The crystal structure of triclinic hen egg-white lysozyme (HEWL) has been refined against diffraction data extending to 0.65 A resolution measured at 100 K using synchrotron radiation. Refinement with anisotropic displacement parameters and with the removal of stereochemical restraints for the well ordered parts of the structure converged with a conventional R factor of 8.39% and an R(free) of 9.52%. The use of full-matrix refinement provided an estimate of the variances in the derived parameters. In addition to the 129-residue protein, a total of 170 water molecules, nine nitrate ions, one acetate ion and three ethylene glycol molecules were located in the electron-density map. Eight sections of the main chain and many side chains were modeled with alternate conformations. The occupancies of the water sites were refined and this step is meaningful when assessed by use of the free R factor. A detailed description and comparison of the structure are made with reference to the previously reported triclinic HEWL structures refined at 0.925 A (at the low temperature of 120 K) and at 0.95 A resolution (at room temperature).

The 19ID undulator beamline of the Structure Biology Center has been designed and built to take full advantage of the high flux, brilliance and quality of X-ray beams delivered by the Advanced Photon Source. The beamline optics are capable of delivering monochromatic X-rays with photon energies from 3.5 to 20 keV (3.5-0.6 A wavelength) with fluxes up to 8-18 x 10(12) photons s(-1) (depending on photon energy) onto cryogenically cooled crystal samples. The size of the beam (full width at half-maximum) at the sample position can be varied from 2.2 mm x 1.0 mm (horizontal x vertical, unfocused) to 0.083 mm x 0.020 mm in its fully focused configuration. Specimen-to-detector distances of between 100 mm and 1500 mm can be used. The high flexibility, inherent in the design of the optics, coupled with a kappa-geometry goniometer and beamline control software allows optimal strategies to be adopted in protein crystallographic experiments, thus maximizing the chances of their success. A large-area mosaic 3 x 3 CCD detector allows high-quality diffraction data to be measured rapidly to the crystal diffraction limits. The beamline layout and the X-ray optical and endstation components are described in detail, and the results of representative crystallographic experiments are presented.

The Structural Biology Center beamline, 19ID, has been designed to take full advantage of the highly intense undulator radiation and very low source emittance available at the Advanced Photon Source. In order to keep the X-ray beam focused onto the pre-sample slits, a novel position-sensitive PIN diode array has been developed. The array consists of four PIN diodes positioned upstream of a 0.5 microm-thick metal foil placed in the X-ray beam. Using conventional difference-over-the-sum techniques, two-dimensional position information is obtained from the metal foil fluorescence. Because the full X-ray beam passes through the metal foil, the true beam center-of-mass is measured. The device is compact, inexpensive to construct, operates in a vacuum and has a working range of 8 mm x 10 mm that can be expanded with design modifications. Measured position sensitivity is 1-2 microm. Although optimized for use in the 5-25 keV energy range, the upper limit can be extended by changing metals or adjusting foil thickness.

SummaryFlhD is a 13.3 kDa transcriptional activator protein of flagellar genes and a global regulator. FlhD activates the transcription of class II operons in the flagellar regulon when complexed with a second protein FlhC (21.5 kDa). FlhD also regulates other expression systems in Escherichia coli. We are seeking to understand this plasticity of FlhD's DNA-binding specificity and, to this end, we have determined the crystal structure of the isolated FlhD protein. The structure was solved by substituting seleno-methionine for natural sulphurmethionine in FlhD, crystallizing the protein and determining the structure factor phases by the method of multiple-energy anomalous dispersion (MAD). The FlhD protein is dimeric. The dimer is tightly coupled, with an intimate contact surface, implying that the dimer does not easily dissociate. The FlhD monomer is predominantly a-helical. The C-termini of both FlhD monomers (residues 83±116) are completely disrupted by crystal packing, implying that this region of FlhD is highly flexible. However, part of the C-terminus structure in chain A (residues 83±98) was modelled using a native FlhD crystal. What is seen in chain A suggests a classic DNA-binding, helix±turn±helix (HTH) motif. FlhD does not bind DNA by itself, so it may be that the DNA-binding HTH motif becomes rigidly defined only when FlhD forms a complex with some other protein, such as FlhC. If this were true, it might explain how FlhD exhibits plasticity in its DNA-binding specificity, as each partner protein with which it forms a complex could allosterically affect the binding specificity of its HTH motif. A disulphide bridge is seen between the unique cysteine residues (Cys-65) of FlhD native homodimers. Alanine substitution at Cys-65 does not affect FlhD transcription activator activity, suggesting that the disulphide bond is not necessary for either dimer stability or this function of FlhD. Electrostatic potential analysis indicates that dimeric FlhD has a negatively charged surface.

