of bnding to ¶7 RNA polymerase. Binding of T7 RNA polymerase inhibits amidase activity.Although named for its lytic activity, T7 lysozyme also binds specifically to T7 RNA polymerase and inhibits transcription (1). This interaction provides a feedback mechanism that shuts off late transcription during 17 infection and also stimulates DNA replication (refs. 1-4; X.Z. and F.W.S., unpublished data). The ability to inhibit transcription has made 17 lysozyme widely used for controlling basal expression in a gene expression system based on T7 RNA polymerase (5, 6). ¶7 lysozyme differs from the previously well-studied egg-white and phage T4 lysozymes not only in having an interaction with T7 RNA polymerase but also in the chemistry of lysis: it cuts the amide bond between N-acetylmuramic acid and L-alanine rather than the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer of bacterial cell walls (7). We expected that knowledge ofthe structure ofT7 lysozyme would help us to understand how its dual activities are accommodated in a protein of only 150 amino acids (8).EXPERIMENTAL PROCEDURES Expression, Purification, and Crystallization. Base pairs 10,706-11,161 of T7 DNA code for T7 lysozyme (8). Sequencing oflysozyme clones revealed that nucleotides 11,061 and 11,062 of 17 DNA are TG instead of GT, a result confirmed in wild-type T7 DNA. Therefore, amino acid 118 ofT7 lysozyme is predicted to be valine instead ofglycine, as also observed in the crystal structure. An Ava II-Ase I fragment (base pairs 10,164) was cloned between the *10 promoter and To terminator for T7 RNA polymerase in the pET-3 expression vector (6) to give plasmid pAR4593, the source of wild-type lysozyme. The AK6 mutation, which deletes amino acids 2-5 of wild-type lysozyme, was made by joining a Dra I-EcoRI fragment coding for amino acids 6-150 ofT7 lysozyme (from pAR4593) to the Nhe I-EcoRI fragment that contains the T71ac promoter and slO translation start from pET-lid (6), producing pAR4617. The Nhe I site of the latter fragment was partly filled in, using G and A, and the protruding A and G were trimmed with mung bean nuclease...
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