John R. Ruble

Lower dimer impurity incorporation may result in higher perfection of HEWL crystals grown in microgravity: A case study

Crystals of tetragonal hen egg white lysozyme (HEWL) grown on a series of space missions and their terrestrial counterparts were analyzed by gel electrophoresis and X-ray diffraction. The crystals were produced by vapor-diffusion and dialysis methods. The microgravity and terrestrial grown HEWL crystals were found to have effective partitioning coefficients (K eff ) for an oxidatively formed covalent dimer impurity (MW 28 K) of 2 and 9, respectively, i.e. the latter contain 4.5 times more dimers. The microgravity grown crystals allowed the collection of 24% more useful reflections and improved the resolution from 1.6 to 1.35 A. Other improvements were also noted including lower isotropic B-factors of 16.9. versus 23.8 A 2 for their terrestrial counterparts. High-resolution laser interferometry was applied quantitatively to evaluate the influence of dimer impurity on growth kinetics. It is shown that the growth of the (1 0 1) face from solution into which I % dimers were introduced decelerates with increasing solution flow rate and the growth stops at a flow rate of about 0.2 mm/s. This effect occurs faster than in ultrapure solutions. The covalently bound dimers essentially increase the amplitudes of the striation-inducing growth rate fluctuations. The effect is ascribed to the enhanced transport of growth inhibiting HEWL dimer to the interface. Theoretical analysis shows that a stagnant solution around a growing crystal is strongly depleted with respect to impurity by about 60% for the measured growth parameters as compared to the solution bulk. Thus, a crystal in microgravity grows from essentially purer solution than the ones in the presence of convection flows. Therefore, it traps less stress inducing impurity and should be more perfect. For crystal/impurity systems where K eff is small enough microgravity should have an opposite effect.

Structural studies of several clinically important oncology drugs in complex with human serum albumin.

BACKGROUNDnSerum albumin is a major pharmacokinetic effector of drugs. To gain further insight into albumin binding chemistry, the crystal structures of six oncology agents were determined in complex with human serum albumin at resolutions of 2.8 to 2.0Å: camptothecin, 9-amino-camptothecin, etoposide, teniposide, bicalutamide and idarubicin.nnnMETHODSnProtein crystal growth and low temperature X-ray crystallographynnnRESULTSnThese large, complex drugs are all bound within the subdomain IB binding region which can be described as a hydrophobic groove formed by α-helices h7, h8 and h9 covered by the extended polypeptide L1. L1 creates a binding cavity with two access sites, one between loop L1 and α-helices h7 and h8 (distal site: IBd) and the other between L1 and α-helix h9 (proximal site: IBp). Camptothecin (2.4Å) and 9 amino camptothecin (2.0Å) are clearly bound as the open lactone form (IBp). Idarubicin (2.8Å) binds in a DNA like dimer complex via an intermolecular π stacking arrangement in IBd. Bicalutamide (2.4Å) is bound in a folded intramolecular π stacking arrangement between two aromatic rings in IBd similar to idarubicin. Teniposide (2.7Å) and etoposide (2.7Å), despite small chemical differences, are bound in two distinctly different sites at or near IB. Teniposide is internalized via primarily hydrophobic interactions and spans through both openings (IBp-d). Etoposide is bound between the exterior of IB and IIA and exhibits an extensive hydrogen bonding network.nnnCONCLUSIONSnSubdomain IB is a major binding site for complex heterocyclic molecules.nnnGENERAL SIGNIFICANCEnThe structures have important implications for drug design and development. This article is part of a Special Issue entitled Serum Albumin.

Structure of human ferritin L chain

Ferritin is the major iron-storage protein present in all cells. It generally contains 24 subunits, with different ratios of heavy chain (H) to light chain (L), in the shape of a hollow sphere hosting up to 4500 ferric Fe atoms inside. H-rich ferritins catalyse the oxidation of iron(II), while L-rich ferritins promote the nucleation and storage of iron(III). Several X-ray structures have been determined, including those of L-chain ferritins from horse spleen (HoSF), recombinant L-chain ferritins from horse (HoLF), mouse (MoLF) and bullfrog (BfLF) as well as recombinant human H-chain ferritin (HuHF). Here, structures have been determined of two crystal forms of recombinant human L-chain ferritin (HuLF) obtained from native and perdeuterated proteins. The structures show a cluster of acidic residues at the ferrihydrite nucleation site and at the iron channel along the threefold axis. An ordered Cd2+ structure is observed within the iron channel, offering further insight into the route and mechanism of iron transport into the capsid. The loop between helices D and E, which is disordered in many other L-chain structures, is clearly visible in these two structures. The crystals generated from perdeuterated HuLF will be used for neutron diffraction studies.

