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Insertion sequence (IS) elements are the simplest autonomous transposable elements found in prokaryotic genomes1. We recently discovered that IS110 family elements encode a recombinase and a non-coding bridge RNA (bRNA) that confers modular specificity for target DNA and donor DNA through two programmable loops2. Here we report the cryo-electron microscopy structures of the IS110 recombinase in complex with its bRNA, target DNA and donor DNA in three different stages of the recombination reaction cycle. The IS110 synaptic complex comprises two recombinase dimers, one of which houses the target-binding loop of the bRNA and binds to target DNA, whereas the other coordinates the bRNA donor-binding loop and donor DNA. We uncovered the formation of a composite RuvC-Tnp active site that spans the two dimers, positioning the catalytic serine residues adjacent to the recombination sites in both target and donor DNA. A comparison of the three structures revealed that (1) the top strands of target and donor DNA are cleaved at the composite active sites to form covalent 5-phosphoserine intermediates, (2) the cleaved DNA strands are exchanged and religated to create a Holliday junction intermediate, and (3) this intermediate is subsequently resolved by cleavage of the bottom strands. Overall, this study reveals the mechanism by which a bispecific RNA confers target and donor DNA specificity to IS110 recombinases for programmable DNA recombination. more...

LAT1 (SLC7A5) transports large neutral amino acids and plays pivotal roles in cancer proliferation, immune response and drug delivery. Despite recent advances in structural understanding of LAT1, how it discriminates substrates and inhibitors including the clinically relevant drugs remains elusive. Here we report six structures of LAT1 across three conformations with bound ligands, elucidating its substrate transport and inhibitory mechanisms. JPH203 (also known as nanvuranlat or KYT-0353), an anticancer drug in clinical trials, traps LAT1 in an outward-facing state with a U-shaped conformer, with its amino-phenylbenzoxazol moiety pushing against transmembrane helix 3 (TM3) and bending TM10. Physiological substrates like ʟ-Phe lack such effects, whereas melphalan poses steric hindrance, explaining its inhibitory activity. The "classical" system L inhibitor BCH induces an occluded state critical for transport, confirming its substrate-like behavior. These findings provide a structural basis for substrate recognition and inhibition of LAT1, guiding future drug design. Lee et al. report six cryo-EM structures of LAT1 in complex with various substrates and inhibitors, including nanvuranlat (JPH203) under clinical trials. These structures provide insights into the mechanisms of amino acid transport and inhibition, aiding future drug design. [ABSTRACT FROM AUTHOR] more...

Metals are essential components for the structure and function of many proteins. However, accurate modelling of their coordination environments remains a challenge due to the complexity and diversity of metal‐coordination geometries. To address this, a method is presented for extracting and analysing coordination information, including bond lengths and angles, from the Crystallography Open Database. By using these data, comprehensive descriptions of metal‐containing components are generated. A stereochemical information generator for a particular component within a specific macromolecule leverages an example PDB/mmCIF file containing the component to account for the actual surrounding environment. A matching process has been developed and implemented to align the derived metal structures with idealized coordinates from a coordination geometry library. Additionally, various strategies, depending on the quality of the matches, were employed to compile distance and angle statistics for the refinement of macromolecular structures. The developed methods were implemented in a new program, MetalCoord, that classifies and utilizes the metal‐coordination geometry. The effectiveness of the developed algorithms was tested using metal‐containing components from the PDB. As a result, metal‐containing components from the CCP4 monomer library have been updated. The updated monomer dictionaries, in concert with the derived restraints, can be used in most structural biology computations, including macromolecular crystallography, single‐particle cryo‐EM and even molecular mechanics. [ABSTRACT FROM AUTHOR] more...

Author(s): Wataru Shihoya [sup.1] , Keiichi Inoue [sup.2] [sup.3] [sup.4] [sup.5] , Manish Singh [sup.2] , Masae Konno [sup.2] , Shoko Hososhima [sup.2] , Keitaro Yamashita [sup.1] [sup.6] , Kento [...], Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics.sup.1. They are widely present in bacteria, archaea, algae and algal viruses.sup.2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the [beta]-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins. A crystal structure of Thermoplasmatales archaeon heliorhodopsin at 2.4 Å resolution shows that it adopts a similar fold to that of type I rhodopsin--despite the low sequence identity--but there are also several marked differences that provide insights into heliorhodopsin function. more...

