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Conformational dynamics are essential for proteins to function. We adapted time-resolved serial crystallography developed at x-ray lasers to visualize protein motions using synchrotrons. We recorded the structural changes in the light-driven proton-pump bacteriorhodopsin over 200 milliseconds in time. The snapshot from the first 5 milliseconds after photoactivation shows structural changes associated with proton release at a quality comparable to that of previous x-ray laser experiments. From 10 to 15 milliseconds onwards, we observe large additional structural rearrangements up to 9 angstroms on the cytoplasmic side. Rotation of leucine-93 and phenylalanine-219 opens a hydrophobic barrier, leading to the formation of a water chain connecting the intracellular aspartic acid–96 with the retinal Schiff base. The formation of this proton wire recharges the membrane pump with a proton for the next cycle. [ABSTRACT FROM AUTHOR]

Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space group C2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed. [ABSTRACT FROM AUTHOR]

In chemical synthesis, the widely used Birch reduction of aromatic compounds to cyclic dienes requires alkali metals in ammonia as extremely low-potential electron donors. An analogous reaction is catalyzed by benzoyl-coenzyme A reductases (BCRs) that have a key role in the globally important bacterial degradation of aromatic compounds at anoxic sites. Because of the lack of structural information, the catalytic mechanism of enzymatic benzene ring reduction remained obscure. Here, we present the structural characterization of a dearomatizing BCR containing an unprecedented tungsten cofactor that transfers electrons to the benzene ring in an aprotic cavity. Substrate binding induces proton transfer from the bulk solvent to the active site by expelling a Zn2+ that is crucial for active site encapsulation. Our results shed light on the structural basis of an electron transfer process at the negative redox potential limit in biology. They open the door for biological or biomimetic alternatives to a basic chemical synthetic tool. [ABSTRACT FROM AUTHOR]

Rhodopsin is a G‐protein‐coupled receptor that detects light and initiates the intracellular signalling cascades that underpin vertebrate vision. Light sensitivity is achieved by covalent linkage to 11‐cis retinal, which isomerizes upon photo‐absorption. Serial femtosecond crystallography data collected from rhodopsin microcrystals grown in the lipidic cubic phase were used to solve the room‐temperature structure of the receptor. Although the diffraction data showed high completeness and good consistency to 1.8 Å resolution, prominent electron‐density features remained unaccounted for throughout the unit cell after model building and refinement. A deeper analysis of the diffraction intensities uncovered the presence of a lattice‐translocation defect (LTD) within the crystals. The procedure followed to correct the diffraction intensities for this pathology enabled the building of an improved resting‐state model. The correction was essential to both confidently model the structure of the unilluminated state and interpret the light‐activated data collected after photo‐excitation of the crystals. It is expected that similar cases of LTD will be observed in other serial crystallography experiments and that correction will be required in a variety of systems. [ABSTRACT FROM AUTHOR]

The coenzyme A (CoA)-dependent aerobic benzoate metabolic pathway uses an unprecedented chemical strategy to overcome the high aromatic resonance energy by forming the non-aromatic 2,3-epoxybenzoyl-CoA. The crucial dearomatizing reaction is catalyzed by three enzymes, BoxABC, where BoxA is an NADPH-dependent reductase, BoxB is a benzoyl-CoA 2,3-epoxidase, and BoxC is an epoxide ring hydrolase. We characterized the key enzyme BoxB from Azoarcus evansii by structural and Mössbauer spectroscopic methods as a new member of class I diiron enzymes. Several family members were structurally studied with respect to the diiron center architecture, but no structure of an intact diiron enzyme with its natural substrate has been reported. X-ray structures between 1.9 and 2.5 Å resolution were determined for BoxB in the diferric state and with bound substrate benzoyl-CoA in the reduced state. The substrate-bound reduced state is distinguished from the diferric state by increased iron-ligand distances and the absence of directly bridging groups between them. The position of benzoyl-CoA inside a 20 Å long channel and the position of the phenyl ring relative to the diiron center are accurately defined. The C2 and C3 atoms of the phenyl ring are closer to one of the irons. Therefore, one oxygen of activated O2 must be ligated predominantly to this proximate iron to be in a geometrically suitable position to attack the phenyl ring. Consistent with the observed iron/phenyl geometry, BoxB stereoselectively should form the 2S,3R-epoxide. We postulate a reaction cycle that allows a charge delocalization because of the phenyl ring and the electron-withdrawing CoA thioester. [ABSTRACT FROM AUTHOR]