Diffusion-controlled crystallization apparatus for microgravity (DCAM): flight and ground-based applications

Abstract A versatile microdialysis counterdiffusion apparatus was developed in response to special exploratory opportunities for long-duration protein crystal growth experiments presented by the US and Russian Space Program. Challenged to reduce man-tended dependence during this phase, the hardware was designed to eliminate in-flight activation or deactivation. In addition, a disposable interface was incorporated in the design to allow for improved logistics and handling during the pre-flight loading and post-flight distribution of the flight samples for each experiment. The hardware is referred to as the diffusion-controlled crystallization apparatus for microgravity or DCAM (pronounced “dee-cam”) which utilizes the dialysis method and allows the equilibration rate of each individual experiment to be passively controlled from several days to several months. Precision control of the rate of approach to supersaturation has routinely produced macro (5xa0mm to 1.25xa0cm) crystals for a variety of proteins in this hardware. A description of the hardware and preliminary results from a series of US Shuttle/Mir flight experiments are presented.

PCAM: a multi-user facility-based protein crystallization apparatus for microgravity

A facility-based protein crystallization apparatus for microgravity (PCAM) has been constructed and flown on a series of Space Shuttle Missions. The hardware development was undertaken largely because of the many important examples of quality improvements gained from crystal growth in the diffusion-limited environment in space. The concept was based on the adaptation for microgravity of a commonly available crystallization tray to increase sample density, to facilitate co-investigator participation and to improve flight logistics and handling. A co-investigator group representing scientists from industry, academia, and government laboratories has been established. Microgravity applications of the hardware have produced improvements in a number of structure-based crystallographic studies and include examples of enabling research. Additionally, the facility has been used to support fundamental research in protein crystal growth which has delineated factors contributing to the effect of microgravity on the growth and quality of protein crystals.

Polycapillary optic–source combinations for protein crystallography

The work in this paper is a systematic study of the application of collimating and slightly focusing polycapillary optics to the X-ray crystallographic structure determination of egg-white lysozyme using two different sources: a standard rotating anode source and a low-power table-top microfocusing source. For the rotating anode source, a series of measurements comparing duplicate data sets obtained from the same individual crystal are summarized. Intensity and data quality are discussed for measurements with a pinhole collimator, a collimating polycapillary optic and a focusing polycapillary optic. The collected data were analyzed using conventional analysis software; limitations of the use of conventional analysis software for focused beams are discussed. Two data sets were collected using the low-power source and collimating optics, and three data sets using a lower-power source and focusing optics with three different limiting apertures.

Neutron structure of monoclinic lysozyme crystals produced in microgravity

Crystals of the monoclinic form of lysozyme (space group P2(1) with a = 28.00 Angstrom, b = 62.88 Angstrom, c = 60.30 Angstrom, beta = 90.68 degrees) were grown under microgravity conditions on the Mir station and brought back to earth on the US Space Shuttle. Counter-diffusion methods developed specifically for application in microgravity have been utilized to produce several examples of macroscopic crystals. Large crystals are of great importance for neutron diffraction studies of bio-macromolecules, which can reveal key details of the hydrogen atom structure of biological molecules at medium resolution. The structure of the monoclinic crystal form described here has been determined to 2.1 Angstrom by neutron diffraction and contains two molecules in the asymmetric unit. Details of the structure and refinement are presented

Convergent-beam method in macromolecular crystallography

A data-collection method for macromolecular crystals using convergent sources is described here. Because of the unique characteristics of the diffraction patterns, a software package CBMPRO has been developed specifically for processing data images collected with the convergent beam method (CBM). The resulting data sets from crystals with two different sets of unit-cell parameters are presented and compared. There is good agreement between data sets from the same type of crystals under slightly different experimental conditions and data sets collected and processed with CBM also agree well with those from conventional oscillation methods, marking an important step to establishing CBM as a viable alternate data-collection method for macromolecular crystals.

Low-power protein crystallography using polycapillary optics

This paper describes the design and performance of a low power protein crystallography system using polycapillary optics. The characterization of the source and polycapillary optics are presented. Three optic types: collimating, slightly focusing and strongly focusing optics have been used in low power source-optic combination systems. The source-collimating optic and source- slightly focusing optic systems were used to collect data sets for chicken egg-white Lysozyme with conventional sample oscillation during data collection. The data sets show high quality by analysis with a conventional software package, DENZO. Still diffraction patterns without oscillation have also been obtained by a low power source-strongly focusing optic combination. These patterns have been analyzed by developing software for processing diffraction patterns obtained with strongly convergent X-ray beams. The directions of future work and system improvements are also discussed.

A simple method for motorized alignment of Osmic confocal optics

A system for the remote alignment of X-ray optics has been designed to address safety considerations related to manual optic adjustment. The described system also addresses some of the common problems associated with the alignment process, such as binding of components and the effects of external forces due to user contact that lead to irreproducibility of the process. The system as described should be applicable to most home laboratories.