Colin Nave

Molecular models and structural comparisons of native and mutant class I filamentous bacteriophages : Ff (fd, f1, M13), If1 and IKe

The filamentous bacteriophages are flexible rods about 1 to 2 microns long and 6 nm in diameter, with a helical shell of protein subunits surrounding a DNA core. The approximately 50-residue coat protein subunit is largely alpha-helix and the axis of the alpha-helix makes a small angle with the axis of the virion. The protein shell can be considered in three sections: the outer surface, occupied by the N-terminal region of the subunit, rich in acidic residues that interact with the surrounding solvent and give the virion a low isoelectric point; the interior of the shell, including a 19-residue stretch of apolar side-chains, where protein subunits interact mainly with each other; and the inner surface, occupied by the C-terminal region of the subunit, rich in basic residues that interact with the DNA core. The fact that virtually all protein side-chain interactions are between different subunits in the coat protein array, rather than within subunits, makes this a useful model system for studies of interactions between alpha-helix subunits in a macromolecular assembly. We describe molecular models of the class I filamentous bacteriophages. This class includes strains fd, f1, M13 (these 3 very similar strains are members of the Ff group), If1 and IKe. Our model of fd has been refined to fit quantitative X-ray fibre diffraction data to 30 A resolution in the meridional direction and 7 A resolution in the equatorial direction. A simulated 3.3 A resolution diffraction pattern from this model has the same general distribution of intensity as the experimental diffraction pattern. The observed diffraction data at 7 A resolution are fitted much better by the calculated diffraction pattern of our molecular model than by that of a model in which the alpha-helix subunit is represented by a rod of uniform density. The fact that our fd model explains the fd diffraction data is only part of our structure analysis. The atomic details of the model are supported by non-diffraction data, in part previously published and in part newly reported here. These data include information about permitted or forbidden side-chain replacements, about the effect of chemical modification, and about spectroscopic experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

The organisation of collagen fibrils in the human corneal stroma: A synchrotron X-ray diffraction study

The low angle equatorial diffraction pattern from the human corneal stroma shows that the collagen fibrils have two preferred orientations: inferior-superior and medial-lateral. We have not observed this effect in any other animal species. This arrangement, which was found to be more pronounced in the posterior than in the anterior stroma, was maintained until the last 1 to 2 millimetres before the limbus at which point uniaxial orientation was observed along the circumference. Our interpretation of this result is that most collagen fibrils wrap around the circumference of the cornea and relatively few continue radially into the limbus where uniformity of collagen fibril diameters is lost.

Towards an understanding of radiation damage in cryocooled macromolecular crystals

Interest in radiation damage is growing rapidly owing to the surge in macromolecular crystallography experiments carried out at modern brilliant synchrotron macromolecular crystallography beamlines. Work on the characterization of radiation damage in cryocooled protein crystals is starting to have some impact on our understanding of the problem and of how damage might be affecting both the process of structure solution and the actual structure obtained. A brief review of the most recent developments is given together with an assessment of the remaining problems. Although progress is being made, the understanding of radiation damage is far from complete. Methods for recognizing the damage and treating the data are being made available but they are still at an early stage of development.

Automation of the collection and processing of X-ray diffraction data - a generic approach

With modern detectors and synchrotron sources, it is now routine to collect complete data sets in 10-30 min. To make the most efficient use of these resources, it is desirable to automate the collection and processing of the diffraction data, ideally to a level at which multiple data sets can be acquired without any intervention. A scheme is described to allow fully automated data collection and processing. The design is modular, so that it can easily be interfaced with different beamline-control programs and different data-processing programs. An expert system provides a communication path between the data-processing software and the beamline-control software and takes decisions about the data collection based on project information provided by the user and experimental data provided by the data-processing program.

Pf1 Inovirus. Electron density distribution calculated by a maximum entropy algorithm from native fibre diffraction data to 3 A resolution and single isomorphous replacement data to 5 A resolution.

We have calculated the electron density distribution of the Pf1 strain of filamentous bacteriophage by a maximum entropy method. In the calculation we included native X-ray fibre diffraction data extending to 3 A resolution in the meridional direction on 60 layerlines that are resolved to 4 A in the equatorial direction, and lower resolution data from a single isomorphous derivative iodinated on the Tyr25 residue. The electron density map indicates that the 46-residue protein subunit is a single, gently curved stretch of alpha-helix with its axis at an angle of about 20 degrees to the axis of the virion. The alpha-helix subunit curves around the virion axis by about 1/6 turn, and decreases from about 27 A radius to about 13 A radius in the virion as the amino acid sequence of the subunit runs from the N terminus to the C terminus. Nearest-neighbour alpha-helical subunits are about 10 A apart along their length, and the axis of each subunit makes an unexpected negative angle with its nearest neighbours in the virion. To confirm the validity of the maximum entropy calculation, we have varied the constraints on the calculation. All variations result in either a map that is close to the original map or a map that cannot be interpreted in terms of secondary structure: we find only one map that makes structural sense.