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines. [ABSTRACT FROM AUTHOR]

The article presents a summary of research published online at http://dx.doi.org/10.1126/science.aat0094 on the use of x-ray free-electron lasers (XFEL) radiation studying retinal isomerization structural dynamics in light-driven proton-pump bacteriorhodopsin (bR). It states that high-resolution x-ray diffraction data was gathered from bR microcrystals after the retinal chromophore was exited by optical laser pulses. It talks about the role of charge transfer in retinal isomerization.

A correction to the article "Structural basis of enzymatic benzene ring reduction" is presented.

A correction to the article "Structural basis of enzymatic benzene ring reduction" in the previous issue is presented.

A correction to the article "Fast native-SAD phasing for routine macromolecular structure determination," by Vincent Olieric, Lirong Chen, and Hua Zhang that was published online on December 15, 2014 is presented.

We present the structure of a photoactivated animal (6-4) photolyase in its radical pair state, captured by serial crystallography. We observe how a conserved asparigine moves towards the semiquinone FAD chromophore and stabilizes it by hydrogen bonding. Several amino acids around the final tryptophan radical rearrange, opening it up to the solvent. The structure explains how the protein environment stabilizes the radical pair state, which is crucial for function of (6-4) photolyases and cryptochromes. [ABSTRACT FROM AUTHOR]

Chloride transport by microbial rhodopsins is an essential process for which molecular details such as the mechanisms that convert light energy to drive ion pumping and ensure the unidirectionality of the transport have remained elusive. We combined time-resolved serial crystallography with time-resolved spectroscopy and multiscale simulations to elucidate the molecular mechanism of a chloride-pumping rhodopsin and the structural dynamics throughout the transport cycle. We traced transient anion-binding sites, obtained evidence for how light energy is used in the pumping mechanism, and identified steric and electrostatic molecular gates ensuring unidirectional transport. An interaction with the p-electron system of the retinal supports transient chloride ion binding across a major bottleneck in the transport pathway. These results allow us to propose key mechanistic features enabling finely controlled chloride transport across the cell membrane in this light-powered chloride ion pump. [ABSTRACT FROM AUTHOR]

The anaerobic oxidation of methane (AOM) with sulphate, an area currently generating great interest in microbiology, is accomplished by consortia of methanotrophic archaea (ANME) and sulphate-reducing bacteria. The enzyme activating methane in methanotrophic archaea has tentatively been identified as a homologue of methyl-coenzyme M reductase (MCR) that catalyses the methane-forming step in methanogenic archaea. Here we report an X-ray structure of the 280?kDa heterohexameric ANME-1 MCR complex. It was crystallized uniquely from a protein ensemble purified from consortia of microorganisms collected with a submersible from a Black Sea mat catalysing AOM with sulphate. Crystals grown from the heterogeneous sample diffract to 2.1?Å resolution and consist of a single ANME-1 MCR population, demonstrating the strong selective power of crystallization. The structure revealed ANME-1 MCR in complex with coenzyme M and coenzyme B, indicating the same substrates for MCR from methanotrophic and methanogenic archaea. Differences between the highly similar structures of ANME-1 MCR and methanogenic MCR include a F430 modification, a cysteine-rich patch and an altered post-translational amino acid modification pattern, which may tune the enzymes for their functions in different biological contexts. [ABSTRACT FROM AUTHOR]

The article focuses on time-resolved crystallography capturing the light-driven DNA repair mechanism by photolyase enzymes. Topics discussed include the enzyme's structural dynamics, flavin adenine dinucleotide (FAD) binding mode, disruption of water networks, and stepwise product release involving thymine base ejection from the active site.

