Thomas R. Schneider

Serial crystallography on in vivo grown microcrystals using synchrotron radiation

The structure solution of T. brucei cathepsin B from 80 in vivo grown crystals with an average volume of 9u2005µm3 obtained by serial synchrotron crystallography at a microfocus beamline is reported.

Structure of catalase HPII from Escherichia coli at 1.9 A resolution.

Catalase HPII from Escherichia coli, a homotetramer of subunits with 753 residues, is the largest known catalase. The structure of native HPII has been refined at 1.9 Å resolution using X‐ray synchrotron data collected from crystals flash‐cooled with liquid nitrogen. The crystallographic agreement factors R and Rfree are respectively 16.6% and 21.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222‐point group symmetry. The structure of the central part of the HPII subunit gives a root mean square deviation of 1.5 Å for 477 equivalencies with beef liver catalase. Most of the additional 276 residues of HPII are located in either an extended N‐terminal arm or in a C‐terminal domain organized with a flavodoxin‐like topology. A small number of mostly hydrophilic interactions stabilize the relative orientation between the C‐terminal domain and the core of the enzyme. The heme component of HPII is a cis‐hydroxychlorin γ‐spirolactone in an orientation that is flipped 180°with respect to the orientation of the heme found in beef liver catalase. The proximal ligand of the heme is Tyr415 which is joined by a covalent bond between its Cβ atom and the Nδ atom of His392. Over 2,700 well‐defined solvent molecules have been identified filling a complex network of cavities and channels formed inside the molecule. Two channels lead close to the distal side heme pocket of each subunit suggesting separate inlet and exhaust functions. The longest channel, that begins in an adjacent subunit, is over 50 Å in length, and the second channel is about 30 Å in length. A third channel reaching the heme proximal side may provide access for the substrate needed to catalyze the heme modification and His‐Tyr bond formation. HPII does not bind NADPH and the equivalent region to the NADPH binding pocket of bovine catalase, partially occluded in HPII by residues 585–590, corresponds to the entrance to the second channel. The heme distal pocket contains two solvent molecules, and the one closer to the iron atom appears to exhibit high mobility or low occupancy compatible with weak coordination. Proteins 1999;34:155–166.

The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design

Insights into proteasome inhibition Proteasomes are large protein complexes that degrade and remove proteins to maintain proper cellular physiology and growth. Proteasomes are a validated target for anticancer therapy, but drug design has been hampered by poor understanding of how inhibitors interact with the active site. Schrader et al. succeeded in crystallizing various proteasome-inhibitor complexes. They subsequently obtained crystal structures for the native human proteasome and eight different inhibitor complexes at resolutions between 1.9 and 2.1 Å. The inhibitors sampled include drugs that are approved or in trial for cancer treatment. Science, this issue p. 594 High-resolution structures of human 20S proteasomes reveal chemical principles for next-generation drug design. The proteasome is a validated target for anticancer therapy, and proteasome inhibition is employed in the clinic for the treatment of tumors and hematological malignancies. Here, we describe crystal structures of the native human 20S proteasome and its complexes with inhibitors, which either are drugs approved for cancer treatment or are in clinical trials. The structure of the native human 20S proteasome was determined at an unprecedented resolution of 1.8 angstroms. Additionally, six inhibitor-proteasome complex structures were elucidated at resolutions between 1.9 and 2.1 angstroms. Collectively, the high-resolution structures provide new insights into the catalytic mechanisms of inhibition and necessitate a revised description of the proteasome active site. Knowledge about inhibition mechanisms provides insights into peptide hydrolysis and can guide strategies for the development of next-generation proteasome-based cancer therapeutics.

X-ray crystallography reveals stringent conservation of protein fold after removal of the only disulfide bridge from a stabilized immunoglobulin variable domain

BACKGROUNDnImmunoglobulin domains owe a crucial fraction of their conformational stability to an invariant central disulfide bridge, the closure of which requires oxidation. Under the reducing conditions prevailing in cell cytoplasm, accumulation of soluble immunoglobulin is prohibited by its inability to acquire and maintain the native conformation. Previously, we have shown that disulfide-free immunoglobulins can be produced in Escherichia coli and purified from cytoplasmic extracts.nnnRESULTSnImmunoglobulin REIv is the variable domain of a human kappa light chain. The disulfide-free variant REIv-C23V/Y32H was crystallized and its structure analyzed by X-ray crystallography (2.8 A resolution). The conformation of the variant is nearly identical to that of the wild-type protein and the conformationally stabilized variant REIv-T39K. This constitutes the first crystal structure of an immunoglobulin fragment without a disulfide bridge. The lack of the disulfide bridge produces no obvious local change in structure (compared with the wild type), whereas the Y32H mutation allows the formation of an additional hydrogen bond. There is a further change in the structure that is seen in the dimer in which Tyr49 has flipped out of the dimer interface in the mutant.nnnCONCLUSIONSnImmunoglobulin derivatives without a central disulfide bridge but with stringently conserved wild-type conformation can be constructed in a practical two-step approach. First, the protein is endowed with additional folding stability by the introduction of one or more stabilizing amino acid exchanges; second, the disulfide bridge is destroyed by substitution of one of the two invariant cysteines. Such derivatives can be accumulated in soluble form in the cytoplasmic compartment of the E. coli cell. Higher protein yields and evolutionary refinement of catalytic antibodies by genetic complementation are among the possible advantages.

