Eleanor J. Dodson

Overview of the CCP4 suite and current developments

An overview of the CCP4 software suite for macromolecular crystallography is given.

Efficient anisotropic refinement of macromolecular structures using FFT

This paper gives the equations for the use of fast Fourier transformations in individual atomic anisotropic refinement. Restraints on bonded atoms, on the sphericity of each atom and between non-crystallographic symmetry related atoms are described. These have been implemented in the program REFMAC and its performance with several examples is analysed. All the tests show that anisotropic refinement not only reduces the R value and Rfree but also improves the fit to geometric targets, indicating that this parameterization is valuable for improving models derived from experimental data. The computer time taken is comparable to that for isotropic refinements.

A graphical user interface to the CCP4 program suite.

CCP4i is a graphical user interface that makes running programs from the CCP4 suite simpler and quicker. It is particularly directed at inexperienced users and tightly linked to introductory and scientific documentation. It also provides a simple project-management system and visualization tools. The system is readily extensible and not specific to CCP4 software.

Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes of several bacilli by binding single-stranded RNA. The binding sequence is composed of eleven triplet repeats, predominantly GAG, separated by two or three non-conserved nucleotides. Here we present the crystal structure of a complex of TRAP and a 53-base single-stranded RNA containing eleven GAG triplets, revealing that each triplet is accommodated in a binding pocket formed by β-strands. In the complex, the RNA has an extended structure without any base-pairing and binds to the protein mostly by specific protein–base interactions. Eleven binding pockets on the circular TRAP 11-mer form a belt with a diameter of about 80 Å. This simple but elegant mechanism of arresting the RNA segment by encircling it around a protein disk is applicable to both transcription, when TRAP binds the nascent RNA, and to translation, when TRAP binds the same sequence within a non-coding leader region of the messenger RNA.

The structure of the saccharide-binding site of concanavalin A.

A complex of concanavalin A with methyl alpha‐D‐mannopyranoside has been crystallized in space group P212121 with a = 123.9 A, b = 129.1 A and c = 67.5 A. X‐ray diffraction intensities to 2.9 A resolution have been collected on a Xentronics/Nicolet area detector. The structure has been solved by molecular replacement where the starting model was based on refined coordinates of an I222 crystal of saccharide‐free concanavalin A. The structure of the saccharide complex was refined by restrained least‐squares methods to an R‐factor value of 0.19. In this crystal form, the asymmetric unit contains four protein subunits, to each of which a molecule of mannoside is bound in a shallow crevice near the surface of the protein. The methyl alpha‐D‐mannopyranoside molecule is bound in the C1 chair conformation 8.7 A from the calcium‐binding site and 12.8 A from the transition metal‐binding site. A network of seven hydrogen bonds connects oxygen atoms O‐3, O‐4, O‐5 and O‐6 of the mannoside to residues Asn14, Leu99, Tyr100, Asp208 and Arg228. O‐2 and O‐1 of the mannoside extend into the solvent. O‐2 is hydrogen‐bonded through a water molecule to an adjacent asymmetric unit. O‐1 is not involved in any hydrogen bond and there is no fixed position for its methyl substituent.

Structural Framework for DNA Translocation Via the Viral Portal Protein

Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X‐ray structure of the bacteriophage SPP1 portal protein in its isolated 13‐subunit form and the pseudoatomic structure of a 12‐subunit assembly. The first defines the DNA‐interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions. Structural observations together with geometrical constraints dictate that in the portal–DNA complex, the loops form an undulating belt that fits and tightly embraces the helical DNA, suggesting that DNA translocation is accompanied by a ‘mexican wave’ of positional and conformational changes propagating sequentially along this belt.

The crystal structure of a cyanogenic β-glucosidase from white clover, a family 1 glycosyl hydrolase

BACKGROUND beta-glucosidases occur in a variety of organisms and catalyze the hydrolysis of aryl and alkyl-beta-D-glucosides as well as glucosides with only a carbohydrate moiety (such as cellobiose). The cyanogenic beta-glucosidase from white clover (subsequently referred to as CBG) is responsible for the cleavage of cyanoglucosides. Both CBG and the cyanoglucosides occur within the plant cell wall where they are found in separate compartments and only come into contact when the leaf tissue experiences mechanical damage. This results in the eventual production of hydrogen cyanide which acts as a deterrent to grazing animals. beta-glucosidases have been assigned to particular glycosyl hydrolase families on the basis of sequence similarity; this classification has placed CBG in family 1 (there are a total of over 40 families) for which a three-dimensional structure has so far not been determined. This is the first report of the three-dimensional structure of a glycosyl hydrolase from family 1. RESULTS The crystal structure of CBG has been determined using multiple isomorphous replacement. The final model has been refined at 2.15 A resolution to an R factor of 18.9%. The overall fold of the molecule is a (beta/alpha)8 [or (alpha/beta)8] barrel (in common with a number of glycosyl hydrolases) with all residues located in a single domain. CONCLUSIONS Sequence comparisons between beta-glucosidases of the same family show that residues Glu183 and Glu397 are highly conserved. Both residues are positioned at the end of a pocket located at the C terminus of the barrel and have been assigned the respective roles of proton donor and nucleophile on the basis of inhibitor-binding and mutagenesis experiments. These roles are consistent with the environments of the two residues. The pocket itself is typical of a sugar-binding site as it contains a number of charged, aromatic and polar groups. In support of this role, we present crystallographic data on a possible product complex between CBG and glucose, resulting from co-crystallization of the native enzyme with its natural substrate, linamarin.

Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to methylation at the O6 position of guanine in DNA. O6‐methylguanine directs the incorporation of either thymine or cytosine without blocking DNA replication, resulting in GC to AT transition mutations. In prokaryotic and eukaryotic cells antimutagenic repair is effected by direct reversal of this DNA damage. A suicidal methyltransferase repair protein removes the methyl group from DNA to one of its own cysteine residues. The resulting self‐methylation of the active site cysteine renders the protein inactive. Here we report the X‐ray structure of the 19 kDa C‐terminal domain of the Escherichia coli ada gene product, the prototype of these suicidal methyltransferases. In the crystal structure the active site cysteine is buried. We propose a model for the significant conformational change that the protein must undergo in order to bind DNA and effect methyl transfer.

The crystal structure of two macrolide glycosyltransferases provides a blueprint for host cell antibiotic immunity

Glycosylation of macrolide antibiotics confers host cell immunity from endogenous and exogenous agents. The Streptomyces antibioticus glycosyltransferases, OleI and OleD, glycosylate and inactivate oleandomycin and diverse macrolides including erythromycin, respectively. The structure of these enzyme–ligand complexes, in tandem with kinetic analysis of site-directed variants, provide insight into the interaction of macrolides with their synthetic apparatus. Erythromycin binds to OleD and the 23S RNA of its target ribosome in the same conformation and, although the antibiotic contains a large number of polar groups, its interaction with these macromolecules is primarily through hydrophobic contacts. Erythromycin and oleandomycin, when bound to OleD and OleI, respectively, adopt different conformations, reflecting a subtle effect on sugar positioning by virtue of a single change in the macrolide backbone. The data reported here provide structural insight into the mechanism of resistance to both endogenous and exogenous antibiotics, and will provide a platform for the future redesign of these catalysts for antibiotic remodelling.

Penicillin V acylase crystal structure reveals new Ntn-hydrolase family members.

414 nature structural biology ¥ volume 6 number 5 ¥ may 1999 Two enzyme types, penicillin V acylases (PVA) and penicillin G acylases (PGA), with distinct substrate preferences, account for all the enzymic industrial production of 6-aminopenicillanic acid 1,2. This b-lactam compound is then elaborated into a range of semi-synthetic penicillins. Although their industrial substrates are very similar, representative examples of the two enzyme types differ widely in molecular properties. PVA from Bacillus sphaericus is tetrameric with a monomer M r of 35,000 while PGA from Escherichia coli is a heterodimer of M r 90,000. Furthermore, they have no detectable sequence homology. These differences, which exist in spite of the similarity of their industrial substrates, provoked us to determine the crystal structure of PVA to establish the nature of its catalytic mechanism and to identify any biochemical and structural relationships with PGA and other Ntn (N-terminal nucleophile) hydrolases. The PVA molecule is a well-defined tetramer with 222 organization made up of two obvious dimers (A and D) and (B and C), which generate a flat disc-like assembly (Fig. 1a). The X-ray analysis revealed that the PVA monomer contains two central anti-parallel b-sheets above and below which is a pair of anti-parallel helices (Fig. 1b). There are two extensions , one from the upper pair of helices and the other at the C-terminal segment, that interact with other monomers in the tetramer and help stabilize it. The b-sheet and helix organization and connectivity are characteristic of members of the Ntn hydrolase family, which have an N-terminal catalytic residue that is often created by autocatalytic processing 3,4. In the PVA structure, cysteine was observed as the N-terminal residue, whereas the gene sequence predicts an N-terminal sequence of Met-Leu-Gly-Cys 5. This finding shows that three amino acids are processed from the precursor N-terminus to unmask a nucleophile with a free a-amino group. Since PVA is an Ntn hydro-lase, we can deduce that the N-terminal cysteine in PVA is the catalytic residue. The PVA and PGA enzymes thus share a distinctive structural core but are otherwise unrelated in primary sequence, including the active site residue. Both PGA and PVA have approximately the same angle (+30°) between the b-strands of the two b-sheets, which are decorated by the active site residues in Ntn hydro-lases. Using these b-sheets for structural alignment reveals that the catalytic regions of PVA and PGA overlap (Fig. 1c) with a root …