Recently, strategies to reduce primary radiation damage have been proposed which depend on focusing X-rays to dimensions smaller than the penetration depth of excited photoelectrons. For a line focus as used here the penetration depth is the maximum distance from the irradiated region along the X-ray polarization direction that the photoelectrons penetrate. Reported here are measurements of the penetration depth and distribution of photoelectron damage excited by 18.6 keV photons in a lysozyme crystal. The experimental results showed that the penetration depth of ~17.35 keV photoelectrons is 1.5 ± 0.2 µm, which is well below previous theoretical estimates of 2.8 µm. Such a small penetration depth raises challenging technical issues in mitigating damage by line-focus mini-beams. The optimum requirements to reduce damage in large crystals by a factor of 2.0-2.5 are Gaussian line-focus mini-beams with a root-mean-square width of 0.2 µm and a distance between lines of 2.0 µm. The use of higher energy X-rays (> 26 keV) would help to alleviate some of these requirements by more than doubling the penetration depth. It was found that the X-ray dose has a significant contribution from the crystal's solvent, which initially contained 9.0%(w/v) NaCl. The 15.8 keV photoelectrons of the Cl atoms and their accompanying 2.8 keV local dose from the decay of the resulting excited atoms more than doubles the dose deposited in the X-ray-irradiated region because of the much greater cross-section and higher energy of the excited atom, degrading the mitigation of radiation damage from 2.5 to 2.0. Eliminating heavier atoms from the solvent and data collection far from heavy-atom absorption edges will significantly improve the mitigation of damage by line-focus mini-beams.

A room-temperature determination of the absolute structure factor for the forbidden (222) reflection in silicon has been conducted at the University of Missouri Research Reactor with 103-keV gamma rays. The measured structure factor of F(222) =1.456 +0.008 is in excellent agreement with five of the earlier intensity measurements, and is significantly different from any value determined using Pendellosung techniques. An increase in accuracy over previous intensity measurements by a factor of between 2 and 10 has been achieved and is made possible through the use of monoenergetic, shortwavelength gamma rays, which allow absolute measurements to be made in Laue geometry on relatively thick crystals (-1 mm) without encountering extinction problems.

Reported here are measurements of the penetration depth and spatial distribution of photoelectron (PE) damage excited by 18.6 keV X-ray photons in a lysozyme crystal with a vertical submicrometre line-focus beam of 0.7 µm full-width half-maximum (FWHM). The experimental results determined that the penetration depth of PEs is 5 ± 0.5 µm with a monotonically decreasing spatial distribution shape, resulting in mitigation of diffraction signal damage. This does not agree with previous theoretical predication that the mitigation of damage requires a peak of damage outside the focus. A new improved calculation provides some qualitative agreement with the experimental results, but significant errors still remain. The mitigation of radiation damage by line focusing was measured experimentally by comparing the damage in the X-ray-irradiated regions of the submicrometre focus with the large-beam case under conditions of equal exposure and equal volumes of the protein crystal, and a mitigation factor of 4.4 ± 0.4 was determined. The mitigation of radiation damage is caused by spatial separation of the dominant PE radiation-damage component from the crystal region of the line-focus beam that contributes the diffraction signal. The diffraction signal is generated by coherent scattering of incident X-rays (which introduces no damage), while the overwhelming proportion of damage is caused by PE emission as X-ray photons are absorbed.

It is normally assumed that a commercial gaseous nitrogen cold‐stream provides a sample environment near 100 K and that the force of the cold‐stream does not induce movement in the sample. As might be expected, the reality is much more complex. Here, an investigation of one cold‐stream, starting with the temperature profile, is presented. Using silicon single crystals and flexible mounting loops, an approximate force/vibration profile of the cold‐stream is obtained. Results indicate that the center of the temperature profile is offset from the position suggested by the manufacturer‐supplied alignment tool and coincides with the area within the cold‐stream that has the most consistent force profile. Tests indicate that this region is only about one‐third of the width of the cold‐stream nozzle opening. To verify that the results were relevant to protein crystallographic data collection, the impact of cold‐stream position on the final data quality for lysozyme crystals was analyzed. On the basis of the observations it is recommended that users perform a temperature profile of their cold‐streams to ensure proper alignment instead of relying only on the alignment tool for setup. In addition, suggestions are made on what users can look for in data processing to identify problems with loop movement and what users can do to minimize the impact of these problems on their experiments.