The article focuses on a research which report the crystal structure of light-sensitive G protein coupled receptors (GPCRs) rhodopsin bound to an engineered mini-G protein. It mentions rhodopsin seems to adopt predominantly one thermodynamically stable active conformation and analysis of the well-defined GPCR–G protein interface suggests that the precise position of the carboxyl-terminal. It also mentions receptor is a significant determinant in selective G protein activation.

Nucleotidyl cyclases, includingmembrane-integral and soluble adenylyl and guanylyl cyclases, are central components in a wide range of signaling pathways. These proteins are architecturally diverse, yet many of them share a conserved feature, a helical region that precedes the catalytic cyclase domain. The role of this region in cyclase dimerization has been a subject of debate. Although mutations within this region in various cyclases have been linked to genetic diseases, the molecular details of their effects on the enzymes remain unknown. Here, we report an X-ray structure of the cytosolic portion of the membrane-integral adenylyl cyclase Cya from Mycobacterium intracellulare in a nucleotide-bound state. The helical domains of each Cya monomer form a tight hairpin, bringing the two catalytic domains into an active dimerized state. Mutations in the helical domain of Cya mimic the disease-related mutations in human proteins, recapitulating the profiles of the corresponding mutated enzymes, adenylyl cyclase-5 and retinal guanylyl cyclase-1. Our experiments with fulllength Cya and its cytosolic domain link the mutations to protein stability, and the ability to induce an active dimeric conformation of the catalytic domains. Sequence conservation indicates that this domain is an integral part of cyclase machinery across protein families and species. Our study provides evidence for a role of the helical domain in establishing a catalytically competent dimeric cyclase conformation. Our results also suggest that the disease-associated mutations in the corresponding regions of human nucleotidyl cyclases disrupt the normal helical domain structure. [ABSTRACT FROM AUTHOR]

Summary Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4 Å resolution. We combined X-ray crystallography, single-particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in immunoglobulin homology domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity. [ABSTRACT FROM AUTHOR]

Here, a method for presenting crystals of soluble and membrane proteins growing in the lipid cubic or sponge phase for in situ in situ diffraction data collection at cryogenic temperatures is introduced. The method dispenses with the need for the technically demanding and inefficient crystal-harvesting step that is an integral part of the lipid cubic phase or in meso in meso method of growing crystals. Crystals are dispersed in a bolus of mesophase sandwiched between thin plastic windows. The bolus contains tens to hundreds of crystals, visible with an in-line microscope at macromolecular crystallography synchrotron beamlines and suitably disposed for conventional or serial crystallographic data collection. Wells containing the crystal-laden boluses are removed individually from hermetically sealed glass plates in which crystallization occurs, affixed to pins on goniometer bases and excess precipitant is removed from around the mesophase. The wells are snap-cooled in liquid nitrogen, stored and shipped in Dewars, and manually or robotically mounted on a goniometer in a cryostream for diffraction data collection at 100 K, as is performed routinely with standard, loop-harvested crystals. The method is a variant on the recently introduced in meso in situ in meso in situ serial crystallography (IMISX) method that enables crystallographic measurements at cryogenic temperatures where crystal lifetimes are enormously enhanced whilst reducing protein consumption dramatically. The new approach has been used to generate high-resolution crystal structures of a G-protein-coupled receptor, α-helical and β-barrel transporters and an enzyme as model integral membrane proteins. Insulin and lysozyme were used as test soluble proteins. The quality of the data that can be generated by this method was attested to by performing sulfur and bromine SAD phasing with two of the test proteins. [ABSTRACT FROM AUTHOR]

The Parallel Robotics Inspired Goniometer (PRIGo) is a novel compact and high-precision goniometer providing an alternative to (mini-)kappa, traditional three-circle goniometers and Eulerian cradles used for sample reorientation in macromolecular crystallography. Based on a combination of serial and parallel kinematics, PRIGo emulates an arc. It is mounted on an air-bearing stage for rotation around ω and consists of four linear positioners working synchronously to achieve x, y, z translations and χ rotation (0-90°), followed by a ϕ stage (0-360°) for rotation around the sample holder axis. Owing to the use of piezo linear positioners and active correction, PRIGo features spheres of confusion of <1 µm, <7 µm and <10 µm for ω, χ and ϕ, respectively, and is therefore very well suited for micro-crystallography. PRIGo enables optimal strategies for both native and experimental phasing crystallographic data collection. Herein, PRIGo hardware and software, its calibration, as well as applications in macromolecular crystallography are described. [ABSTRACT FROM AUTHOR]

