S. Michael Soltis

A novel evolutionarily conserved domain of cell-adhesion GPCRs mediates autoproteolysis.

The G protein‐coupled receptor (GPCR) Proteolysis Site (GPS) of cell‐adhesion GPCRs and polycystic kidney disease (PKD) proteins constitutes a highly conserved autoproteolysis sequence, but its catalytic mechanism remains unknown. Here, we show that unexpectedly the ∼40‐residue GPS motif represents an integral part of a much larger ∼320‐residue domain that we termed GPCR‐Autoproteolysis INducing (GAIN) domain. Crystal structures of GAIN domains from two distantly related cell‐adhesion GPCRs revealed a conserved novel fold in which the GPS motif forms five β‐strands that are tightly integrated into the overall GAIN domain. The GAIN domain is evolutionarily conserved from tetrahymena to mammals, is the only extracellular domain shared by all human cell‐adhesion GPCRs and PKD proteins, and is the locus of multiple human disease mutations. Functionally, the GAIN domain is both necessary and sufficient for autoproteolysis, suggesting an autoproteolytic mechanism whereby the overall GAIN domain fine‐tunes the chemical environment in the GPS to catalyse peptide bond hydrolysis. Thus, the GAIN domain embodies a unique, evolutionarily ancient and widespread autoproteolytic fold whose function is likely relevant for GPCR signalling and for multiple human diseases.

Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis.

Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca2+- and Mg2+-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca2+-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca2+ triggering, moves en bloc as Ca2+ influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

New paradigm for macromolecular crystallography experiments at SSRL: automated crystal screening and remote data collection

Through the combination of robust mechanized experimental hardware and a flexible control system with an intuitive user interface, SSRL researchers have screened over 200 000 biological crystals for diffraction quality in an automated fashion. Three quarters of SSRL researchers are using these data-collection tools from remote locations.

Laue diffraction study on the structure of cytochrome c peroxidase compound I

BACKGROUND Cytochrome c peroxidase from yeast is a soluble haem-containing protein found in the mitochondrial electron transport chain where it probably protects against toxic peroxides. The aim of this study was to obtain a reliable structure for the doubly oxidized transient intermediate (termed compound I) in the reaction of cytochrome c peroxidase with hydrogen peroxide. This intermediate contains a semistable free radical on Trp191, and an oxyferryl haem group. RESULTS Compound I was produced in crystals of yeast cytochrome c peroxidase by reacting the crystalline enzyme with hydrogen peroxide in a flow cell. The reaction was monitored by microspectrophotometry and Laue crystallography in separate experiments. A nearly complete conversion to compound I was achieved within two minutes of the addition of hydrogen peroxide, and the concentration of the intermediate remained at similar levels for an additional half an hour. The structure of the intermediate was determined by Laue diffraction. The refined Laue structure for compound I shows clear structural changes at the peroxide-binding site but no significant changes at the radical site. The photographs were processed with a new software package (LEAP), overcoming many of the former problems encountered in extracting structural information from Laue exposures. CONCLUSIONS The geometry of the haem environment in this protein allows structural changes to be extremely small, similar in magnitude to those observed for the Fe2+/Fe3+ transition in cytochrome c. The results suggest that these molecules have evolved to transfer electrons with a minimal need for structural adjustment.

Goniometer-based femtosecond crystallography with X-ray free electron lasers

Significance The extremely short and bright X-ray pulses produced by X-ray free-electron lasers unlock new opportunities in crystallography-based structural biology research. Efficient methods to deliver crystalline material are necessary due to damage or destruction of the crystal by the X-ray pulse. Crystals for the first experiments were 5 µm or smaller in size, delivered by a liquid injector. We describe a highly automated goniometer-based approach, compatible with crystals of larger and varied sizes, and accessible at cryogenic or ambient temperatures. These methods, coupled with improvements in data-processing algorithms, have resulted in high-resolution structures, unadulterated by the effects of radiation exposure, from only 100 to 1,000 diffraction images. The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

Web-Ice: Integrated Data Collection and Analysis for Macromolecular Crystallography

