Clemens Schulze-Briese

The PILATUS 1M detector.

The PILATUS 1M detector is a hybrid pixel array detector with over one million pixels that operate in single photon counting mode. The detector, designed for macromolecular crystallography, is the largest pixel array detector currently in use at a synchrotron. It is a modular system consisting of 18 multichip modules covering an area of 21 cm x 24 cm. The design of the components as well as the manufacturing of the detector including the bump-bonding was performed at the Paul Scherrer Institute (PSI). The use of a single photon counting detector for protein crystallography requires detailed studies of the charge collection properties of the silicon sensor. The 18 modules are read out in parallel, leading to a full frame readout-time of 6.7 ms. This allows crystallographic data to be acquired in fine-varphi-slicing mode with continuous rotation of the sample. The detector was tested in several experiments at the protein crystallography beamline X06SA at the Swiss Light Source at PSI. Data were collected both in conventional oscillation mode using the shutter, as well as in a fine-varphi-slicing mode. After applying all the necessary corrections to data from a thaumatin crystal, the processing of the conventional data led to satisfactory merging R-factors of the order of 8.5%. This allows, for the first time, determination of a refined electron density map of a macromolecular biological crystal using a silicon pixel detector.

The molecular organization of cypovirus polyhedra.

Cypoviruses and baculoviruses are notoriously difficult to eradicate because the virus particles are embedded in micrometre-sized protein crystals called polyhedra. The remarkable stability of polyhedra means that, like bacterial spores, these insect viruses remain infectious for years in soil. The environmental persistence of polyhedra is the cause of significant losses in silkworm cocoon harvests but has also been exploited against pests in biological alternatives to chemical insecticides. Although polyhedra have been extensively characterized since the early 1900s, their atomic organization remains elusive. Here we describe the 2 Å crystal structure of both recombinant and infectious silkworm cypovirus polyhedra determined using crystals 5–12 micrometres in diameter purified from insect cells. These are the smallest crystals yet used for de novo X-ray protein structure determination. We found that polyhedra are made of trimers of the viral polyhedrin protein and contain nucleotides. Although the shape of these building blocks is reminiscent of some capsid trimers, polyhedrin has a new fold and has evolved to assemble in vivo into three-dimensional cubic crystals rather than icosahedral shells. The polyhedrin trimers are extensively cross-linked in polyhedra by non-covalent interactions and pack with an exquisite molecular complementarity similar to that of antigen–antibody complexes. The resulting ultrastable and sealed crystals shield the virus particles from environmental damage. The structure suggests that polyhedra can serve as the basis for the development of robust and versatile nanoparticles for biotechnological applications such as microarrays and biopesticides.

The Molecular Basis of Vitamin E Retention: Structure of Human α-Tocopherol Transfer Protein

Abstract α-Tocopherol transfer protein (α-TTP) is a liver protein responsible for the selective retention of α-tocopherol from dietary vitamin E, which is a mixture of α, β, γ, and δ-tocopherols and the corresponding tocotrienols. The α-TTP-mediated transfer of α-tocopherol into nascent VLDL is the major determinant of plasma α-tocopherol levels in humans. Mutations in the α-TTP gene have been detected in patients suffering from low plasma α-tocopherol and ataxia with isolated vitamin E deficiency (AVED). The crystal structure of α-TTP reveals two conformations. In its closed tocopherol-charged form, a mobile helical surface segment seals the hydrophobic binding pocket. In the presence of detergents, an open conformation is observed, which probably represents the membrane-bound form. The selectivity of α-TTP for RRR-α-tocopherol is explained from the van der Waals contacts occurring in the lipid-binding pocket. Mapping the known mutations leading to AVED onto the crystal structure shows that no mutations occur directly in the binding pocket.

Cryoradiolytic Reduction of Crystalline Heme Proteins: Analysis by Uv-Vis Spectroscopy and X-Ray Crystallography

The X-ray crystallographic analysis of redox-active systems may be complicated by photoreduction. Although radiolytic reduction by the probing X-ray beam may be exploited to generate otherwise short-lived reaction intermediates of metalloproteins, it is generally an undesired feature. Here, the X-ray-induced reduction of the three heme proteins myoglobin, cytochrome P450cam and chloroperoxidase has been followed by on-line UV-Vis absorption spectroscopy. All three systems showed a very rapid reduction of the heme iron. In chloroperoxidase the change of the ionization state from ferric to ferrous heme is associated with a movement of the heme-coordinating water molecule. The influence of the energy of the incident X-ray photons and of the presence of scavengers on the apparent reduction rate of ferric myoglobin crystals was analyzed.

Origin and temperature dependence of radiation damage in biological samples at cryogenic temperatures.

Radiation damage is the major impediment for obtaining structural information from biological samples by using ionizing radiation such as x-rays or electrons. The knowledge of underlying processes especially at cryogenic temperatures is still fragmentary, and a consistent mechanism has not been found yet. By using a combination of single-crystal x-ray diffraction, small-angle scattering, and qualitative and quantitative radiolysis experiments, we show that hydrogen gas, formed inside the sample during irradiation, rather than intramolecular bond cleavage between non-hydrogen atoms, is mainly responsible for the loss of high-resolution information and contrast in diffraction experiments and microscopy. The experiments that are presented in this paper cover a temperature range between 5 and 160 K and reveal that the commonly used temperature in x-ray crystallography of 100 K is not optimal in terms of minimizing radiation damage and thereby increasing the structural information obtainable in a single experiment. At 50 K, specific radiation damage to disulfide bridges is reduced by a factor of 4 compared to 100 K, and samples can tolerate a factor of 2.6 and 3.9 higher dose, as judged by the increase of Rfree values of elastase and cubic insulin crystals, respectively.

PILATUS: a two-dimensional X-ray detector for macromolecular crystallography

A large quantum-limited area X-ray detector for protein crystallography is under development at the Swiss Light Source. The final detector will be 2k � 2k pixels covering 40 � 40 cm 2 : A three-module prototype with 1120 � 157 pixels covering an active area of 24:3 � 3: 4c m 2 has been tested. X-rays above 6 keV with peak count rates exceeding 5 � 10 5 X-ray/pixel/s could be detected in single photon counting mode. Statistics of module production and results of threshold trimming are presented. To demonstrate the potential of this new detector, protein crystal data were collected at beamline 6S of the SLS. r 2002 Elsevier Science B.V. All rights reserved. PACS: 87.64.Bx

Optimal fine φ-slicing for single-photon-counting pixel detectors.

Fine ϕ-slicing substantially improves scaling statistics and anomalous signal for diffraction data collection with hybrid pixel detectors.

Determination of X-ray flux using silicon pin diodes.

Accurate measurement of photon flux from an X-ray source is a parameter required to calculate the dose absorbed by a sample. The development of a model for determining the photon flux incident on pin diodes, and experiments to test this model, are described for incident energies between 4 and 18 keV used in macromolecular crystallography.

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X‐ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK‐860) and Minalrestat (WAY‐509) were determined at atomic resolutions of 0.92 Å and 1.1 Å, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion‐binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestats hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo‐fluorobenzyl group inside the “specificity” pocket. The interactions between Minalrestats bromo‐fluorobenzyl group and the enzyme include the stacking against the side‐chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main‐chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active‐site residue His110. The 1′‐position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nϵ2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H‐atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl‐ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design. Proteins 2004.

The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses

Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the N-terminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be α-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-Å structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.