The optimum conditions to collect X-ray data from very small samples.

A previous paper [Nave & Hill (2005). J. Synchrotron Rad. 12, 299-303] examined the possibility of reduced radiation damage for small crystals (10 microm and below in size) under conditions where the photoelectrons could escape from the sample. The conclusion of this paper was that higher-energy radiation (e.g. 40 keV) could offer an advantage as the photoelectron path length was greater and less energy would be deposited in the crystal. This paper refines these calculations further by including the effects of energy deposited owing to Compton scattering and the energy difference between the incident photon and the emitted photoelectron. An estimate is given for the optimum wavelength for collecting data from a protein crystal of a given size and composition. Another way of reducing radiation damage from a protein crystal is to collect data with a very short pulsed X-ray source where a single image can be obtained before subsequent radiation damage occurs. A comparison of this approach compared with the use of shorter wavelengths is made.

Pf1 filamentous bacteriophage: refinement of a molecular model by simulated annealing using 3.3 A resolution X-ray fibre diffraction data.

The filamentous bacteriophage Pf1 is structurally similar to the well known Ff (fd, fl, M13) strains, but it gives much better X-ray diffraction patterns, enabling a more detailed analysis of the molecular structure. The 46-residue protein subunit can be closely approximated by a single gently curved stretch of alpha-helix. The axes of the subunits are at a small angle to the virion axis, and several thousand subunits form an overlapping inter-digitated helical array surrounding a DNA core. We have derived a detailed model of the virion based on X-ray data and stereochemical constraints. We have considered potential sources of error in the diffraction data, and used the improved data to study regions where the protein subunit of Pf1 may deviate from a continuous alpha-helix. We use simulated annealing to escape from local minima, and various kinds of electron-density maps to guide the model building. Refinement of the model shows that the first few residues at the N terminus are non-helical, and there is a slight discontinuity in the alpha-helix near the middle of the sequence. The model is consistent both with general structural principles derived from high-resolution analysis of other proteins, and with specific chemical and spectroscopic data about Pf1. We apply the same refinement techniques to an alternative model with a non-helical surface loop between residues 13 and 19. Comparative analysis of models with and without a loop shows that the loop model is not supported by 3.3 A resolution X-ray diffraction data.

Radiation damage in protein crystals examined under various conditions by different methods.

Investigation of radiation damage in protein crystals has progressed in several directions over the past couple of years. There have been improvements in the basic procedures such as calibration of the incident X-ray intensity and calculation of the dose likely to be deposited in a crystal of known size and composition with this intensity. There has been increased emphasis on using additional techniques such as optical, Raman or X-ray spectroscopy to complement X-ray diffraction. Apparent discrepancies between the results of different techniques can be explained by the fact that they are sensitive to different length scales or to changes in the electronic state rather than to movement of atoms. Investigations have been carried out at room temperature as well as cryo-temperatures and, in both cases, with the introduction of potential scavenger molecules. These and other studies are leading to an overall description of the changes which can occur when a protein crystal is irradiated with X-rays at both cryo- and room temperatures. Results from crystallographic and spectroscopic radiation-damage experiments can be reconciled with other studies in the field of radiation physics and chemistry.

A Description of Imperfections in Protein Crystals

An analysis is given of the contribution of various crystal imperfections to the rocking widths of reflections and the divergence of the diffracted beams. The crystal imperfections are the angular spread of the mosaic blocks in the crystal, the size of the mosaic blocks and the variation in cell dimensions between blocks. The analysis has implications for improving crystal perfection, defining data-collection requirements and for data-processing procedures. Measurements on crystals of tetragonal lysozyme at room temperature and 100 K were made in order to illustrate how parameters describing the crystal imperfections can be obtained. At 100 K, the dominant imperfection appeared to be a variation in unit-cell dimensions in the crystal.

The Protein Information Management System (PiMS): a generic tool for any structural biology research laboratory

The Protein Information Management System (PiMS) is described together with a discussion of how its features make it well suited to laboratories of all sizes.