Macromolecular crystallography involves four stages: growth of crystals; collection of data;
structure determination and refinement; structure analysis. These images illustrate this
process:
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Crystals of the ligand-free form of LuxP/LuxQ (see
Neiditch et al 2005)
were initially found by the hanging drop vapor diffusion method (the photo
at left is from a sitting drop experiment, using larger volumes).
The hanging drop method is
compact and fast, and in conjunction with the commercially available factorial and sparse-matrix crystallization screens
(e.g. Crystal Screen from Hampton Research Inc) quite a large amount of "crystallization space" can be covered
with a modest consumption of protein. Nevertheless the requirements for a complete macromolecular structure determination typically
ranges from a few mg to several tens of mg of material.
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With suitable crystals, collection of X-ray diffraction images is a fairly routine matter. Crystals are typically
flash-cooled in a stream of nitrogen gas at 100K which dramatically reduces the incidence of radiation damage.
Data can be collected on relatively automated diffraction apparatus in-house (RAXIS-IV++, RAXIS-IIc) or at
local ultra-bright synchrotron X-ray beamlines (Brookhaven National Lab, CHESS at Cornell University). This diffraction
pattern was one of several collected at Brookhaven during data collection on the MC159 DED domain project (see
Li et al
2005).
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Since phase information is invariably lost during the data collection process, we must reconstruct the image
of the electron density for each molecule by computational means. For molecules for which structures of homologs
are available from the PDB,
the molecular replacement technique can often be a fast method of structure determination. For novel
structures, we typically use the multi-wavelength anomalous dispersion (MAD) technique, often in conjunction
with proteins that have seleno-methionine substituted for methionine during expression. The electron density map at left shows part
of an experimental map calculated using the MAD method from SeMet-substituted MC159 crystals, superimposed upon the final optimized structure. The electron
density map (cyan wire-frame contoured at 1 sigma) closely matches the atomic
model.
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Crystal structures themselves are fascinating in their complexity, and especially powerful in their ability to
provide a framework on which to interpret biological data and design new experiments. The structure of the
ligand-bound LuxP/LuxQ complex led to a new theory of action for that complex and for homologous proteins,
confirmed by experiments designed in light of this crystal structure. The "cartoon" representation of the
LuxP/LuxQ complex at left, optimized to show its secondary structure, belies the wealth of information that
exists within each three-dimensional macromolecular structure.
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