P13, the EMBL macromolecular crystallography beamline at the low-emittance PETRA III ring for high- and low-energy phasing with variable beam focusing

The P13 macromolecular crystallography beamline, based on the low-emittance source PETRA III, enables X-ray diffraction experiments on macromolecular crystals over a wide wavelength range (0.7–3.1u2005Å). The beam has a variable focus size and a small divergence enabling data collection on micrometre-sized crystals.

Terahertz radiation induces non-thermal structural changes associated with Fröhlich condensation in a protein crystal

Whether long-range quantum coherent states could exist in biological systems, and beyond low-temperature regimes where quantum physics is known to be applicable, has been the subject to debate for decades. It was proposed by Fröhlich that vibrational modes within protein molecules can order and condense into a lowest-frequency vibrational mode in a process similar to Bose-Einstein condensation, and thus that macroscopic coherence could potentially be observed in biological systems. Despite the prediction of these so-called Fröhlich condensates almost five decades ago, experimental evidence thereof has been lacking. Here, we present the first experimental observation of Fröhlich condensation in a protein structure. To that end, and to overcome the challenges associated with probing low-frequency molecular vibrations in proteins (which has hampered understanding of their role in proteins function), we combined terahertz techniques with a highly sensitive X-ray crystallographic method to visualize low-frequency vibrational modes in the protein structure of hen-egg white lysozyme. We found that 0.4 THz electromagnetic radiation induces non-thermal changes in electron density. In particular, we observed a local increase of electron density in a long α-helix motif consistent with a subtle longitudinal compression of the helix. These observed electron density changes occur at a low absorption rate indicating that thermalization of terahertz photons happens on a micro- to milli-second time scale, which is much slower than the expected nanosecond time scale due to damping of delocalized low frequency vibrations. Our analyses show that the micro- to milli-second lifetime of the vibration can only be explained by Fröhlich condensation, a phenomenon predicted almost half a century ago, yet never experimentally confirmed.

A novel β-xylosidase structure from Geobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

Geobacillus thermoglucosidasius is a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding a G. thermoglucosidasius β-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed in Escherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme-substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of the G. thermoglucosidasius β-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.

Synchrotron radiation: micrometer-sized x-ray beams as fine tools for macromolecular crystallography.

Structural data play a central role in understanding biological function at the molecular level. At present, the majority of high‐resolution structural data about biological macromolecules and their complexes originates from crystallography. In crystal structure determination, the major hurdle to overcome is the production of crystals of sufficient size and quality. High‐flux x‐ray beams with diameters of a few micrometers or less help to alleviate this problem as small beams allow the use of small crystals or scanning of large crystals for regions of acceptable diffraction. Using sophisticated x‐ray optics and mechanics with submicrometer precision, Riekel et al. [Acta Crystallogr., Sect. D: Biol. Crystallogr., 64, 158–166 (2008)], have recently demonstrated that an x‐ray beam of 1 μm can be used to determine the crystal structure of a protein to a resolution of 1.5 Å. The smallest volume from which usable diffraction data were collected amounted to 20 μm3, corresponding to not more than 2×108 unit cells. In a diffraction volume of micrometer dimensions, radiation damage is expected to be reduced with respect to large volumes as a significant fraction of the photoelectrons produced by the incident radiation escapes from the diffracting volume before dissipating their energy. The possibility to make use of small and/or inhomogeneous crystals in combination with a possible reduction in radiation damage due to size effects has the potential to make many more systems amenable to crystal structure analysis.

Data collection with a tailored X‐ray beam size at 2.69 Å wavelength (4.6 keV): sulfur SAD phasing of Cdc23Nterm

Data collection with a tailored 50u2005µm diameter X-ray beam at 4.6u2005keV (λ = 2.69u2005Å) on the newly established EMBL beamline P13 at PETRA III allowed the crystal structure determination of the Cdc23Nterm homodimer (65.4u2005kDa; 12 Cys and ten Met residues) by sulfur SAD phasing at 3.1u2005Å resolution while overcoming crystal twinning.

A multicrystal diffraction data-collection approach for studying structural dynamics with millisecond temporal resolution

In situ crystallization using a Kapton sandwich assembly allows diffraction data to be recorded from multiple protein crystals at room temperature with millisecond temporal resolution at high-brilliance synchrotron X-ray radiation sources.