Author(s): Yoon Seok Kim [sup.1] , Hideaki E. Kato [sup.2] [sup.3] , Keitaro Yamashita [sup.4] , Shota Ito [sup.5] , Keiichi Inoue [sup.3] [sup.5] [sup.6] , Charu Ramakrishnan [sup.1] , [...], The naturally occurring channelrhodopsin variant anion channelrhodopsin-1 (ACR1), discovered in the cryptophyte algae Guillardia theta, exhibits large light-gated anion conductance and high anion selectivity when expressed in heterologous settings, properties that support its use as an optogenetic tool to inhibit neuronal firing with light. However, molecular insight into ACR1 is lacking owing to the absence of structural information underlying light-gated anion conductance. Here we present the crystal structure of G. theta ACR1 at 2.9 Å resolution. The structure reveals unusual architectural features that span the extracellular domain, retinal-binding pocket, Schiff-base region, and anion-conduction pathway. Together with electrophysiological and spectroscopic analyses, these findings reveal the fundamental molecular basis of naturally occurring light-gated anion conductance, and provide a framework for designing the next generation of optogenetic tools.The crystal structure of anion channelrhodopsin-1 (ACR1) from the algae Guillardia theta provides insights into the basis of anion conductance. more...

Hydrogen (H) atoms are abundant in macromolecules and often play critical roles in enzyme catalysis, ligand‐recognition processes and protein–protein interactions. However, their direct visualization by diffraction techniques is challenging. Macromolecular X‐ray crystallography affords the localization of only the most ordered H atoms at (sub‐)atomic resolution (around 1.2 Å or higher). However, many H atoms of biochemical significance remain undetectable by this method. In contrast, neutron diffraction methods enable the visualization of most H atoms, typically in the form of deuterium (2H) atoms, at much more common resolution values (better than 2.5 Å). Thus, neutron crystallography, although technically demanding, is often the method of choice when direct information on protonation states is sought. REFMAC5 from the Collaborative Computational Project No. 4 (CCP4) is a program for the refinement of macromolecular models against X‐ray crystallographic and cryo‐EM data. This contribution describes its extension to include the refinement of structural models obtained from neutron crystallographic data. Stereochemical restraints with accurate bond distances between H atoms and their parent atom nuclei are now part of the CCP4 Monomer Library, the source of prior chemical information used in the refinement. One new feature for neutron data analysis in REFMAC5 is refinement of the protium/deuterium (1H/2H) fraction. This parameter describes the relative 1H/2H contribution to neutron scattering for hydrogen isotopes. The newly developed REFMAC5 algorithms were tested by performing the (re‐)refinement of several entries available in the PDB and of one novel structure (FutA) using either (i) neutron data only or (ii) neutron data supplemented by external restraints to a reference X‐ray crystallographic structure. Re‐refinement with REFMAC5 afforded models characterized by R‐factor values that are consistent with, and in some cases better than, the originally deposited values. The use of external reference structure restraints during refinement has been observed to be a valuable strategy, especially for structures at medium–low resolution. [ABSTRACT FROM AUTHOR] more...

Many organisms capture light energy and information using the rhodopsin family of proteins, which comprise the heptahelical transmembrane (7-TM) proteins called opsins covalently linked to retinal. Based on their primary [...], Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven [Na.sup.+] pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves [Na.sup.+] transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate [Na.sup.+] transport. Together with the structure-based engineering of the first light-driven [K.sup.+] pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics. more...

PSII is a multi-subunit pigment-protein complex embedded in the thylakoid membranes of higher plants, green algae and cyanobacteria, and is the only molecular machine capable of oxidizing water by use [...], Photosynthesis converts light energy into biologically useful chemical energy vital to life on Earth. The initial reaction of photosynthesis takes place in photosystem II (PSII), a 700-kilodalton homodimeric membrane protein complex that catalyses photo-oxidation of water into dioxygen through an S-state cycle of the oxygen evolving complex (OEC). The structure of PSII has been solved by X-ray diffraction (XRD) at 1.9 angstrom resolution, which revealed that the OEC is a [Mn.sub.4]Ca[O.sub.5]-cluster coordinated by a well defined protein environment (1). However, extended X-ray absorption fine structure (EXAFS) studies showed that the manganese cations in the OEC are easily reduced by X-ray irradiation (2), and slight differences were found in the Mn-Mn distances determined by XRD (1), EXAFS (3-7) and theoretical studies (8-14). Here we report a 'radiation-damage-free' structure of PSII from Thermosynechococcus vulcanusin the [S.sub.1] state at a resolution of 1.95 angstroms using femtosecond X-ray pulses of the SPring-8 angstrom compact free-electron laser (SACLA) and hundreds of large, highly isomorphous PSII crystals. Compared with the structure from XRD, the OEC in the X-ray free electron laser structure has Mn-Mn distances that are shorter by 0.1-0.2 angstroms. The valences of each manganese atom were tentatively assigned as Mn1D(m), Mn2C(IV), Mn3B(IV) and Mn4A(III), based on the average Mn-ligand distances and analysis of the Jahn-Teller axis on Mn(III). One of the oxo-bridged oxygens, O5, has significantly longer distances to Mn than do the other oxo-oxygen atoms, suggesting that O5 is a hydroxide ion instead of a normal oxygen dianion and therefore may serve as one of the substrate oxygen atoms. These findings provide a structural basis for the mechanism of oxygen evolution, and we expect that this structure will provide a blueprint for the design of artificial catalysts for water oxidation. more...