Optically controlled chemical reagents, termed "photopharmaceuticals," are powerful tools for precise spatiotemporal control of proteins particularly when genetic methods, such as knockouts or optogenetics are not viable options. However, current photopharmaceutical scaffolds, such as azobenzenes are intolerant of GFP/YFP imaging and are metabolically labile, posing severe limitations for biological use. We rationally designed a photoswitchable "SBT" scaffold to overcome these problems, then derivatized it to create exceptionally metabolically robust and fully GFP/YFP-orthogonal "SBTub" photopharmaceutical tubulin inhibitors. Lead compound SBTub3 allows temporally reversible, cell-precise, and even subcellularly precise photomodulation of microtubule dynamics, organization, and microtubule-dependent processes. By overcoming the previous limitations of microtubule photopharmaceuticals, SBTubs offer powerful applications in cell biology, and their robustness and druglikeness are favorable for intracellular biological control in in vivo applications. We furthermore expect that the robustness and imaging orthogonality of the SBT scaffold will inspire other derivatizations directed at extending the photocontrol of a range of other biological targets. • SBTub3 photocontrols microtubule dynamics, organization, and dependent processes • Microtubule photocontrol is cell and sub-cellularly precise and temporally reversible • SBT photocontrol is orthogonal to GFP/YFP imaging and SBTs are metabolically stable • The SBT scaffold is promising for photopharmaceuticals for other protein targets Photocontrollable reagents have unique potential as high spatiotemporal precision modulators of biological systems. Here, Gao et al. demonstrate a GFP-orthogonal and metabolically stable photoswitch that allows optical control over microtubule dynamics and architecture with subcellular resolution. The photoswitch scaffold also offers new possibilities for photopharmaceutical design against other targets. [ABSTRACT FROM AUTHOR]

De novo membrane protein structure determination is often limited by the availability of large crystals and the difficulties in obtaining accurate diffraction data for experimental phasing. Here we present a method that combines in situ serial crystallography with de novo phasing for fast, efficient membrane protein structure determination. The method enables systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without the need for direct crystal harvesting. The requisite data quality for experimental phasing is achieved by accumulating diffraction signals from isomorphous crystals identified post-data collection. The method works in all experimental phasing scenarios and is particularly attractive with fragile, weakly diffracting microcrystals. The automated serial data collection approach can be readily adopted at most microfocus macromolecular crystallography beamlines. Huang et al. report a fully automated method for collecting and merging data from thousands of in meso-grown microcrystals. This in situ approach provides a fast and efficient way to determine the structures of membrane proteins from fragile microcrystals. [ABSTRACT FROM AUTHOR]

Summary The dynamic instability of microtubules plays a key role in controlling their organization and function, but the cellular mechanisms regulating this process are poorly understood. Here, we show that cytoplasmic linker-associated proteins (CLASPs) suppress transitions from microtubule growth to shortening, termed catastrophes, including those induced by microtubule-destabilizing agents and physical barriers. Mammalian CLASPs encompass three TOG-like domains, TOG1, TOG2, and TOG3, none of which bind to free tubulin. TOG2 is essential for catastrophe suppression, whereas TOG3 mildly enhances rescues but cannot suppress catastrophes. These functions are inhibited by the C-terminal domain of CLASP2, while the TOG1 domain can release this auto-inhibition. TOG2 fused to a positively charged microtubule-binding peptide autonomously accumulates at growing but not shrinking ends, suppresses catastrophes, and stimulates rescues. CLASPs suppress catastrophes by stabilizing growing microtubule ends, including incomplete ones, preventing their depolymerization and promoting their recovery into complete tubes. TOG2 domain is the key determinant of these activities. [ABSTRACT FROM AUTHOR]