X-ray or gamma ray systems or devices – Specific application – Diffraction – reflection – or scattering analysis
Reexamination Certificate
2002-05-10
2003-08-19
Dunn, Drew A. (Department: 2882)
X-ray or gamma ray systems or devices
Specific application
Diffraction, reflection, or scattering analysis
C378S208000
Reexamination Certificate
active
06608883
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to X-ray crystallography, and, in particular, to methods and apparatus for mounting and aligning of samples for X-ray crystallographic analysis.
2. Discussion of the Art
X-ray crystallography is an established, well-studied technique for providing a three-dimensional representation of the appearance of a molecule in a crystal. Scientists have employed X-ray crystallography to determine the crystal structures of many molecules.
In order to perform an X-ray crystallographic analysis, a sample of the crystal must be mounted onto a positioning device, then carefully aligned so that the entire crystal is within the diameter of the X-ray beam, and X-ray diffraction data collected at a number of rotational angles. Because the typical sizes of crystals and the diameter of the X-ray beam are in the range of 100 to 400 micrometers, the alignment requires a high degree of precision. In addition, to ensure the integrity of crystals, the crystals must be stored under liquid nitrogen and maintained at temperatures near that of liquid nitrogenduring the entire mounting, aligning, and data collecting processes. Currently, mounting and aligning of samples is performed manually.
A typical X-ray crystallography apparatus comprises an X-ray generator, a detector, and a rotating spindle onto which a finely adjustable head of the positioning device is mounted. Raw diffraction data collected by the detector are input into a computer for processing. The head of the positioning device allows minute adjustments in two axes that are perpendicular to one another and to the axis of the spindle. Some heads of positioning devices also allow for angular input into a computer for processing. The head of the positioning device allows minute adjustments in two axes that are perpendicular to one another and to the axis of the spindle. Some heads of positioning devices also allow for angular adjustments in one or more axes. A third axis of adjustment is provided by translation of the rotating spindle in a direction that is orthogonal to the two axes of the head of the positioning device. The sample mount position of the head of the positioning device is positioned so that when mounted, the sample is near the centerline of the X-ray beam. A CCD camera is mounted so that a magnified image of the mounted sample can be displayed on a video monitor. Cross-hairs on the video display indicate the desired position of the sample, corresponding to the intersection of the center of the X-ray beam with the axis of the spindle. In order to maintain the sample at a sufficiently low temperature once it is mounted, a stream of cold nitrogen gas is directed at the sample mount position.
The procedure for mounting and aligning a sample manually is described below. An operator places a sample into a small canister of liquid nitrogen and then maneuvers the canister near to the sample mount position on the head of the positioning device. As quickly as possible, the operator withdraws the sample and mounts it onto the head of the positioning device. Using the video image on the monitor, the operator turns adjustment screws controlling the “X”, “Y”, and “Z” axes until the sample is centered within the X-ray beam and spindle axes (as indicated by the cross-hairs on the video display). After the sample has been centered, analysis of the sample by X-ray diffraction is begun. The procedure is described in detail in Garman, et al., “Macromolecular Cryocrystallography”, J. Appl. Cryst. (1997) 30, 211-237 (hereinafter “Garman et al.”), incorporated herein by reference.
According to Garman et al., there are numerous problems involved in manual procedures for X-ray-diffraction data collection from macromolecular crystals at cryogenic temperatures. According to Garman, prerequisites for starting a cryogenic data collection are a reliable cryostat, the ability to maintain an ice-free environment, some crystal-mounting equipment, a sufficient number of crystals, and some manual dexterity for smooth and rapid operation on the part of the operator. An important part of a cryocrystallographic data collection is the method of crystal mounting and the hardware associated with it. Macromolecular crystals require special treatment compared to crystals of small molecules, because macromolecular crystals have a liquid content ranging from approximately 5 to 70%. The current most widely used technique is the loop method, wherein a loop is used to suspend a crystal by surface tension in a thin film of cryoprotected buffer. The first loops were made of gold-plated tungsten wire. These metal loops were replaced by loops made from various fine (10-50 &mgr;m diameter) fibers that do not absorb and scatter X-rays to the same extent as metal, such as hair, fibers of glass, nylon, rayon, fly-fishing threads, unwaxed dental floss, cotton, surgical thread and mohair wool.
There are several ways of connecting the loop-supporting pin to the head of the positioning device. Two widely used methods are insertion of a pin directly into the hole in the head of the positioning device and attachment of a magnet to the head of the positioning device, to which a magnetic pin-holder is attracted and rigidly held.
Evaporation from the film suspended in the loop is very rapid because of its large surface-to-volume ratio. Therefore, one of the most critical parameters in a cryocrystallographic experiment is the time between picking up the crystal and flash cooling it. This time should be as short as possible, ideally less than one second, otherwise the crystal can dehydrate or components of the buffer can precipitate. According to Garman et al., all manipulations and motions should be practised on several dry runs with nothing in the loop, to ensure smooth and rapid operation later on. No time should be wasted in viewing the crystal within the loop, since flash cooling an empty loop is less harmful than losing crystals before cooling by stopping to check whether they really are in the loop.
For most protein crystals, flash cooling in a gas stream is perfectly adequate and represents the safest and simplest option. From a practical standpoint, for gas-stream flash cooling it is helpful at first to have a second operator present who can divert the cold gas stream by holding a piece of card over it as soon as the “fisher” signals that the crystal is caught. Once the crystal is positioned, the card is then swiftly whipped away ensuring rapid and reproducible cooling. Experienced cryocrystallographers tend to divert the cold gas stream themselves or do not divert it at all while placing the crystal on the head of the positioning device, success depending on the quickness and certainty of their action.
The most common difficulty experienced by experimenters starting to use cryotechniques is ice around, near, on, and/or in the crystal. There are several reasons for ice forming around the crystal. The end of the cryonozzle may be positioned too far from the crystal: ideally it should be as close as possible since the temperature profile of the cold nitrogen stream is very sharp (the temperature rises from 100° K. to room temperature over a few millimeters for most open-flow systems). In addition, further away from the nozzle the gas stream becomes dissipated and is thus more susceptible to the effects of turbulence and drafts. If placing the cryonozzle near the crystal results in a shadow on the X-ray detector, thought should be given to changing the angle of approach of the stream. If this proves impossible, the shadow can be masked out during data processing.
A question that often arises concerns the optimum angle of incidence of the cold stream on the crystal. This is not an important factor in a draft-free and carefully monitored experiment. However, most cold streams operate better with the gas flowing downwards. Also, experimental constraints must be taken into account. For instance, for crystal storage enough space must be available to allow cryovial access.
In general, a major reason for ice formation is
Greer Jonathan
Jones Ronald B.
Muchmore Steven W.
Nienaber Vicki L.
Olson Jeffrey A.
Abbott Laboratories
Dunn Drew A.
Weinstein David L.
Yun Jurie
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