In the crystal structure of bovine mitochondrial F1-ATPase determined at 2.8 A resolution, the three catalytic beta-subunits differ in conformation and in the bound nucleotide. The structure supports a catalytic mechanism in intact ATP synthase in which the three catalytic subunits are in different states of the catalytic cycle at any instant. Interconversion of the states may be achieved by rotation of the alpha 3 beta 3 subassembly relative to an alpha-helical domain of the gamma-subunit.

With a size of 372kDa, the F~ ATPase particle is the largest asymmetric structure solved to date. lsomorphous differences arising from reacting the crystals with methyl-mercury nitrate at two concentrations allowed the structure determination. Careful data collection and data processing were essential in this process as well as a new form of electron-density modification, 'solvent flipping'. The most important feature of this new procedure is that the electron density in the solvent region is inverted rather than set to a constant value, as in conventional solvent flat&ening. All non-standard techniques and variations on new techniques which were employed in the structure determination are described.

Antithrombin, a plasma serpin, is relatively inactive as an inhibitor of the coagulation proteases until it binds to the heparan side chains that line the microvasculature. The binding specifically occurs to a core pentasaccharide present both in the heparans and in their therapeutic derivative heparin. The accompanying conformational change of antithrombin is revealed in a 2.9-Å structure of a dimer of latent and active antithrombins, each in complex with the high-affinity pentasaccharide. Inhibitory activation results from a shift in the main sheet of the molecule from a partially six-stranded to a five-stranded form, with extrusion of the reactive center loop to give a more exposed orientation. There is a tilting and elongation of helix D with the formation of a 2-turn helix P between the C and D helices. Concomitant conformational changes at the heparin binding site explain both the initial tight binding of antithrombin to the heparans and the subsequent release of the antithrombin-protease complex into the circulation. The pentasaccharide binds by hydrogen bonding of its sulfates and carboxylates to Arg-129 and Lys-125 in the D-helix, to Arg-46 and Arg-47 in the A-helix, to Lys-114 and Glu-113 in the P-helix, and to Lys-11 and Arg-13 in a cleft formed by the amino terminus. This clear definition of the binding site will provide a structural basis for developing heparin analogues that are more specific toward their intended target antithrombin and therefore less likely to exhibit side effects.Heparin, a sulfated polysaccharide, is second only to insulin as a natural therapeutic agent and is the initial-choice anticoagulant in the treatment and prevention of thromboembolic disease. It functions in life as a component of the heparans that line the inner walls of the microvascular system (1), but heparin as a drug is a heterogeneous animal extract administered by injection to circulate in the bloodstream. Both heparin and the natural heparans contain a specific pentasaccharide fragment (2, 3) that binds and activates the plasma proteinase inhibitor antithrombin.In nature, this binding to heparans substantially localizes the function of antithrombin to inhibition of the serine proteases of the coagulation cascade within the bloodstream, allowing their coagulant activity in damaged tissue outside the vascular system. The heparans and the longer-chain heparins (4) activate the inhibition of thrombin by antithrombin by bringing them into close apposition, but there is also a direct activation of inhibition due to an overall conformational change (5) induced by the binding to the core pentasaccharide present in both heparin and heparans. This pentasaccharide-induced change alters the conformation of the reactive site loop of antithrombin (6, 7) and gives a 300-fold increase in inhibitory activity against the key coagulation protease factor Xa. Linked to this is a change in affinity at the heparin binding site (see Fig. 1), and as antithrombin contacts the pentasaccharide, it moves from an initial low-affinit...

Crystallography of nanocrystalline materials has witnessed a true revolution in the past 10 years, thanks to the introduction of protocols for 3D acquisition and analysis of electron diffraction data. This method provides single-crystal data of structure solution and refinement quality, allowing the atomic structure determination of those materials that remained hitherto unknown because of their limited crystallinity. Several experimental protocols exist, which share the common idea of sampling a sequence of diffraction patterns while the crystal is tilted around a noncrystallographic axis, namely, the goniometer axis of the transmission electron microscope sample stage. This Outlook reviews most important 3D electron diffraction applications for different kinds of samples and problematics, related with both materials and life sciences. Structure refinement including dynamical scattering is also briefly discussed.

The reactive site loop of the serpin family of serine proteinase inhibitors is flexible and can adopt a number of diverse conformations. A 2.9 A resolution structure of alpha 1-antitrypsin-the principal proteinase inhibitor in human plasma-shows the loop in a stable canonical conformation matching that found in all other families of serine proteinase inhibitors. This unexpected finding in the absence of loop insertion into the body of the molecule favours a two-stage mechanism of inhibition and provides a model for the heparin activation of antithrombin. The beta-pleated strand conformation of the loop also accounts for the polymerization of the serpins in disease and for their association with other beta-sheet structures, most notably the beta-amyloid of Alzheimer's disease.

The structures of the bacterial F1-ATPase alpha and beta subunits are very similar to their counterparts in the mitochondrial enzyme, suggesting a common catalytic mechanism. The study presented here allows an analysis of the different conformations adopted by the alpha and beta subunits and may ultimately further our understanding of this mechanism.

When protein crystals are submicrometre-sized, X-ray radiation damage precludes conventional diffraction data collection. For crystals that are of the order of 100 nm in size, at best only single-shot diffraction patterns can be collected and rotation data collection has not been possible, irrespective of the diffraction technique used. Here, it is shown that at a very low electron dose (at most 0.1 e À Å À2 ), a Medipix2 quantum area detector is sufficiently sensitive to allow the collection of a 30-frame rotation series of 200 keV electrondiffraction data from a single $100 nm thick protein crystal. A highly parallel 200 keV electron beam ( = 0.025 Å ) allowed observation of the curvature of the Ewald sphere at low resolution, indicating a combined mosaic spread/beam divergence of at most 0.4 . This result shows that volumes of crystal with low mosaicity can be pinpointed in electron diffraction. It is also shown that strategies and data-analysis software (MOSFLM and SCALA) from X-ray protein crystallography can be used in principle for analysing electron-diffraction data from three-dimensional nanocrystals of proteins.

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