New software tools are introduced to facilitate diffraction experiments involving large numbers of crystals. While existing programs have long provided a framework for lattice indexing, Bragg spot integration, and symmetry determination, these initial data processing steps often require significant manual effort. This limits the timely availability of data analysis needed for high-throughput procedures, including the selection of the best crystals from a large sample pool, and the calculation of optimal data collection parameters to assure complete spot coverage with minimal radiation damage. To make these protocols more efficient, we developed a network of software applications and application servers, collectively known as Web-Ice. When the package is installed at a crystallography beamline, a programming interface allows the beamline control software (e.g., Blu-Ice / DCSS) to trigger data analysis automatically. Results are organized based on a list of samples that the user provides, and are examined within a Web page, accessible both locally at the beamline or remotely. Optional programming interfaces permit the user to control data acquisition through the Web browser. The system as a whole is implemented to support multiple users and multiple processors, and can be expanded to provide additional scientific functionality. Web-Ice has a distributed architecture consisting of several stand-alone software components working together via a well defined interface. Other synchrotrons or institutions may integrate selected components or the whole of Web-Ice with their own data acquisition software. Updated information about current developments may be obtained at http://smb.slac.stanford.edu/research/developments/webice.

N6-methyladenosine in mRNA disrupts tRNA selection and translation elongation dynamics

N6-methylation of adenosine (forming m6A) is the most abundant post-transcriptional modification within the coding region of mRNA, but its role during translation remains unknown. Here, we used bulk kinetic and single-molecule methods to probe the effect of m6A in mRNA decoding. Although m6A base-pairs with uridine during decoding, as shown by X-ray crystallographic analyses of Thermus thermophilus ribosomal complexes, our measurements in an Escherichia coli translation system revealed that m6A modification of mRNA acts as a barrier to tRNA accommodation and translation elongation. The interaction between an m6A-modified codon and cognate tRNA echoes the interaction between a near-cognate codon and tRNA, because delay in tRNA accommodation depends on the position and context of m6A within codons and on the accuracy level of translation. Overall, our results demonstrate that chemical modification of mRNA can change translational dynamics.

Structure of the proton-gated urea channel from the gastric pathogen Helicobacter pylori.

Half the world’s population is chronically infected with Helicobacter pylori, causing gastritis, gastric ulcers and an increased incidence of gastric adenocarcinoma. Its proton-gated inner-membrane urea channel, HpUreI, is essential for survival in the acidic environment of the stomach. The channel is closed at neutral pH and opens at acidic pH to allow the rapid access of urea to cytoplasmic urease. Urease produces NH3 and CO2, neutralizing entering protons and thus buffering the periplasm to a pH of roughly 6.1 even in gastric juice at a pH below 2.0. Here we report the structure of HpUreI, revealing six protomers assembled in a hexameric ring surrounding a central bilayer plug of ordered lipids. Each protomer encloses a channel formed by a twisted bundle of six transmembrane helices. The bundle defines a previously unobserved fold comprising a two-helix hairpin motif repeated three times around the central axis of the channel, without the inverted repeat of mammalian-type urea transporters. Both the channel and the protomer interface contain residues conserved in the AmiS/UreI superfamily, suggesting the preservation of channel architecture and oligomeric state in this superfamily. Predominantly aromatic or aliphatic side chains line the entire channel and define two consecutive constriction sites in the middle of the channel. Mutation of Trp 153 in the cytoplasmic constriction site to Ala or Phe decreases the selectivity for urea in comparison with thiourea, suggesting that solute interaction with Trp 153 contributes specificity. The previously unobserved hexameric channel structure described here provides a new model for the permeation of urea and other small amide solutes in prokaryotes and archaea.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography

X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

Concentric-flow electrokinetic injector enables serial crystallography of ribosome and photosystem II

We describe a concentric-flow electrokinetic injector for efficiently delivering microcrystals for serial femtosecond X-ray crystallography analysis that enables studies of challenging biological systems in their unadulterated mother liquor. We used the injector to analyze microcrystals of Geobacillus stearothermophilus thermolysin (2.2-Å structure), Thermosynechococcus elongatus photosystem II (<3-Å diffraction) and Thermus thermophilus small ribosomal subunit bound to the antibiotic paromomycin at ambient temperature (3.4-Å structure).