An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin.

Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.

A redox switch allows binding of Fe(II) and Fe(III) ions in the cyanobacterial iron-binding protein FutA from Prochlorococcus.

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.

A versatile approach to high-density microcrystals in lipidic cubic phase for room-temperature serial crystallography.

Serial crystallography has emerged as an important tool for structural studies of integral membrane proteins. The ability to collect data from micrometre-sized weakly diffracting crystals at room temperature with minimal radiation damage has opened many new opportunities in time-resolved studies and drug discovery. However, the production of integral membrane protein microcrystals in lipidic cubic phase at the desired crystal density and quantity is challenging. This paper introduces VIALS (versatile approach to high-density microcrystals in lipidic cubic phase for serial crystallography), a simple, fast and efficient method for preparing hundreds of microlitres of high-density microcrystals suitable for serial X-ray diffraction experiments at both synchrotron and free-electron laser sources. The method is also of great benefit for rational structure-based drug design as it facilitates in situ crystal soaking and rapid determination of many co-crystal structures. Using the VIALS approach, room-temperature structures are reported of (i) the archaerhodopsin-3 protein in its dark-adapted state and 110 ns photocycle intermediate, determined to 2.2 and 1.7 Å, respectively, and (ii) the human A2A adenosine receptor in complex with two different ligands determined to a resolution of 3.5 Å.

Dose-resolved serial synchrotron and XFEL structures of radiation-sensitive metalloproteins.

An approach is demonstrated to obtain, in a sample- and time-efficient manner, multiple dose-resolved crystal structures from room-temperature protein microcrystals using identical fixed-target supports at both synchrotrons and X-ray free-electron lasers (XFELs). This approach allows direct comparison of dose-resolved serial synchrotron and damage-free XFEL serial femtosecond crystallography structures of radiation-sensitive proteins. Specifically, serial synchrotron structures of a heme peroxidase enzyme reveal that X-ray induced changes occur at far lower doses than those at which diffraction quality is compromised (the Garman limit), consistent with previous studies on the reduction of heme proteins by low X-ray doses. In these structures, a functionally relevant bond length is shown to vary rapidly as a function of absorbed dose, with all room-temperature synchrotron structures exhibiting linear deformation of the active site compared with the XFEL structure. It is demonstrated that extrapolation of dose-dependent synchrotron structures to zero dose can closely approximate the damage-free XFEL structure. This approach is widely applicable to any protein where the crystal structure is altered by the synchrotron X-ray beam and provides a solution to the urgent requirement to determine intact structures of such proteins in a high-throughput and accessible manner.

Resolving polymorphs and radiation-driven effects in microcrystals using fixed-target serial synchrotron crystallography.

The ability to determine high-quality, artefact-free structures is a challenge in micro-crystallography, and the rapid onset of radiation damage and requirement for a high-brilliance X-ray beam mean that a multi-crystal approach is essential. However, the combination of crystal-to-crystal variation and X-ray-induced changes can make the formation of a final complete data set challenging; this is particularly true in the case of metalloproteins, where X-ray-induced changes occur rapidly and at the active site. An approach is described that allows the resolution, separation and structure determination of crystal polymorphs, and the tracking of radiation damage in microcrystals. Within the microcrystal population of copper nitrite reductase, two polymorphs with different unit-cell sizes were successfully separated to determine two independent structures, and an X-ray-driven change between these polymorphs was followed. This was achieved through the determination of multiple serial structures from microcrystals using a high-throughput high-speed fixed-target approach coupled with robust data processing.

A natural, single-residue substitution yields a less active peptaibiotic: the structure of bergofungin A at atomic resolution.

Bergofungin is a peptide antibiotic that is produced by the ascomycetous fungus Emericellopsis donezkii HKI 0059 and belongs to peptaibol subfamily 2. The crystal structure of bergofungin A has been determined and refined to 0.84 Šresolution. This is the second crystal structure of a natural 15-residue peptaibol, after that of samarosporin I. The amino-terminal phenylalanine residue in samarosporin I is exchanged to a valine residue in bergofungin A. According to agar diffusion tests, this results in a nearly inactive antibiotic peptide compared with the moderately active samarosporin I. Crystals were obtained from methanol solutions of purified bergofungin mixed with water. Although there are differences in the intramolecular hydrogen-bonding scheme of samarosporin I, the overall folding is very similar for both peptaibols, namely 310-helical at the termini and α-helical in the middle of the molecules. Bergofungin A and samarosporin I molecules are arranged in a similar way in both lattices. However, the packing of bergofungin A exhibits a second solvent channel along the twofold axis. This latter channel occurs in the vicinity of the N-terminus, where the natural substitution resides.

Polyhedra structures and the evolution of the insect viruses.

Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽ 1.5 Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These structures illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule's evolution so that surface residues are almost as conserved as the internal residues.

Structure of CPV17 polyhedrin determined by the improved analysis of serial femtosecond crystallographic data.

The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared with a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis.

Visualization of membrane protein crystals in lipid cubic phase using X-ray imaging.

The focus in macromolecular crystallography is moving towards even more challenging target proteins that often crystallize on much smaller scales and are frequently mounted in opaque or highly refractive materials. It is therefore essential that X-ray beamline technology develops in parallel to accommodate such difficult samples. In this paper, the use of X-ray microradiography and microtomography is reported as a tool for crystal visualization, location and characterization on the macromolecular crystallography beamlines at the Diamond Light Source. The technique is particularly useful for microcrystals and for crystals mounted in opaque materials such as lipid cubic phase. X-ray diffraction raster scanning can be used in combination with radiography to allow informed decision-making at the beamline prior to diffraction data collection. It is demonstrated that the X-ray dose required for a full tomography measurement is similar to that for a diffraction grid-scan, but for sample location and shape estimation alone just a few radiographic projections may be required.

Picornavirus uncoating intermediate captured in atomic detail.

It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release.

The crystal structure of samarosporin I at atomic resolution.

The atomic resolution structures of samarosporin I have been determined at 100 and 293 K. This is the first crystal structure of a natural 15-residue peptaibol. The amino acid sequence in samarosporin I is identical to emerimicin IV and stilbellin I. Samarosporin is a peptide antibiotic produced by the ascomycetous fungus Samarospora rostrup and belongs to peptaibol subfamily 2. The structures at both temperatures are very similar to each other adopting mainly a 310-helical and a minor fraction of α-helical conformation. The helices are significantly bent and packed in an antiparallel fashion in the centered monoclinic lattice leaving among them an approximately 10-Å channel extending along the crystallographic twofold axis. Only two ordered water molecules per peptide molecule were located in the channel. Comparisons have been carried out with crystal structures of subfamily 2 16-residue peptaibols antiamoebin and cephaibols. The repercussion of the structural analysis of samarosporin on membrane function is discussed.

Outrunning free radicals in room-temperature macromolecular crystallography.

A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A(2A) adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macromolecular crystallography.

Four complete turns of a curved 310-helix at atomic resolution: the crystal structure of the peptaibol trichovirin I-4A in a polar environment suggests a transition to α-helix for membrane function.

The first crystal structure of a member of peptaibol antibiotic subfamily 4, trichovirin I-4A (14 residues), has been determined by direct methods and refined at atomic resolution. The monoclinic unit cell has two molecules in the asymmetric unit. Both molecules assume a 310 right-handed helical conformation and are significantly bent. The molecules pack loosely along the crystallographic twofold axis, forming two large tunnels between symmetry-related molecules in which no ordered solvent could be located. Carbonyl O atoms which are not involved in intramolecular hydrogen bonding participate in close van der Waals interactions with apolar groups. The necessary amphipathicity for biological activity of peptaibols is not realised in the crystal structure. Hence, a structural change of trichovirin to an α-helical conformation is proposed for membrane integration and efficient water/ion transportation across the lipid bilayer.

How baculovirus polyhedra fit square pegs into round holes to robustly package viruses.

Natural protein crystals (polyhedra) armour certain viruses, allowing them to survive for years under hostile conditions. We have determined the structure of polyhedra of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), revealing a highly symmetrical covalently cross-braced robust lattice, the subunits of which possess a flexible adaptor enabling this supra-molecular assembly to specifically entrap massive baculoviruses. Inter-subunit chemical switches modulate the controlled release of virus particles in the unusual high pH environment of the target insect's gut. Surprisingly, the polyhedrin subunits are more similar to picornavirus coat proteins than to the polyhedrin of cytoplasmic polyhedrosis virus (CPV). It is, therefore, remarkable that both AcMNPV and CPV polyhedra possess identical crystal lattices and crystal symmetry. This crystalline arrangement must be particularly well suited to the functional requirements of the polyhedra and has been either preserved or re-selected during evolution. The use of flexible adaptors to generate a powerful system for packaging irregular particles is characteristic of the AcMNPV polyhedrin and may provide a vehicle to sequester a wide range of objects such as biological nano-particles.